PI: Jiang, Mengxi Title: Intersection of polyomavirus infection and host cellular responses

Received: 06/02/2015 FOA: PA13-302 Council: 01/2016

Competition ID: FORMS-C FOA Title: RESEARCH PROJECT GRANT (PARENT R01)

1 R01 AI123162-01 Dual: Accession Number: 3825771

IPF: 1288803 Organization: UNIVERSITY OF ALABAMA AT BIRMINGHAM

Former Number: Department: Microbiology

IRG/SRG: VIRB AIDS: N Expedited: N

Subtotal Direct Costs 

(excludes consortium F&A) 

Year 1:

Year 2:

Year 3:

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Animals: N 

Humans: N 

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Current HS Code: 10 

HESC: N

New Investigator: Y

Early Stage Investigator: Y

Senior/Key Personnel: Organization: Role Category:

Mengxi Jiang UNIVERSITY OF ALABAMA AT 

BIRMINGHAM

PD/PI 

University of Alabama at Birmingham Co-Investigator

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Intersection of polyomavirus infection and host cellular responses

12. PROPOSED PROJECT

Start Date* Ending Date*

04/01/2016 03/31/2021

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AL-007
Contact PD/PI: Jiang, Mengxi 

Tracking Number: GRANT11922341 Funding Opportunity Number: PA-13-302 . Received Date:

2015-06-02T17:13:17.000-04:00

SF 424 (R&R) APPLICATION FOR FEDERAL ASSISTANCE Page 2

14. PROJECT DIRECTOR/PRINCIPAL INVESTIGATOR CONTACT INFORMATION

Prefix: First Name*: Mengxi Middle Name: Last Name*: Jiang Suffix: 

Position/Title: Assistant Professor

Organization Name*: UNIVERSITY OF ALABAMA AT BIRMINGHAM

Department: Microbiology

Division: School of Medicine

Street1*: BBRB 834, 845 19th Street South

Street2:

City*: Birmingham

County:

State*: AL: Alabama

Province:

Country*: USA: UNITED STATES

ZIP / Postal Code*: 352942170

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❍ PROGRAM HAS NOT BEEN SELECTED BY STATE FOR

REVIEW

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Position/Title*: Director

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Department: Office of Sponsored Programs

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Street1*: 1720 2nd Avenue South

Street2: AB 1170

City*: Birmingham

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State*: AL: Alabama

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Country*: USA: UNITED STATES

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06/02/2015

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424 R&R and PHS-398 Specific

Table Of Contents Page Numbers

SF 424 R&R Cover Page----------------------------------------------------------------------------------------- 1

 Table of Contents------------------------------------------------------------------------- 3

Performance Sites--------------------------------------------------------------------------------------------- 4

Research & Related Other Project Information------------------------------------------------------------------ 5

 Project Summary/Abstract(Description)----------------------------------------------------- 6

 Project Narrative------------------------------------------------------------------------- 7

 Facilities & Other Resources-------------------------------------------------------------- 8

 Equipment--------------------------------------------------------------------------------- 10

Research & Related Senior/Key Person-------------------------------------------------------------------------- 11

PHS398 Cover Page Supplement---------------------------------------------------------------------------------- 22

PHS 398 Modular Budget---------------------------------------------------------------------------------------- 24

 Personnel Justification------------------------------------------------------------------- 30

PHS 398 Research Plan----------------------------------------------------------------------------------------- 31

 Specific Aims----------------------------------------------------------------------------- 32

 Research Strategy------------------------------------------------------------------------- 33

 Bibliography & References Cited----------------------------------------------------------- 45

 Letters Of Support------------------------------------------------------------------------ 50

 

Contact PD/PI: Jiang, Mengxi 

Table of Contents Page 3
	
Contact PD/PI: Jiang, Mengxi 

OMB Number: 4040-0010

Expiration Date: 06/30/2016

Tracking Number: GRANT11922341 Funding Opportunity Number: PA-13-302. Received Date:

2015-06-02T17:13:17.000-04:00

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Tracking Number: GRANT11922341 Funding Opportunity Number: PA-13-302. Received Date:

2015-06-02T17:13:17.000-04:00

OMB Number: 4040-0001

Expiration Date: 06/30/2016

RESEARCH & RELATED Other Project Information

1. Are Human Subjects Involved?* ❍ Yes ● No

1.a. If YES to Human Subjects

 Is the Project Exempt from Federal regulations? ❍ Yes ❍ No

If YES, check appropriate exemption number: 1 2 3 4 5 6

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Is the IACUC review Pending? ❍ Yes ❍ No

IACUC Approval Date:

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3. Is proprietary/privileged information included in the application?* ❍ Yes ● No

4.a. Does this project have an actual or potential impact - positive or negative - on the environment?* ❍ Yes ● No

4.b. If yes, please explain:

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environmental assessment (EA) or environmental impact statement (EIS) been performed?

4.d. If yes, please explain:

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collaborators?*

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7. Project Summary/Abstract* Project_Abstract_Final.pdf Mime Type: application/pdf

8. Project Narrative* Project_Narrative_Final.pdf Mime Type: application/pdf

9. Bibliography & References Cited References_Final.pdf Mime Type: application/pdf

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Contact PD/PI: Jiang, Mengxi 

 Page 5
	
PROJECT SUMMARY/ABSTRACT 

Polyomaviruses cause a variety of severe human diseases particularly in immunocompromised individuals. 

No specific anti-viral treatments or prophylactic approaches exist to target this family of viruses. There are 

several critical gaps in our current knowledge of the molecular mechanism of viral replication and 

tumorigenesis. Our long-term goals are to identify how these viruses subvert normal host cellular processes to 

facilitate viral replication, and how these interactions may result in oncogenesis. 

Our previous studies revealed an intricate balanced relationship between viral replication and virus-induced 

host genomic instability. These results lead to our central hypothesis that an activated cellular DNA damage 

response (DDR) is important for facilitating viral replication and maintaining host genome stability during 

polyomavirus infection. Towards this hypothesis, we have identified host mismatch repair system and 

replicating viral DNA as novel factors contributing to DDR activation. We have also discovered that the ability 

of polyomavirus to cause host genomic DNA damage is linked to its ability to replicate viral DNA. Guided by 

strong preliminary data, we propose to pursue three Specific Aims to characterize DDR activation mechanism 

and how the DDR ties together viral replication and host genomic stability: (1) To define the role of host 

mismatch repair proteins in polyomavirus replication and polyomavirus-induced DDR activation. (2) To 

determine the viral DNA triggers that activate the DDR upon polyomavirus infection. (3) To elucidate the 

molecular mechanism by which polyomavirus induces host genome instability. 

Collectively, our proposed research will broadly impact the field by characterizing the essential roles that 

the DDR plays in promoting viral replication and maintaining host genome stability. These studies will have the 

potential to uncover novel molecular mechanisms underlying polyomavirus replication as well as viral 

oncogenesis. These findings may be extrapolated to other DNA viruses and to our understanding of normal 

cellular processes. 

 

 

Contact PD/PI: Jiang, Mengxi 

Project Summary/Abstract Page 6
	
PROJECT NARRATIVE 

 Polyomaviruses are a family of viruses associated with severe human diseases and a subset of them can 

also cause cancers. Our proposed studies aim to understand the interactions between polyomaviruses and the 

host DNA damage response, a cellular pathway important for both viral replication and host genome 

maintenance. This research will have the potential to reveal novel therapeutic host targets to treat 

polyomavirus-related diseases. 

 

Contact PD/PI: Jiang, Mengxi 

Project Narrative Page 7
	
 

Contact PD/PI: Jiang, Mengxi 

FACILITIES AND OTHER RESOURCES

Jiang Laboratory: 

Laboratory: 

Approximately 1000 square feet of laboratory space are dedicated for use by Dr. Jiang and affiliated personnel

on the 8th floor of the Bevill Biomedical Sciences Research Building. The laboratory is operated at biosafety 

level II (BSL-2) containment level and is equipped for cell culture, molecular biology, biochemical, and cell 

biology techniques. There are two 4-ft biosafety cabinets and two CO2 cell culture incubators that are suited for 

working with infectious viruses. There are two common equipment rooms on the 4th and 7th floor and include 

all major equipment needed. 

Biohazards:

All of the BSL-2 work proposed in the studies will be performed in the Jiang laboratory. There are designated 

and approved areas in the laboratory for handling infectious agents including BK polyomavirus and lentivirus. 

Approval from the UAB Occupational Health and Safety Office will be obtained for all the studies involving 

infectious agents. An annual laboratory audit will be performed by UAB to ensure compliance with the safety 

requirements. All members of the lab have been trained to safely handle infectious agents and are re-trained 

on a yearly basis. 

Office: 

Dr. Jiang’s office is located directly next to the lab space. It is a 105 square foot office with a window opening 

into the laboratory allowing ease of communication with lab members throughout the day. 

Computer: 

A Dell PC Desktop connected to both LaserJet and color printers is readily accessible in the Jiang lab. In 

addition, Dr. Jiang has a PC laptop and a Macbook Pro laptop. All computers are outfitted with a wide suite of 

software including Microsoft Office, Adobe Creative Suite, DNA sequence and proteomics analysis software, 

statistics, and graphic design software. The computers are linked to the Microbiology network, providing 

access to additional software, a color printer/scanner, the Internet, and a storage server for data backup. 

Telecommunication is available allowing Dr. Jiang to communicate with other investigators in the field of 

polyomavirus research on a regular basis to seek advice and collaborations if needed.

Administrative Support: 

Dr. Jiang’s research is supported by the administrative offices of the Department of Microbiology at UAB. Full 

time personnel are available for grant management and general administrative services.

Laboratory: 

Laboratory: 

 has 1,800 square feet of newly renovated lab space on the first floor of Volker Hall. The lab is fully 

equipped with standard laboratory items and sufficient bench space for 10 individuals. Additional tissue culture 

rooms, common use space with major equipment, and adjacent conference rooms are readily accessible. 

Office: 

 has a 420 square foot office 

, with full secretarial and business office support staff. Dr. has 

a Mac Quad-Core computer, a color laser printer, and scanner/fax machine in her office in the Department 

office suite, which is immediately adjacent to the lab. 

Other Resources: 

University core facilities: 

Numerous shared core facilities are maintained at the UAB. These include: UAB high resolution imaging core

capable of 3D and 2D Confocal Laser Microscopy, Electron Microscopy (EM) including transmission EM and 

Cryo-EM; flow cytometry facilities equipped with BSL-3 live cell sorting; UAB mass spectrometry/proteomics 

consortium performing both high resolution and quantitative analyses of small molecules and selected 

peptides; the Heflin Center for Genomic Sciences provide Next Generation Sequence analysis and standard 

Sanger sequencing analysis; state of the art animal facilities; biostatistics and bioinformatics cores; X-ray 

crystallography and NMR facilities; tissue procurement and biobanking cores; hybridoma cores for monoclonal 

antibody production; biofermentation cores for recombinant protein production. All core facilities are within 

close proximity from the PI’s laboratory space. 

Facilities & Other Resources Page 8
	
Department support: 

The Department of Microbiology is extremely supportive of Dr. Jiang’s career development as a junior 

independent scientist. At least 80% of Dr. Jiang’s effort will be protected for research activities for the next five 

years. The department chair also helped Dr. Jiang to form a mentoring committee consisting of senior faculty 

from both within and outside of the department to guide Dr. Jiang on research, publications, grantsmanship, 

and laboratory management. There is annual faculty retreat and department retreat, providing Dr. Jiang and 

her trainees opportunities to present research and seek collaborations. Please see attached Department Chair 

Letter of Support. 

Collaborative Research Environment at UAB: 

UAB is a highly collaborative and collegial research university. There are 25 University-wide Interdisciplinary 

Research Centers, including an NCI designated Comprehensive Cancer Center (CCC), an NIH-designated 

Center for AIDS Research (CFAR), Center for Clinical and Translational Science (CCTS), and the Center for 

Emerging Drug Discovery (which serves as the operational base for UAB participation in the Alabama Drug 

Discovery Alliance). As such, UAB provides a rich intellectual environment in which basic, translational and 

clinical investigators collaborate.

There are about 40 faculty including 15 virologists in the Department of Microbiology with most of them located 

in the same building as Dr. Jiang. Additionally, there are virologists in the Departments of Biochemistry, 

Pediatrics, Internal Medicine, and the CFAR. Scientific discussion and communication occur on a daily basis 

among faculty. This open environment has greatly facilitated research collaborations with our colleagues 

including Drs. Luo, Chow, Broker, Xu. There are weekly seminar series in the department, CFAR, and Cancer 

Center by invited outside speakers. Dr. Jiang holds a monthly joint meeting with Dr. Guangxiang Luo’s (a 

senior virologist in the Department of Microbiology) group in addition to the weekly lab meeting to stimulate 

idea exchanges. There is also a virology discussion group at UAB consisting of faculty members, postdoctoral 

fellows, and students from virology labs on campus. This group meets weekly with rotating group presentations. 

All of these activities greatly enhance the quality and excellence of our research and education and therefore 

will facilitate the successful completion of our proposed studies. 

 

Contact PD/PI: Jiang, Mengxi 

Facilities & Other Resources Page 9
	
 

Contact PD/PI: Jiang, Mengxi 

EQUIPMENT

Major equipment available in the Jiang laboratory: Two tissue culture incubators, a liquid nitrogen storage 

tank, -80°C and -20°C freezers, a 4°C refrigerator, an inverted microscope, and a refrigerated centrifuge with 

a swinging bucket rotor and a Fiberlite rotor are located within Dr. Jiang’s lab space for cell culture 

experiments. Two biosafety hoods and one chemical fume hood are installed. We also have all the necessary 

equipment for molecular biology, including a refrigerated microfuge, two regular microfuges, a thermocycler, 

electrophoresis power supplies and apparatus, analytical balances, water baths and incubators, and many 

small laboratory items. A walk-in cold room is located right across from the Jiang lab space for biochemical 

experiments and storage of reagents. 

Major equipment available in the laboratory: Instrumentation of 2-D gels and DNA fiber analyses 

are available in the lab. The lab is also equipped with biosafety cabinets, incubators, water baths, 

phase microscopes and coulter counter. The facility also contains humidified CO2 incubators for 384 wellformatted plates, a MultiDrop Combi for liquid /media dispensing, CLARIOstar plate reader (BMG labtech) 

with 5 primary detection modes (absorbance, fluorescence intensity, fluorescence polarization, time-resolved 

fluorescence, luminescence); and an ABI QuantStudio 7 Flex sequence detection system for RT-PCR. 

Additional common use space includes baculovirus and bacterial culture facilities, immunofluorescent 

microscopes, dark room, and other large pieces of equipment, including RT-PCR machines, fluorescent plate 

readers, -20 ̊C and -80 ̊C freezers, and liquid N2 storage units for cell line storage.

Shared Equipment in the Department of Microbiology: A number of shared resources are available in the 

Department of Microbiology, which are all readily accessible within the same building as Dr. Jiang’s lab. 

These include: ultra-, medium, and low speed centrifuges, fluorescence microscope, real-time PCR, 

nanodrop spectrophotometer, gel documentation and Odyssey imaging system, phosphorimager, scintillation 

counters, shaker incubators, dark room with film processor, autoclaves, and dishwashers. Other centralized 

resources and equipment are described in the Facilities and Other Resources section. 

Overall, all the necessary equipment to successfully carry out the proposed studies are in place either in the 

Jiang and labs, or as part of a well-organized shared program and UAB core facilities. 

Equipment Page 10
	
Contact PD/PI: Jiang, Mengxi 

OMB Number: 4040-0001

Expiration Date: 06/30/2016

Tracking Number: GRANT11922341 Funding Opportunity Number: PA-13-302 . Received Date:

2015-06-02T17:13:17.000-04:00

RESEARCH & RELATED Senior/Key Person Profile (Expanded)

PROFILE - Project Director/Principal Investigator

Prefix:  First Name*: Mengxi Middle Name  Last Name*: Jiang Suffix: 

Position/Title*: Assistant Professor

Organization Name*: UNIVERSITY OF ALABAMA AT BIRMINGHAM

Department: Microbiology

Division: School of Medicine

Street1*: BBRB 834, 845 19th Street South

Street2:

City*: Birmingham

County:

State*: AL: Alabama

Province:

Country*: USA: UNITED STATES

Zip / Postal Code*: 352942170

Phone

Number*: 

Fax Number:  E-Mail*: 

Credential, e.g., agency login: 

Project Role*:  PD/PI Other Project Role Category: 

Degree Type:  Ph.D. Degree Year:  2006

File Name Mime Type

Attach Biographical Sketch*: Jiang_Biosketch_Final.pdf application/pdf

Attach Current & Pending Support:

 Page 11 
Contact PD/PI: Jiang, Mengxi 

Tracking Number: GRANT11922341 Funding Opportunity Number: PA-13-302 . Received Date:

2015-06-02T17:13:17.000-04:00

PROFILE - Senior/Key Person

Prefix:  First Name*:  Middle Name  Last Name*:  Suffix: 

Position/Title*:

Organization Name*: University of Alabama at Birmingham

Department: Pharmacology and Toxicology

Division:

Street1*:

Street2:

City*:

County:

State*:

Province:

Country*:

Zip / Postal Code*:

Phone

Number*: 

Fax Number:  E-Mail*: 

Credential, e.g., agency login: 

Project Role*:  Co-Investigator Other Project Role Category: 

Degree Type:  Degree Year: 

File Name Mime Type

Attach Biographical Sketch*: lication/pdf

Attach Current & Pending Support:

 Page 12 
 

Contact PD/PI: Jiang, Mengxi 

OMB No. 0925-0001/0002 (Rev. 08/12 Approved Through 8/31/2015)

BIOGRAPHICAL SKETCH

Provide the following information for the Senior/key personnel and other significant contributors.

Follow this format for each person. DO NOT EXCEED FIVE PAGES.

NAME: Jiang, Mengxi

eRA COMMONS USER NAME (agency login): 

POSITION TITLE: Assistant Professor

EDUCATION/TRAINING (Begin with baccalaureate or other initial professional education, such as nursing,

include postdoctoral training and residency training if applicable.)

INSTITUTION AND LOCATION DEGREE

(if applicable)

Completion

Date 

MM/YYYY

FIELD OF STUDY

Fudan University BS 06/2001 Life Sciences

University of Michigan, Ann Arbor,

MI

PHD 08/2006 Molecular, Cellular & Developmental

Biology

University of Michigan, Ann Arbor,

MI

Postdoctoral

Fellow

01/2012 Microbiology & Immunology

A. Personal Statement

The goal of the proposed research is to elucidate how the host DNA damage response (DDR) regulates viral

replication and host genomic stability during polyomavirus infection. I have the technical expertise and

leadership experience to perform the proposed studies. My graduate work focused on ribosome biogenesis in

E. coli, during which I pioneered the usage of powerful quantitative proteomic methodologies to analyze

defective ribosomal particles and to identify novel ribosome-associated proteins. As a postdoctoral fellow in Dr.

Michael Imperiale’s lab, my research focused on the molecular characterization of various aspects of host-BK

polyomavirus interactions, including viral entry, viral replication, and host nuclear architecture rearrangements.

I discovered that polyomavirus replication and host genome stability rely on an activated host DDR pathway

during infection, which laid the groundwork for this proposal. I have extensive experience in polyomavirus

molecular virology and cell biology, and have been leading and designing research projects throughout my

research career. Since I started my independent research career at UAB, my laboratory has discovered that

mismatch repair proteins are required for polyomavirus replication and that viral DNA replication drives the

activation of the DDR. The current application builds logically on my prior work and these novel findings.

Additionally, I have teamed up with as a co-investigator to provide expertise in DNA

damage and repair for this project. The characterization of the important functions of the DDR for both viral

replication and host genome maintenance has the potential to broadly impact the DNA virus and tumor virology

field. Collectively, I have demonstrated both research skills and productivity to be the principal investigator to

address these important and fundamental questions regarding the intersection of polyomavirus infections and

host cellular responses.

1. Jiang M, Abend JR, Tsai B, Imperiale MJ. Early events during BK virus entry and disassembly. J Virol.

2009 Feb;83(3):1350-8. PubMed PMID: 19036822; PubMed Central PMCID: PMC2620883. 

2. Jiang M, Entezami P, Gamez M, Stamminger T, Imperiale MJ. Functional reorganization of

promyelocytic leukemia nuclear bodies during BK virus infection. MBio. 2011;2(1):e00281-10. PubMed

PMID: 21304169; PubMed Central PMCID: PMC3039439. 

3. Jiang M, Zhao L, Gamez M, Imperiale MJ. Roles of ATM and ATR-mediated DNA damage responses

during lytic BK polyomavirus infection. PLoS Pathog. 2012;8(8):e1002898. PubMed PMID: 22952448; 

PubMed Central PMCID: PMC3431332. 

4. Verhalen B, Justice JL, Imperiale MJ, Jiang M. Viral DNA replication-dependent DNA damage response

activation during BK polyomavirus infection. J Virol. 2015 May;89(9):5032-9. PubMed PMID: 25694603; 

PubMed Central PMCID: PMC4403456. 

Biosketches Page 13
	
B. Positions and Honors

Positions and Employment

2001 - 2005 Graduate Student Instructor, University of Michigan, Department of Molecular, Cellular & 

Developmental Biology, Ann Arbor, MI 

2001 - 2006 Graduate Research Assistant, University of Michigan, Department of Molecular, Cellular & 

Developmental Biology, Ann Arbor, MI 

2006 - 2012 Research Fellow, University of Michigan, Department of Microbiology & Immunology, Ann 

Arbor, MI 

2012 - 2013 Research Investigator, University of Michigan, Department of Microbiology & Immunology, Ann 

Arbor, MI 

2014 - Assistant Professor, University of Alabama at Birmingham, Department of Microbiology, 

Birmingham, AL 

Other Experience and Professional Memberships

2008 - Full Member, American Society for Virology 

2010 - 2012 Member, Sigma Xi, The Scientific Research Society 

2011 - Ad Hoc Reviewer, PloS One, Journal of Virology, International Journal of Tropical Disease & 

Health, Antiviral Research, American Journal of Transplantation, Journal of Cardiovascular 

Disease Research, Applied and Environmental Microbiology

2014 - Member, American Heart Association 

2014 - Member, American Society for Microbiology 

2014 - Member, UAB Comprehensive Cancer Center 

2014 - Member, UAB Center for AIDS Research 

2014 - Editorial Board Member, Clinical Journal of Microbiology & Pathology 

Honors

1997 Monsanto Fellowship, Fudan University 

2001 Outstanding student fellowships, First Prize, Fudan University 

2005 Arnold Ravin-Muriel Rogers Fellowship, National Science Foundation 

2006 Rackham Graduate Student Travel Fellowship, University of Michigan 

2008 Postdoctoral Travel Award, American Society for Virology 

2008 Postdoctoral Fellowship, American Heart Association 

2009 Postdoctoral Travel Award, American Society for Virology 

2011 Postdoctoral Travel Award, University of Michigan, Department of Microbiology & Immunology 

2013 Early Career Investigator Travel Fellowship, PML Consortium 

2014 Annual Transplant Symposium Travel Grant, UAB Comprehensive Transplant Institute/Emory 

Transplant Center 

2014 Faculty Development Grant, UAB 

2014 CFAR Research Day People’s Choice Poster Award, UAB 

2015 Travel Grant, International Center for Genetic Engineering and Biotechnology 

C. Contribution to Science

1. I have demonstrated the importance of the host DNA damage response (DDR) for productive polyomavirus 

life cycle and host genome stability during polyomavirus infection: Host DDR had been implicated in 

polyomavirus life cycle but the detailed roles of the two key players in the DDR pathway, namely ATR and 

ATM, had not been clearly understood. My work has demonstrated that both ATR and ATM are required 

for optimal viral replication. Additionally, in the absence of either kinase, the host genome becomes 

susceptible to polyomavirus-induced damage. I have further demonstrated that the activation of these two 

kinases is mainly dependent on an active viral DNA replication during polyomavirus infection. These 

findings have contributed to our basic knowledge of how polyomaviruses replicate and the molecular basis 

for polyomavirus-induced genome instability. I anticipate that in the long run these discoveries may 

 

Contact PD/PI: Jiang, Mengxi 

Biosketches Page 14
	
facilitate the development of novel inhibitors to combat polyomavirus infection or to treat polyomavirusinduced tumors. 

a. Jiang M, Zhao L, Gamez M, Imperiale MJ. Roles of ATM and ATR-mediated DNA damage responses 

during lytic BK polyomavirus infection. PLoS Pathog. 2012;8(8):e1002898. PubMed PMID: 22952448; 

PubMed Central PMCID: PMC3431332. 

b. Justice JL, Verhalen B, Jiang M. Polyomavirus interaction with the DNA damage response. Virol Sin. 

2015 Apr;30(2):122-9. PubMed PMID: 25910481. 

c. Verhalen B, Justice JL, Imperiale MJ, Jiang M. Viral DNA replication-dependent DNA damage 

response activation during BK polyomavirus infection. J Virol. 2015 May;89(9):5032-9. PubMed PMID: 

25694603; PubMed Central PMCID: PMC4403456. 

2. I have identified essential intracellular trafficking pathways and inhibitors for polyomavirus entry: The early 

trafficking pathways of polyomavirus in relevant cell types had not been well defined. I have led research 

projects to show that BK polyomavirus traffics through endosomes, and uses an intact microtubule network 

to reach the endoplasmic reticulum (ER) where viral disassembly occurs. I have also shown that the virus 

hijacks the host ER-associated degradation pathway in order to penetrate the limiting membrane. 

Furthermore, my work has demonstrated that an inhibitor targeting the host Abl kinase is able to block 

polyomavirus entry through down-regulation of cellular receptors for polyomavirus. These studies have 

revealed previously unknown trafficking pathways for polyomavirus in primary cells and will form the basis 

for the development of novel entry inhibitors for polyomavirus infections. 

a. Jiang M, Abend JR, Tsai B, Imperiale MJ. Early events during BK virus entry and disassembly. J Virol. 

2009 Feb;83(3):1350-8. PubMed PMID: 19036822; PubMed Central PMCID: PMC2620883. 

b. Swimm AI, Bornmann W, Jiang M, Imperiale MJ, Lukacher AE, Kalman D. Abl family tyrosine kinases 

regulate sialylated ganglioside receptors for polyomavirus. J Virol. 2010 May;84(9):4243-51. PubMed 

PMID: 20181697; PubMed Central PMCID: PMC2863717. 

c. Bennett SM, Jiang M, Imperiale MJ. Role of cell-type-specific endoplasmic reticulum-associated 

degradation in polyomavirus trafficking. J Virol. 2013 Aug;87(16):8843-52. PubMed PMID: 23740996; 

PubMed Central PMCID: PMC3754070. 

3. I have applied quantitative proteomic methods to identify novel ribosome assembly factors in E. coli: 

Ribosome biogenesis is a complex biological process that requires orchestrated assembly of both 

ribosomal RNAs and proteins. In addition to the core structural proteins, there are also protein components 

that perform chaperone functions to facility the assembly. I was among one of the first researchers to apply 

quantitative proteomic approaches to identify such assembly factors in E. coli. My research provided novel 

insight into how ribosome biogenesis is regulated in bacteria and also demonstrated the power of using 

novel quantitative proteomic approaches to address biological questions. We are now adapting some of 

the technologies to answer polyomavirus-related questions such as host proteomic changes during 

infection. 

a. Jiang M, Datta K, Walker A, Strahler J, Bagamasbad P, Andrews PC, Maddock JR. The Escherichia 

coli GTPase CgtAE is involved in late steps of large ribosome assembly. J Bacteriol. 2006 

Oct;188(19):6757-70. PubMed PMID: 16980477; PubMed Central PMCID: PMC1595513. 

b. Jiang M, Sullivan SM, Walker AK, Strahler JR, Andrews PC, Maddock JR. Identification of novel 

Escherichia coli ribosome-associated proteins using isobaric tags and multidimensional protein 

identification techniques. J Bacteriol. 2007 May;189(9):3434-44. PubMed PMID: 17337586; PubMed 

Central PMCID: PMC1855874. 

c. Jiang M, Sullivan SM, Wout PK, Maddock JR. G-protein control of the ribosome-associated stress 

response protein SpoT. J Bacteriol. 2007 Sep;189(17):6140-7. PubMed PMID: 17616600; PubMed 

Central PMCID: PMC1951942. 

Complete List of Published Work in My Bibliography: 

http://www.ncbi.nlm.nih.gov/myncbi/browse/collection/47959635/?sort=date&direction=ascending

 

Contact PD/PI: Jiang, Mengxi 

Biosketches Page 15
	
 

Contact PD/PI: Jiang, Mengxi 

D. Research Support

Ongoing Research Support

 

 

 

 

 

 

 

Completed Research Support

 

 

Biosketches Page 16
	
 

Contact PD/PI: Jiang, Mengxi 

OMB No. 0925-0001/0002 (Rev. 08/12 Approved Through 8/31/2015)

 

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Contact PD/PI: Jiang, Mengxi 

 

, 

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Biosketches Page 21
	
Tracking Number: GRANT11922341 Funding Opportunity Number: PA-13-302. Received Date:

2015-06-02T17:13:17.000-04:00

PHS 398 Cover Page Supplement

OMB Number: 0925-0001

1. Project Director / Principal Investigator (PD/PI)

Prefix:

First Name*: Mengxi

Middle Name:

Last Name*: Jiang

Suffix:

2. Human Subjects

Clinical Trial? ● No ❍ Yes

Agency-Defined Phase III Clinical Trial?* ❍ No ❍ Yes

3. Permission Statement*

If this application does not result in an award, is the Government permitted to disclose the title of your proposed project, and the name,

address, telephone number and e-mail address of the official signing for the applicant organization, to organizations that may be

interested in contacting you for further information (e.g., possible collaborations, investment)?

● Yes ❍ No

4. Program Income*

Is program income anticipated during the periods for which the grant support is requested? ❍ Yes ● No

If you checked "yes" above (indicating that program income is anticipated), then use the format below to reflect the amount and source(s).

Otherwise, leave this section blank.

Budget Period* Anticipated Amount ($)* Source(s)*

   

   

   

   

   

Contact PD/PI: Jiang, Mengxi 

 Page 22
	
Tracking Number: GRANT11922341 Funding Opportunity Number: PA-13-302. Received Date:

2015-06-02T17:13:17.000-04:00

PHS 398 Cover Page Supplement

5. Human Embryonic Stem Cells

Does the proposed project involve human embryonic stem cells?* ● No ❍ Yes

If the proposed project involves human embryonic stem cells, list below the registration number of the specific cell line(s) from the following

list: http://grants.nih.gov/stem_cells/registry/current.htm. Or, if a specific stem cell line cannot be referenced at this time, please check the box

indicating that one from the registry will be used:

Cell Line(s): ❏ Specific stem cell line cannot be referenced at this time. One from the registry will be used.

6. Inventions and Patents (For renewal applications only)

Inventions and Patents*: ❍ Yes ❍ No

If the answer is "Yes" then please answer the following:

Previously Reported*: ❍ Yes ❍ No

7. Change of Investigator / Change of Institution Questions

❏ Change of principal investigator / program director

Name of former principal investigator / program director:

Prefix:

First Name*:

Middle Name:

Last Name*:

Suffix:

❏ Change of Grantee Institution

Name of former institution*:

Contact PD/PI: Jiang, Mengxi 

 Page 23
	
Contact PD/PI: Jiang, Mengxi 

Tracking Number: GRANT11922341 Funding Opportunity Number: PA-13-302. Received Date: 2015-06-02T17:13:17.000-04:00

OMB Number: 0925-0001

PHS 398 Modular Budget

Budget Period: 1

Start Date: 04/01/2016 End Date: 03/31/2017

A. Direct Costs Funds Requested ($)

Direct Cost less Consortium F&A*

Consortium F&A

Total Direct Costs*

B. Indirect Costs

Indirect Cost Type Indirect Cost Rate (%) Indirect Cost Base ($) Funds Requested ($)

1. Modified Total Direct Costs

2.

3.

4.

Cognizant Agency

(Agency Name, POC Name and Phone Number)

DHHS, Steven Zuraf, 301-492-4855

Indirect Cost Rate Agreement Date 09/25/2014 Total Indirect Costs

C. Total Direct and Indirect Costs (A + B) Funds Requested ($)

 Page 24 
Contact PD/PI: Jiang, Mengxi 

Tracking Number: GRANT11922341 Funding Opportunity Number: PA-13-302. Received Date: 2015-06-02T17:13:17.000-04:00

PHS 398 Modular Budget

Budget Period: 2

Start Date: 04/01/2017 End Date: 03/31/2018

A. Direct Costs Funds Requested ($)

Direct Cost less Consortium F&A*

Consortium F&A

Total Direct Costs*

B. Indirect Costs

Indirect Cost Type Indirect Cost Rate (%) Indirect Cost Base ($) Funds Requested ($)

1. Modified Total Direct Costs

2.

3.

4.

Cognizant Agency

(Agency Name, POC Name and Phone Number)

DHHS, Steven Zuraf, 301-492-4855

Indirect Cost Rate Agreement Date 09/25/2014 Total Indirect Costs

C. Total Direct and Indirect Costs (A + B) Funds Requested ($)

 Page 25 
Contact PD/PI: Jiang, Mengxi 

Tracking Number: GRANT11922341 Funding Opportunity Number: PA-13-302. Received Date: 2015-06-02T17:13:17.000-04:00

PHS 398 Modular Budget

Budget Period: 3

Start Date: 04/01/2018 End Date: 03/31/2019

A. Direct Costs Funds Requested ($)

Direct Cost less Consortium F&A*

Consortium F&A

Total Direct Costs*

B. Indirect Costs

Indirect Cost Type Indirect Cost Rate (%) Indirect Cost Base ($) Funds Requested ($)

1. Modified Total Direct Costs

2.

3.

4.

Cognizant Agency

(Agency Name, POC Name and Phone Number)

DHHS, Steven Zuraf, 301-492-4855

Indirect Cost Rate Agreement Date 09/25/2014 Total Indirect Costs

C. Total Direct and Indirect Costs (A + B) Funds Requested ($)

 Page 26 
Contact PD/PI: Jiang, Mengxi 

Tracking Number: GRANT11922341 Funding Opportunity Number: PA-13-302. Received Date: 2015-06-02T17:13:17.000-04:00

PHS 398 Modular Budget

Budget Period: 4

Start Date: 04/01/2019 End Date: 03/31/2020

A. Direct Costs Funds Requested ($)

Direct Cost less Consortium F&A*

Consortium F&A

Total Direct Costs*

B. Indirect Costs

Indirect Cost Type Indirect Cost Rate (%) Indirect Cost Base ($) Funds Requested ($)

1. Modified Total Direct Costs

2.

3.

4.

Cognizant Agency

(Agency Name, POC Name and Phone Number)

DHHS, Steven Zuraf, 301-492-4855

Indirect Cost Rate Agreement Date 09/25/2014 Total Indirect Costs

C. Total Direct and Indirect Costs (A + B) Funds Requested ($)

 Page 27 
Contact PD/PI: Jiang, Mengxi 

Tracking Number: GRANT11922341 Funding Opportunity Number: PA-13-302. Received Date: 2015-06-02T17:13:17.000-04:00

PHS 398 Modular Budget

Budget Period: 5

Start Date: 04/01/2020 End Date: 03/31/2021

A. Direct Costs Funds Requested ($)

Direct Cost less Consortium F&A*

Consortium F&A

Total Direct Costs*

B. Indirect Costs

Indirect Cost Type Indirect Cost Rate (%) Indirect Cost Base ($) Funds Requested ($)

1. Modified Total Direct Costs

2.

3.

4.

Cognizant Agency

(Agency Name, POC Name and Phone Number)

DHHS, Steven Zuraf, 301-492-4855

Indirect Cost Rate Agreement Date 09/25/2014 Total Indirect Costs

C. Total Direct and Indirect Costs (A + B) Funds Requested ($)

 Page 28 
Contact PD/PI: Jiang, Mengxi 

Tracking Number: GRANT11922341 Funding Opportunity Number: PA-13-302. Received Date: 2015-06-02T17:13:17.000-04:00

PHS 398 Modular Budget

Cumulative Budget Information

1. Total Costs, Entire Project Period

Section A, Total Direct Cost less Consortium F&A for Entire Project Period ($)

Section A, Total Consortium F&A for Entire Project Period ($) 0.00

Section A, Total Direct Costs for Entire Project Period ($)

Section B, Total Indirect Costs for Entire Project Period ($)

Section C, Total Direct and Indirect Costs (A+B) for Entire Project Period ($)

2. Budget Justifications

Personnel Justification Budget_Justification_Final.pdf

Consortium Justification

Additional Narrative Justification

 Page 29 
 

Contact PD/PI: Jiang, Mengxi 

BUDGET JUSTIFICATION

Senior/Key Personnel: 

Mengxi Jiang, Ph.D., (Principal Investigator; 4.8 calendar months) is an Assistant Professor in the Department 

of Microbiology at UAB. As Principal Investigator, Dr. Jiang will lead and direct the studies. She will help plan 

and perform experiments, assist in interpreting results, be responsible for all manuscripts, and ensure that the 

scientific goals and milestones of the project are achieved. She will work directly with all the members involved 

in the project to ensure open communication and facilitate progress in the project. She will meet with members 

of her group individually each week in addition to organizing weekly meetings with the entire laboratory staff in 

order to evaluate and analytically critique the data to establish future experiments and troubleshoot potential 

problems. 

She has extensive experience in dissecting the molecular interactions required for the cytotoxic action of 

experimental therapeutics, such as topotecan and inhibitors of TOR signaling, using genetics, biochemistry 

and mammalian cell culture. She also has expertise in the biochemical and genetic characterization of defects 

in DNA replication and checkpoint signaling. For this proposal, she will coordinate with Dr. Jiang on 2-D gel 

and DNA fiber assays. In these studies, she will direct the activities of the Research Associate, Dr. Wright, on 

the proposed analyses of viral DNA replication intermediates, in 2-D gels and DNA fiber assays. She will assist 

with data analyses and manuscript preparation.

Personnel for the project:

Joshua Justice, B.S., (Graduate Assistant; 12.0 calendar months) is a second year Microbiology Graduate 

theme student in the UAB Graduate Biomedical Sciences program. He joined the Jiang lab in May, 2014. He 

has demonstrated exceptional critical thinking and technical skills in the lab. He has presented his work at the 

2014 DNA Tumor Virus meeting and will be presenting again at 2015 American Society for Virology Annual 

conference. He has one first-author publication from his undergraduate research. Since he joined the Jiang 

lab, he has already published one first-author review article and has contributed to one research paper. He will 

be responsible for Specific Aim 1 to examine the role of the host mismatch repair complex during polyomavirus 

infection. He will also be involved in parts of Specific Aim 3 to examine whether polyomavirus-induced host 

DNA damage is coupled with mitosis. 

Brandy Verhalen, Ph.D., (Research Assistant; 6.0 calendar months) has received her Ph.D. degree from 

SUNY Upstate Medical University and completed her postdoctoral training at Vanderbilt University. She has 

been in the Jiang lab for over one year. Brandy has extensive molecular biology and biochemistry experience 

as evidenced by her 7 publications in high-impact journals over the last four years. She will be responsible for 

Specific Aim 2 to identify viral DNA triggers that lead to DDR activation. She will also work with Dr. Wright on 

Specific Aim 3 to determine whether large T antigen causes replication stress in host cells. 

Christine Wright, Ph.D., (Research Associate; 2.4 calendar months) has extensive expertise in the studies of 

cytotoxic chemotherapeutics. For the past 8 years, she has worked in the lab on various aspects of 

cellular responses to DNA damage and replicative stress, and has acquired considerable experience in the 

study of DNA replication intermediates. She will undertake the mechanistic studies of viral DNA replication 

intermediates and fork progression using 2-D gel and DNA fiber technology and will work closely with Drs. 

 and Jiang in discussions of date interpretation and study design.

Personnel Justification Page 30
	
 

Tracking Number: GRANT11922341 Funding Opportunity Number: PA-13-302. Received Date:

2015-06-02T17:13:17.000-04:00

PHS 398 Research Plan

Please attach applicable sections of the research plan, below. OMB Number: 0925-0001

1. Introduction to Application

(for RESUBMISSION or REVISION only)

2. Specific Aims  Specific_Aims_Final.pdf

3. Research Strategy*  Research_Strategy_Final.pdf

4. Progress Report Publication List

Human Subjects Sections

5. Protection of Human Subjects

6. Inclusion of Women and Minorities

7. Inclusion of Children

Other Research Plan Sections

8. Vertebrate Animals

9. Select Agent Research

10. Multiple PD/PI Leadership Plan

11. Consortium/Contractual Arrangements  

12. Letters of Support  Letters_of_Support_Final.pdf

13. Resource Sharing Plan(s)

Appendix (if applicable)

14. Appendix

Contact PD/PI: Jiang, Mengxi 

 Page 31
	
SPECIFIC AIMS 

 Polyomaviruses are ubiquitous in human populations and cause serious life-threatening diseases including 

cancer, particularly in immunocompromised individuals. Currently there are no specific treatments or 

prophylactic approaches to target this family of viruses and their related diseases. Our long-term goals are to 

elucidate the fundamental mechanisms of polyomavirus replication, to understand how these viruses hijack 

and subvert normal host cellular processes to facilitate viral replication, and to dissect how these interactions 

may result in polyomavirus-induced oncogenesis. 

One of the emerging concepts in the polyomavirus field is that cellular DNA damage response (DDR), 

which is a network of cellular pathways required for maintaining genome integrity, is essential for viral 

replication. The molecular details of DDR manipulation by polyomaviruses and the exact functions of the DDR 

during viral replication are not well characterized. On the host side, polyomavirus infection—in particular the 

virally-encoded T antigens—have been shown to cause host cell genomic instability, which could ultimately 

lead to oncogenesis. Currently, the underlying molecular source of such genomic instability remains unclear. 

 Using BK polyomavirus (BKPyV) and a primary renal 

proximal tubule epithelial cell culture infection model, we 

have recently demonstrated that (a) BKPyV activates and 

hijacks the DDR to promote viral replication, and that (b) 

BKPyV infection induces host chromosome damage, but 

only in the absence of viral DNA replication or DDR 

activation. These findings lead us to propose a novel 

balanced model to link virus replication, DDR activation and 

host genome instability together (Fig. 1). The central 

hypothesis of this model is that an activated DDR is 

important for both facilitating virus replication and 

maintaining host genomic stability during polyomavirus 

infection. We propose that viral DNA replication actively 

drives productive infection by inducing the host DDR, 

thereby stabilizing viral replication forks and preventing 

replication-induced DNA damage on the viral genome. We 

also propose that the DDR is essential to prevent host 

genome instability during polyomavirus infection through G2/M cell cycle arrest. To test this central hypothesis, 

we propose the following three specific aims (Fig. 1): 

Aim 1. To define the role of host mismatch repair proteins in polyomavirus replication and 

polyomavirus-induced DDR activation. Our recent quantitative nuclear proteomic analysis identified that the 

host mismatch repair (MMR) proteins, in particular the hMutSα complex, are required for BKPyV replication 

and virus-induced DDR activation. We hypothesize that the MMR proteins serve as novel signaling molecules 

to facilitate DDR activation during polyomavirus infection. The goal of this aim is to determine how the MMR 

proteins drive productive viral infection through DDR activation by examining viral replication products, 

interactions between the large T antigen (TAg) and DDR proteins, and recruitment of DDR proteins to viral 

DNA. 

Aim 2. To determine the viral DNA triggers that activate the DDR upon polyomavirus infection. The 

current view in the polyomavirus field is that viral oncogenes activate the DDR. In contrast, our results suggest 

that a full DDR activation is dependent on viral DNA synthesis. We hypothesize that replicating viral DNA 

serves as a major trigger for DDR activation during infection. In this aim we will identify viral DNA structures 

that are recognized by host cells as damage signals to induce DDR activation. 

Aim 3. To elucidate the molecular mechanism by which polyomavirus induces host genome instability.

We hypothesize that via G2/M cell cycle arrest, the DDR is able to prevent host genome instability caused by 

TAg-induced host DNA replication stress. The goal of this aim is to investigate how TAg affects host cell DNA 

replication using a novel replication stress assay and to determine whether the cellular DNA damage caused 

by TAg is coupled with mitotic entry. 

Together, our proposed studies will have a broad impact on the field by dissecting the crucial roles that the 

DDR play in promoting viral replication and maintaining host genome stability. In the long term, these studies 

may reveal novel therapeutic host targets to treat polyomavirus-related diseases. 

Figure 1. Model of BKPyV replication, DDR 

activation, and host genome instability. See text for 

details. The numbering refers to the three specific aims 

that will be pursued in this proposal. 

 

Contact PD/PI: Jiang, Mengxi 

Specific Aims Page 32
	
RESEARCH STRATEGY

A. SIGNIFICANCE 

 Polyomaviruses are a family of small DNA tumor viruses with a ~5kb circular double-stranded DNA 

genome. The first two human polyomaviruses, BK polyomavirus (BKPyV) and JC polyomavirus (JCPyV), were 

discovered in 1971 (1, 2). Both viruses cause severe disease under certain immunosuppressed conditions (3). 

In the setting of kidney transplantation, BKPyV reactivates in up to 10% of transplant recipients, which can lead 

to severe polyomavirus-associated nephropathy and subsequent graft loss (3). Since a total of 13,299 kidney 

transplants were performed in US alone in 2014 (National Kidney Foundation), BKPyV-related disease 

represents a significant problem. While not proven, there have also been reports linking BKPyV infection to the 

development of urothelial and renal tubular malignancies in these patients (4-11). JCPyV is responsible for 

causing progressive multifocal leukoencephalopathy, a major life-threatening complication in patients with 

several underlying immunosuppressive conditions (12). In the past decade—due to significant improvements in 

DNA amplification and sequencing—a number of new polyomaviruses were discovered from various human 

tissues and samples (13). Among them, Merkel cell polyomavirus (MCPyV) has now been confirmed to be the 

causative agent for most human Merkel cell carcinomas, an aggressive human skin cancer (14-16). So far, no 

specific anti-viral treatments or vaccines have been developed for this group of medically important viruses. 

There are several critical gaps in our knowledge of the basic biology of these viruses. First, how exactly are 

these viruses replicated in host cells? Over the years, Simian Virus 40 (SV40) DNA replication has been 

pursued as a model system to understand eukaryotic chromosome replication, and the bidirectional replication 

mechanism is considered a common feature between viral and eukaryotic cell DNA replication (17). In spite of 

the many similarities, polyomavirus DNA replication is not a sheer mimic of host cell DNA replication but 

exhibits several unique characteristics. One emerging concept in the polyomavirus field is that the virus 

manipulates the host DNA damage response (DDR) pathways to promote a productive viral replication. The 

DDR is composed of cellular signaling cascades that help maintain genome integrity and repair various lesions 

occurring on the DNA. The molecular functions of the DDR during viral replication, however, are not fully 

understood. Second, do these viruses cause genomic instability, and if yes, how? Recently, genomic instability 

induced by polyomavirus has been postulated as a potential mechanism for virally encoded large T antigen 

(TAg)-induced oncogenesis (18-21). The source of such TAg-induced genome instability, however, remains 

elusive in the field. Our research has now identified a link between productive viral infection and host genome 

instability through the regulation of the DDR. The major goals of this proposal are to define the importance of 

the DDR in polyomavirus replication and host genome stability, as well as to elucidate DDR activation 

mechanisms by both host and viral factors. We think that the proposed research is significant because it will 

greatly advance our understanding of polyomavirus biology and may in the long term reveal potential targets to 

prevent or treat diseases that are caused by these viruses. 

Previously, we established a primary human renal proximal tubule epithelial (RPTE) cell culture system for 

BKPyV as a model system to study polyomavirus lytic life cycle (22-25). This is the cell type where BKPyV lytic 

infection is observed in humans (26). Using this system, we have begun to dissect the functional importance of 

DDR during viral lytic replication and we have now identified novel host and viral triggers responsible for 

inducing the DDR. In addition, we have uncovered host genomic instability caused by viral infection. 

Interestingly, this instability only appears when the ability of the virus to replicate its viral DNA or DDR 

activation is compromised (27). Therefore, the proposed work is significant at many levels: 

A1) Our proposal investigates novel mechanisms for polyomavirus DNA replication: Using powerful 

quantitative proteomic approaches we have identified host mismatch repair (MMR) proteins, especially the 

hMutSα complex, as novel host factors required for DDR activation during viral infection. Our proposed study 

will determine whether TAg interaction with the MMR proteins results in the recruitment of DDR proteins to 

replicating viral DNA, thus activating the DDR. This will be highly significant as it will be the first 

characterization of the involvement of host MMR system during productive polyomavirus infection through DDR 

activation, which will contribute to our knowledge about the fundamental replication mechanism of 

polyomaviruses. 

A2) Our proposal examines uncharacterized viral DNA triggers for DDR activation: The current view in the 

polyomavirus field is that the viral TAg activates the DDR as shown in several transformed cell lines (18, 19, 

28). In contrast to results obtained from transformed cell lines, our preliminary results provide compelling 

evidence that TAg alone only activates a minimal DDR in primary cells and that a full DDR activation is 

dependent on viral DNA replication (27). These findings point to the uncharacterized role of polyomavirus DNA 

replication and replication-associated DNA damage in triggering DDR. Our proposed studies will determine 

 

Contact PD/PI: Jiang, Mengxi 

Research Strategy Page 33
	
what DNA structures present on viral DNA during replication lead to DDR activation. This is important for two 

reasons: (i) DDR activation appears to be essential to produce infectious viral progeny. Therefore, dissecting 

the activation mechanism will allow us to further understand the requirements for a productive infection. (ii) As 

will be discussed below, DDR inactivation is closely linked with the appearance of host DNA damage caused 

by polyomavirus. A lack of DDR activation may lead to accumulation of detrimental host DNA damage. 

Understanding how DDR is activated in polyomavirus-infected cells will have implications in preventing host 

DNA damage caused by polyomaviruses. 

A3) Our proposal aims to identify the molecular sources of TAg-induced host cell DNA damage: It has been 

postulated that TAg is able to induce replication stress and mitotic defects (29). The effect of TAg on host DNA 

replication has not been directly measured and whether the TAg-induced host DNA damage is associated with 

mitosis has not been determined. This is important because it allows us to understand the molecular basis of 

such DNA damage. This knowledge may also be applied in the future to selectively induce host DNA damage 

in polyomavirus-infected cells to help eliminate infected cells. 

B. INNOVATION

B1) Our central hypothesis is conceptually innovative: we propose that polyomavirus modulates the host DDR 

to facilitate productive viral replication and to prevent host DNA damage. Towards this hypothesis, we have 

identified host and viral factors that are essential for DDR activation. Our study has also revealed a novel 

connection between viral DNA replication, DDR activation, and virus-induced host genome instability. These 

are all original concepts in the field of polyomavirus virology and tumor biology. 

B2) The model system in which we will test our hypothesis is unique and highly relevant. Our proposed 

research will be carried out in our well-characterized primary RPTE cell culture model. This is the best cell 

system to test our hypotheses for several reasons: First, RPTE mimics the environment that is encountered by 

BKPyV in vivo (23). How viruses establish infection and the effects of viral infections on host cells can vary 

greatly depending on the cell type, therefore we believe that results generated using this system are 

pathologically relevant. Second, it is also extremely important to study the DDR and its contribution to viral 

replication in normal cells with intact cell cycle checkpoint regulation, as DDR characteristics are very different 

in transformed cell lines (30). Finally, our system is fully permissive to BKPyV replication and is therefore more 

relevant than the artificial systems that have been developed to study newly identified polyomaviruses 

including MCPyV (31). 

B3) Our approach is technically innovative by bridging together methodologies in virology and DNA damage 

fields. We have teamed up with experts in the DNA damage and repair field to adapt a series of approaches 

including two-dimensional agarose gels and DNA fiber assays to examine viral DNA replication intermediates 

and the effects of viral infection on host DNA replication. All of these innovative methods will allow us to 

address important yet previously unexplored questions related to polyomavirus-induced DNA damage and viral 

replication strategy. 

C. APPROACH

Based on our preliminary results, we propose the following model for the interplay between BKPyV DNA 

replication, DDR activation, and host genome instability (Fig. 1): 1) During productive BKPyV infection, TAg 

expression upregulates the mismatch repair hMutSα complex, which mediates DDR activation. 2) TAgmediated viral DNA replication is the main driving force for DDR activation and the activated DDR helps 

stabilize viral replication forks and repair replication-associated viral DNA damage. 3) In the absence of viral 

DNA replication and its associated DDR activation, TAg-induced host cell DNA replication stress coupled with 

mitosis can lead to genome instability; however, this genome instability is prevented during normal infection 

through G2/M arrest resulting from virus replication-induced DDR activation. These three independent yet 

related specific aims are directed at understanding: (Aim 1) the roles of host mismatch repair proteins in 

polyomavirus replication, DDR activation, and host genome instability; (Aim 2) the viral DNA triggers that lead 

to DDR activation during polyomavirus infection; (Aim 3) the mechanisms of TAg-induced DNA damage and 

how the damage is connected to DDR activation. 

C1) INTRODUCTION 

Polyomavirus DNA replication. Following polyomavirus entry into a host cell, the viral DNA genome is delivered 

into the nucleus where replication occurs. The viral TAg is a multifunctional protein that orchestrates the viral 

replication cycle (32). TAg binds directly to the viral origin of DNA replication through its origin-binding domain 

(OBD) and forms a double-hexamer. TAg also possesses helicase activity that allows it to unwind viral DNA to 

enable viral replication. Because of the small viral genome size and hence limited coding capacity, viral 

 

Contact PD/PI: Jiang, Mengxi 

Research Strategy Page 34
	
replication relies heavily on host replication machinery. In particular, TAg recruits replication protein A (RPA), 

DNA polymerase α-primase, and topoisomerase I to initiate replication of viral DNA. Even though the basic 

replication mechanism is considered similar between viral and eukaryotic DNA replication, viral DNA replication 

possesses its own distinct features. For example, polymerase ε is important for extending the leading strand 

during eukaryotic DNA replication, but it appears to be dispensable for polyomavirus DNA synthesis (33). 

Since TAg itself is a helicase itself, polyomavirus replication is not dependent on the cellular Cdc45/Mcm2-

7/GINS helicase complex (34). Moreover, recent findings suggest that there are additional unknown host 

components, especially the DNA damage sensing and repair proteins, playing important yet undefined roles 

during viral replication. For example, polyomavirus infections have been shown to recruit several proteins 

involved in the DDR, such as Mre11 and Rad51, into TAg-positive viral replication foci in the nucleus (22, 35). 

How these viruses activate the DDR and the precise molecular functions that DDR proteins serve during viral 

replication still remain poorly understood. 

TAg and cellular transformation. It is well established that TAg is capable of inhibiting normal functions of the 

tumor suppressors retinoblastoma protein (pRb) and p53. TAg binding to pRb relieves pRb inhibition of E2F, 

which is a transcription factor for many genes that are important for S phase progression and DNA synthesis 

(36, 37). TAg stabilizes and functionally inactivates p53, thereby avoiding p53-dependent apoptosis (38, 39). 

Although these are important avenues of TAg driving tumorigenesis, inactivating pRb and p53 is not sufficient 

for cellular transformation by TAg (40) and additional oncogenic activities of TAg are required. Recently, a 

number of polyomavirus TAgs have been reported to induce host DNA damage as determined by either comet 

assays or sensitivities to specific-DNA damaging treatments (18-21). Since chromosome instability is a 

hallmark for many cancer cells, DNA damage induction could be another mechanism contributing to TAginduced oncogenesis. 

DNA damage response (DDR). DDR is considered the 

guardian of the genome. There are two major 

phosphatidylinositol 3-kinase-related kinases: ataxiatelangiectasia mutated (ATM), and ATM and Rad-3-related 

(ATR). These kinases govern the activation of the DDR 

pathways (Fig. 2). ATR is activated by single-stranded DNA 

(ssDNA) lesions and is important for resolving replication 

stress from conditions such as stalled replication forks (41). 

ATM responds mainly to double-stranded breaks (DSBs) 

resulting from conditions including ionizing radiation (IR) (42) 

or collapsed replication forks (43). Both ATR and ATM can 

phosphorylate and activate numerous downstream targets 

that are involved in DNA repair and cell cycle arrest. These 

include the checkpoint kinases Chk1 (mostly by ATR) and 

Chk2 (mostly by ATM), ATM itself (from ATM activationinduced auto-phosphorylation), and a histone variant H2AX 

Figure 2. DDR activation mechanism. See text for 

details. Abbreviations: ssDNA: single-stranded DNA;

DSB: double-stranded break; NHEJ: non-homologous

end joining; HR: homologous recombination. 

(termed γH2AX when phosphorylated). γH2AX 

is thought to mark the sites of DSBs (44) and is 

the first step in recruiting and localizing DNA 

repair proteins (45). Activated checkpoint 

kinases, especially Chk1, play essential roles in 

coordinating cellular responses to replication 

stress. These include suppressing inappropriate 

replication origin firing, stabilizing stalled 

replication forks, and triggering G2/M arrest (46, 

47). For the past decade, DDR has become an 

intensive area of research in virology as a 

number of viruses, especially many DNA 

viruses, have been shown to modulate 

components of the DDR pathway thereby 

promoting viral replication or oncogenesis (48, 

49). 

Figure 3. BKPyV activates the DDR and requires the DDR for 

productive infection. (A) RPTE cells were infected with BKPyV at 

an MOI of 5 infectious units (IU)/cell. Whole cell lysates were 

harvested at 1-3 days post infection (dpi) and immunoblotted for ATR 

and ATM activation markers. (B+C) RPTE cells were transfected with 

indicated siRNAs followed by BKPyV infection at an MOI of 0.5 

IU/cell. Cell-associated viral DNA (B) and virus titer (C) were 

determined at 2 dpi by a real-time PCR assay and a fluorescence IU 

assay, respectively. Data were normalized to no siRNA control. *, p< 

0.05; **, p<0.01. 

 

Contact PD/PI: Jiang, Mengxi 

Research Strategy Page 35
	
C2) PRELIMINARY STUDIES 

DDR is important for productive viral infection and host genome maintenance during polyomavirus infection. 

Our preliminary results show that: 1) BKPyV infection is able to activate both ATR- and ATM-dependent DDR 

signaling pathways in RPTE cells as determined by an increase in Chk1-pS317 (ATR activation marker) and 

ATM-pS1981 (ATM activation marker) in BKPyV-infected cells (Fig. 3A, previous page). 2) Using siRNA 

knockdowns, we have shown that double knockdown of ATR and ATM partially inhibited viral DNA replication 

as determined by a real-time PCR assay (Fig. 3B) and more dramatically inhibited infectious viral progeny 

production (Fig. 3C). 3) Since the real-time PCR assay can only measure the quantity of the viral DNA that is 

recognized by the primer pair, it does not reveal whether these viral DNAs are intact genomes that can be 

packaged into virions. To determine whether there 

was a change in the quality of the viral DNA, which 

may explain a greater defect with the infectious 

viral titer, we used Southern blotting with a specific 

viral probe to examine the nature of the viral DNA. 

We observed that inhibition of the DDR by siRNA 

knockdowns resulted in an accumulation of higher 

molecular weight dimeric and concatemeric viral 

DNA products (Fig. 4A). This is consistent with 

published work on SV40 (50), suggesting that the 

DDR functions to maintain viral replication fork 

integrity and prevent the accumulation of abnormal 

replication products. In the absence of the DDR, 

stalled viral replication forks cannot be stabilized 

and therefore are inclined to collapse. Alternatively, 

an unrepaired DSB at one replication fork can leave 

the other fork to replicate unidirectionally. Both of 

these events can lead to an accumulation of 

aberrant viral DNA intermediates (Fig. 4B). 

Since DDR is important to maintain genomic 

stability under many genotoxic conditions, we also 

examined whether DDR inhibition affected host 

genome stability in the context of polyomavirus 

infection. Knockdown of ATR and ATM in BKPyVinfected cells resulted in abnormal nuclear 

orphology as seen by an increase in TAg-positive 

icronuclei formation (Fig. 5A). Micronuclei are 

iomarkers of genotoxic stress and chromosomal 

nstability, and they are usually formed from misegregated chromosomes or chromosome fragments 

51). To further confirm that the observed micronuclei 

epresent true DNA damage, we performed 

etaphase spread experiments to visualize direct 

amage on the host DNA. We consistently observed 

hat a large proportion of the metaphases became 

everely damaged and displayed a “shattered 

etaphase” appearance in BKPyV-infected cells 

hen the DDR was inhibited (Fig. 5B+C). This 

amage was restricted to BKPyV-infected cells and 

as not detected in mock-infected cells that were 

nocked down for ATR and ATM. Based on these 

indings, we conclude that the DDR is important for 

oth productive polyomavirus infection and the 

aintenance of host genome stability during 

nfection. Most of these data have been published 

22). 

Figure 4. DDR maintains replication fork integrity during 

polyomavirus infection. (A) Control cells or cells that were 

knocked down for ATR and ATM were infected with BKPyV at an 

MOI of 0.5 IU/cell. Total DNA were isolated at 2 dpi and subjected 

to Southern blotting using a viral-specific probe. (B) Model for DDR 

contribution to viral replication integrity. TAg (green circle) unwinds 

viral DNA to initiate viral replication. When a replication fork stalls, 

DDR inhibition can lead to either replication fork collapsing or 

unidirectional replication. Both events can cause an accumulation of 

aberrant viral replication intermediate products. 

m

m

b

i

s

(

r

m

d

t

s

m

w

Figure 5. DDR inhibition in BKPyV-infected cells causes host d

DNA damage. (A) RPTE cells were transfected with the indicated w

siRNAs and infected with BKPyV at an MOI of 0.5 IU/cell. Cells k were fixed at 3 dpi and stained for DAPI (blue) and TAg (red). 

Arrowheads point to micronuclei. Bar, 10 μm. (B) Representative f

pictures of normal and shattered chromosomes. (C) Cells were b

transfected and infected as in (A). Shattered metaphases were m

scored at 3 dpi. ***, p<0.001. i

(

 

Contact PD/PI: Jiang, Mengxi 

Research Strategy Page 36
	
Identification of host mismatch repair (MMR) 

proteins as important players for polyoma–

virus replication and DDR activation. To 

determine further which nuclear components 

are required for BKPyV replication, we used a 

quantitative proteomic approach, stable 

isotope labeling by amino acids in cell culture 

(SILAC) coupled with two-dimensional liquid 

chromatography and tandem mass 

spectrometry (2D LC-MS/MS) to specifically 

examine total nuclear proteome changes in 

BKPyV-infected cells compared with mockinfected cells (Fig. 6A). We grew RPTE cells 

in defined medium supplemented with either 

light amino acids (12C, 14N) or heavy amino 

acids (13C, 15N) under conditions where ~98% 

labeling efficiency was achieved. We infected 

one population with BKPyV (either light or 

heavy-labeled cells in two separate 

experiments), isolated the nuclear fractions 

from both mock and infected cells, mixed 

them and subjected the nuclear proteins to

2D LC-MS/MS (Fig. 6A). Identical peptides of 

different stable-isotope composition were 

Figure 6. Quantitative proteomics identifies that host mismatch repair 

proteins are upregulated by BKPyV. (A) SILAC nuclear proteomic 

analysis set up. RPTE cells were labeled with light or heavy media. In two 

independent repeats, differentially labeled cells were infected with BKPyV 

to ensure that any change observed was not due to the differential 

labeling. Nuclear proteins were isolated, mixed, and subjected to 2D LCMS/MS. (B+C) RPTE cells were infected with BKPyV at an MOI of 0.5 

IU/cell (B) or transduced with a lentivirus expressing TAg or an empty 

control lentivirus (C). Total proteins were immunoblotted for indicated 

proteins. differentiated and peak intensity ratios were 

used for quantitation. From the proteomic screen, we 

identified that the host mismatch repair (MMR) proteins are 

among the most significantly upregulated group of proteins 

during BKPyV infection. The MMR system normally corrects 

DNA mismatches during DNA replication and it is highly 

conserved from prokaryotes to humans. MSH6 and MSH2 

form the hMutSα heterodimer, which preferentially 

recognizes base-base mismatches and short 

insertion/deletion (in/del) loops. MSH2 also interacts with 

MSH3 to form the hMutSβ complex that recognizes larger 

in/del loops (52, 53). Our proteomic data show that MSH6 is 

upregulated by 4.3-fold and MSH2 is upregulated by 2.7-

fold by BKPyV infection. We confirmed the upregulation of 

MSH6 and MSH2 in BKPyV-infected cells by Western 

blotting (Fig. 6B), and we further showed that TAg 

expression alone was sufficient to increase MSH6 (Fig. 6C). 

What is more interesting is that in addition to its function in 

mismatch repair, hMutSα, particularly MSH6, has recently 

been shown to be important for DNA damage signaling and 

DNA repair pathways (54). This raises the possibility that 

the host MMR system may also mediate BKPyV-induced 

Figure 7. MSH6 knockdown (kd) inhibits BKPyV 

productive infection and DDR activation. RPTE cells were 

transfected with siRNAs and infected with BKPyV as in 

Fig. 3. (A) Western blots of viral proteins and DDR 

markers. (B+C) Viral DNA and infectious viral titer were 

determined as in Fig. 3. All data were normalized to no 

siRNA control. **, p<0.01. (D) MSH6 kd caused 

micronuclei formation in TAg-positive cells. Bar, 20 μm. 

DDR. 

Since MSH6 is one of the highly upregulated proteins upon BKPyV infection, we decided to focus on the 

role of hMutSα during polyomavirus infection. We used siRNA targeting MSH6 to specifically inactivate the 

hMutSα complex and assessed the effects on BKPyV gene expression, viral DNA level, infectious viral titer, 

and DDR activation (Fig. 7). Our data revealed that inactivation of the hMutSα complex did not significantly 

change either early viral protein TAg or late viral protein VP1 levels (Fig. 7A). Viral DNA levels were not 

affected either (Fig. 7B); however, the amount of infectious viral progeny was greatly reduced with MSH6 

knockdown (Fig. 7C). We also examined whether the hMutSα complex regulates ATR or ATM-mediated DDR 

activation. There is a noticeable decrease in Chk1-pS317 and a slight decrease in ATM-pS1981 upon MSH6 

knockdown, indicative of a defect in DDR activation (Fig. 7A). Additionally, we detected micronuclei and 

 

Contact PD/PI: Jiang, Mengxi 

Research Strategy Page 37
	
aberrant TAg staining patters in BKPyV-infected, MSH6 knocked down cells (Fig. 7D). Both the infectious viral 

titer and the nuclear morphology phenotypes are similar to those seen in infected cells lacking DDR signaling 

(Fig. 3 and 5), suggesting that MSH6 or the hMutSα complex contributes to ATR-mediated, and to a lesser 

extent, ATM-mediated DDR responses during polyomavirus infection. 

DDR activation during polyomavirus infection is depedent on viral DNA replication. Apart from host factors, we 

also went on to determine whether there are any viral components that trigger DDR activation. The current 

view in the field is that TAg is a major inducer for DDR activation, as has been suggested with polyomavirus 

infections in several immortalized cell lines (18, 19, 28). Our results, however, indicate the involvement of viral 

DNA in DDR activation (Fig. 8). 

When we delivered TAg into RPTE 

cells using lentivirus transduction, 

we found that TAg expression alone 

cannot fully activate the ATR 

response as demonstrated by a 

minimum induction of Chk1-pS317 

compared with infection conditions 

that have similar levels of TAg (Fig. 

8A, compare lane 3 with lane 5). 

We cannot determine the 

contribution of TAg itself to ATM 

activation since the empty lentivirus 

also induces an ATM response (Fig. 

8A). To further determine whether 

viral DNA replication is required for 

DDR activation, we constructed a 

TAg mutant virus (D501A) based on 

Figure 8. DDR activation requires viral DNA replication. (A) RPTE cells were either 

transduced with empty or TAg-expressing lentivirus, or infected with BKPyV at the 

designated MOIs for 3 days. Whole cell lysates were immunoblotted for DDR markers.

(B+C) RPTE cells were infected with wild-type or TAg D501A mutant BKPyV at an 

MOI of 0.5 IU/cell. (B) Viral DNA level was measured by qPCR at 3 dpi and 

normalized to input DNA at 1 dpi. ****, p<0.0001. (C) Whole cell lysates were 

harvested at 1 dpi and 3dpi, and immunoblotted for DDR markers. 

a homologous SV40 helicase mutant (55). This mutation abrogates TAg helicase activity, while leaving other 

functions of TAg such as stabilizing p53 intact (55). We were able to grow this mutant virus in the HEK-293TT 

cell line, which expresses a high-level of SV40 TAg that can complement the BKPyV TAg mutation (56, 57). 

When we used this mutant virus to infect RPTE cells, we found that it was unable to replicate viral DNA as 

expected (Fig. 8B). Interestingly, this mutant virus cannot activate either ATR or ATM-mediated DDR 

pathways (Fig. 8C). These data suggest that although the presence of viral oncogenes such as TAg may 

contribute to DDR activation, viral DNA replication is the major trigger for full DDR activation during 

polyomavirus infection. These data were recently published in (27). 

An inverse relationship between DDR activation and host DNA damage during polyomavirus infection. We 

observed an intriguing inverse correlation between viral DNA replication and the presence of host DNA 

damage (Table 1). During normal infection when 

Viral DNA DDR Host DNA DDR is activated, we do not detect host DNA 

damage (22). When the RPTE cells were 

infected with the TAg D501A virus, viral DNA 

could not be replicated and there was no DDR 

activation (Fig. 8C); however, host DNA damage 

accumulated with this mutant virus as shown by 

a comet assay and the formation of micronuclei 

(27). These phenotypes are similar to those seen 

in cells only expressing TAg itself. In those cells 

ATR-mediated DDR was only minimally activated 

(Fig. 8A) and there was an accumulation of 

replication activation damage 

Normal BKPyV + +++ - infection 

Replication- - - + 

deficient (TAg 

D501A) mutant 

virus infection 

TAg expression - + + 

alone 

Table 1. Summary comparison of WT virus infection, TAg D501A 

mutant virus infection, and TAg expression by lentivirus.

micronuclei (Table 1 and data not shown), indicative of host DNA damage. All of these results suggest that 

host DNA damage is only evident when DDR is not fully activated during polyomavirus infection. 

Overall, our preliminary results demonstrate the importance of the DDR for both viral replication and host 

genome stability during polyomavirus infection. We have identified novel host (hMutSα) and viral (replicating 

viral DNA) components that contribute to DDR activation. These studies form the basis for our proposed 

research to examine further the relationship between viral DNA replication, DDR activation, and host genome 

stability. 

 

Contact PD/PI: Jiang, Mengxi 

Research Strategy Page 38
	
 

Contact PD/PI: Jiang, Mengxi 

C3) RESEARCH PLAN

Aim 1. To define the role of host mismatch repair (MMR) proteins in polyomavirus replication and 

polyomavirus-induced DDR activation. The goal of this Aim is to test the hypothesis that the MMR proteins 

promote polyomavirus replication and host genome maintenance through DDR activation. To address this 

hypothesis, we will: (i) determine whether MMR proteins are important to maintain viral replication integrity and 

host genome stability during infection; (ii) determine the mechanism of MMR-mediated DDR activation during 

viral replication. 

Aim 1.1 Determine whether MMR proteins are required for viral replication integrity and host genome 

stability during BKPyV infection. Rationale: One emerging concept in the DNA damage field is that MMR 

proteins, especially the hMutSα complex, are critical for activating certain DDR signaling pathways and for the 

repair of DSBs (54). Both MSH6 and MSH2 have been found to interact directly with ATR and have been 

implicated in the recruitment of ATR to sites of DNA damage to initiate ATR-mediated DDR and checkpoint 

signaling (58-61). These results are consistent with our findings that a deficiency in MSH6 during BKPyV 

infection decreases the Chk1-pS317 level (Fig. 7A). We hypothesize that hMutSα is a key regulator of DDR, 

especially for ATR activation during polyomavirus replication. Since aberrant viral replication products and host 

genome damage are two prominent phenotypes that we observed when BKPyV-infected cells were inhibited 

for DDR, we will first examine whether hMutSα is important for regulating these two processes in infected cells. 

Experimental Design. We will perform siRNA knockdown experiments for either MSH6 or MSH2 followed by 

mock or BKPyV infection. We will then perform the following three experiments (i) isolate total DNAs and 

subject them to traditional one-dimensional electrophoresis and Southern blotting using a viral specific probe to 

determine whether there is an accumulation of aberrant viral DNA; (ii) determine the degree of host DNA 

damage by metaphase spread experiments as in Fig. 5C; (iii) perform cell cycle analysis using propidium 

iodide staining followed by flow cytometry. 

Expected Outcomes. (i) If our hypothesis is correct, we expect to see an accumulation of larger viral DNA 

products in the MMR knockdown cells, which will be similar to those seen in infected but DDR-deficient cells 

(Fig. 4A). If we detect these products, we will examine in further detail the molecular nature of these viral DNA 

molecules. We will then use two-dimensional agarose gel electrophoresis coupled with Southern blotting to 

resolve replication intermediates (62). In this method, restriction enzyme-digested DNAs are first separated by 

mass in the first dimension, followed by separation by topology in the second dimension. This analysis will 

allow for the resolution of various intermediates including unidirectional replication products and collapsed 

replication forks (Fig. 4B). Infected RPTE cells that are knocked down for ATR and ATM will serve as positive 

controls. It is also highly likely that ATR and ATM may contribute differently to viral replication integrity as 

suggested by Fanning’s work (50). Therefore we will perform these 

analyses with single knockdown and different double knockdown 

combinations of hMutSα, ATR, and ATM to establish the individual 

and combinatorial roles of these proteins during virus replication. Dr. 

i of the Department of Pharmacology and 

Toxicology at UAB is a leading expert in the DNA damage field and 

has extensive experience with this technique examining eukaryotic 

DNA replication intermediates, and therefore she will serve as a coinvestigator and assist us to adapt these assays for viral DNA (see 

attached letter). (ii) We also expect to see an increase in DNA 

breaks and gaps as well as “shattered metaphases” of host DNA in 

MMR-deficient infected cells, which will suggest a DDR-deficient 

phenotype. (iii) One of the functional outcomes of DDR activation is 

cell cycle arrest to prevent DNA damage being passed onto 

daughter cells before the cells repair the damage (63). Consistent 

with this, we have detected that super G2 polyploid (>G2) cells 

accumulate during infection and that such accumulation is abolished 

when DDR is inhibited (Fig. 9). If the hMutSα complex is involved in 

DDR signaling, we expect that the G2/M arrest will be abrogated in 

the absence of hMutSα in infected cells. 

If we detect the above described phenotypes which are 

indicative of the involvement of hMutSα in activating the DDR during 

Figure 9. DDR is required for BKPyVinduced G2/M arrest. RPTE cells were 

transfected with indicated siRNAs and 

infected with BKPyV at an MOI of 5 IU/cell. At 

3 dpi, cells were stained with propidium 

iodide (PI) followed by flow cytometry. 

Research Strategy Page 39
	
infection, we will then perform the following experiments: First, we will use lentivirus to introduce an siRNAresistant form of MSH6 or MSH2 into RPTE cells and determine whether the expression of these constructs 

will rescue the phenotype both in virus production and host genome stability. Second, we will determine 

whether the MMR-mediated DDR activation is linked to the mismatch repair function of the hMutSα complex. 

During mismatch repair, after hMutSα recognizes and binds to DNA mismatches, hMutLα (composed of MLH1 

and PMS2) is recruited to DNA. Assembly of the hMutSα and hMutLα complexes activates the endonuclease 

activity of PMS2, which generates single-strand breaks near the mismatch and allow for the exonuclease 

EXO1 to degrade the strand containing the mismatch (64). Defects in hMutLα inhibit mismatch repair (65-67). 

We will perform siRNA knockdown targeting MLH1 and PMS2 and determine whether viral infection and host 

genome stability will be affected using the assays described above. If we do not detect the same defects in 

DDR activation, viral titer, and genome instability, it will suggest that the hMutSα-mediated DDR activation is 

independent of its mismatch repair function. 

Potential Problems and Alternatives. We do not anticipate major technical problems since the knockdowns of 

MMR proteins are previously well described in cell culture (68), and our preliminary data show that MSH6 can 

be effectively knocked down in RPTE cells without affecting cell viability (Fig. 7A and data not shown). 

Southern blotting, metaphase spread, and cell cycle analyses are all well developed in our laboratory (22). One 

potential problem is that since MSH2 knockdown affects both hMutSα and hMutSβ, it is possible that we will 

get different results when we knockdown MSH6 vs. MSH2. MSH3 competes with MSH6 to bind to MSH2 and 

forms the hMutSβ complex, which recognizes larger in/del loops (53). Our proteomic analysis revealed a 

preferential increase in MSH6 compared with MSH3 (data not shown). Therefore, polyomavirus infection 

perhaps tips the balance between the hMutSα and hMutsβ complexes. The role of the hMutSβ in DDR 

activation is less understood. If we observe differences between MSH6 and MSH2 knockdowns, we will also 

include MSH3 knockdown in our studies. 

Although we did not discuss in detail, we are cognizant that we may find that our original hypothesis is 

incorrect: instead of resolving viral replication intermediates, there may be other aspects of the virus life cycle 

affected by MMR. It could be at the particle assembly step, or alternatively, the assembled particles may 

contain damaged DNA in MMR knockdown cells thereby leading to a decrease in infectivity. To test the former 

possibility, we will determine whether viral particles still form in MSH6 or MSH2 knockdown cells by CsCl 

gradient purification of viral particles followed by immunoblotting for the capsid proteins VP1/2/3 and 

transmission electron microscopy. If the particles appear normal, we will measure the amount of viral DNA 

present in these particles using a real-time PCR assay and determine the viral DNA/infectious unit ratios. A 

high ratio will indicate that the DNA packaged in these particles is defective. These steps can also be affected 

during DDR inhibition and have not been previously explored; therefore we will also extend these studies under 

DDR inhibition conditions. 

Aim 1.2 Determine how MMR proteins contribute to DDR activation during polyomavirus replication. 

Rationale: There are two possible mechanisms for hMutSα-dependent DDR activation during infection. The 

first is an indirect activation model that involves mismatch repair-induced accumulation of ssDNA (69, 70). The 

second is a direct activation model where hMutSα directly recruits ATR and ATM to DNA (59). We believe that 

the second model is more probable because MSH2 and, to a greater extent, MSH6 protein levels both 

increase markedly with BKPyV infection (Fig. 6B). Furthermore, the upregulation of MSH6 can be achieved by 

TAg expression alone (Fig. 6C). Although MSH2 and MSH6 transcription is increased upon BKPyV infection 

as determined in a microarray analysis (71), the increase at the protein level is much greater than at the 

transcript level (data not shown), suggesting some degree of post-transcriptional regulation. One possibility is 

that TAg directly binds to MSH6 and MSH2 and stabilizes these proteins. In the context of infection, we 

hypothesize that the interaction between TAg and MMR proteins serves to recruit MMR proteins to sites of viral 

DNA replication, which in turn recruits ATR and ATM and facilitates DDR activation. 

Experimental Design. To test whether hMutSα recruits DDR proteins to sites of viral replication, we will perform 

the following experiments: (i) Co-immunoprecipitation (co-IP) to examine the protein-protein interactions 

between these proteins. We will use a monoclonal antibody, pAb416, that is known to be able to IP TAg (72) 

and determine whether we can pull down hMutSα, ATR, or ATM in BKPyV-infected cells. Isotype-matched IgG 

and mock-infected cells will be used for negative controls. The immunoprecipitated proteins will be eluted from 

the TAg antibody-conjugated protein G beads followed by Western blotting against MSH6, MSH2, and ATR. 

Reciprocal co-IP will also be performed, and antibodies targeting ATR, ATM, MSH6, and MSH2 are all 

commercially available (61, 73). (ii) Chromatin-IP (ChIP) to determine whether DDR proteins are associated 

with viral DNA and whether these associations occur in an hMutSα-dependent manner. We will 

 

Contact PD/PI: Jiang, Mengxi 

Research Strategy Page 40
	
immunoprecipitate ATR or ATM using commercially available antibodies in BKPyV-infected cells, and the 

immunoprecipitated viral DNA will be quantified using real-time PCR. We will perform these experiments both 

in normal cells and in cells that are knocked down for MSH6 or MSH2. 

Expected Outcomes. (i) If our hypothesis is correct, we will find that TAg is able to co-IP with both hMutSα and 

DDR proteins. Since we observe a greater increase in MSH6 levels compared with MSH2 upon BKPyV 

infection, we expect that we will more likely detect an interaction between TAg and MSH6. Likewise, since 

MSH6 knockdown cells have a greater defect in ATR activation compared with ATM activation, we expect that 

TAg or MMR protein co-IP experiments are more likely to pull down ATR. If we can confirm the interactions 

between these proteins through reciprocal co-IP experiments, we will perform the TAg co-IP experiment in 

MSH6 or MSH2 knockdown cells and determine whether a lack of hMutSα will result in a decreased ability of 

TAg to interact with ATR and ATM. We will also conduct these experiments in cells that are transduced with a 

lentivirus expressing TAg (Fig. 8A) or in cells that are infected with a replication-deficient mutant virus (Fig. 

8B) to determine whether the interaction is dependent on viral DNA replication. (ii) For the ChIP analyses, we 

expect to detect an association of ATR and ATM with viral DNA. If our hypothesis that hMutSα recruits DDR 

proteins to viral DNA is correct, we will find a decreased association of viral DNA with ATM, and more likely 

with ATR, in MSH6 or MSH2 knockdown cells. 

Potential Problems and Alternatives. These experiments are straightforward and we have all of the necessary 

reagents and expertise. A potential technical limitation is that the interactions between these proteins may be 

weak and transient. To ensure that nuclear protein-protein interactions are preserved, we will isolate nuclei 

from infected cells first followed by harvesting lysates under relatively mild conditions (low salt and detergent). 

We will also include crosslinkers such as NHS-ester derivatives (Life Technologies) to preserve interactions if 

necessary. 

It is possible that our direct-recruitment model is incorrect; and the results could instead support the indirect 

model in which it is the mismatch repair-induced accumulation of ssDNA that activates the ATR (69, 70). In this 

case, we may not detect an interaction between hMutSα and DDR proteins by co-IP even though we may still 

detect a decreased association of viral DNA with DDR proteins by ChIP assays. If we get this result, we will 

test the indirect model by siRNA knockdowns of EXO1, which is the exonuclease required to generate ssDNA 

(74), or MLH1, the EXO1-recruiting protein during mismatch repair (75). If the indirect model is correct, we 

expect to see that DDR activation will be diminished with EXO1 or MLH1 knockdown in infected cells and that 

there will be a decreased association of viral DNA with DDR proteins. It is also possible that hMutSα is 

involved in other aspects of DDR function other than recruiting the proteins to viral DNA. In this case, we may 

detect protein-protein interactions between TAg, hMutSα, and DDR through co-IPs, but we will not detect 

changes in viral DNA association with DDR by ChIP. If we get this result, we will investigate the involvement of 

DNA repair pathways such as non-homologous end joining (NHEJ) or homologous recombination (HR) during 

BKPyV replication. We will examine whether these pathways are affected by hMutSα during infection, as 

hMutSα has previously been shown to regulate both pathways (68, 76, 77). Finally, if we do not detect any 

interaction between hMutSα and TAg using the co-IP approach, we will determine whether these proteins at 

least interact with the replicating viral DNA independent of each other, using methods described in Aim 2. 

Aim 2. To determine the viral DNA triggers that activate the DDR upon polyomavirus infection. In this 

Aim we design a series of experiments to test the hypothesis that replicating viral DNA is in fact the major 

trigger for DDR activation during polyomavirus infection. These studies will challenge the current paradigm that 

TAg is the main driver of DDR activation during polyomavirus infection. To our knowledge, this will be the first 

study to directly examine potential viral DNA damage during polyomavirus replication in cells with intact cell 

cycle checkpoint control. 

Aim 2.1 Is viral DNA replication sufficient to activate the DDR? Rationale: Our results with TAg-expressing 

lentivirus and replication-deficient virus establish that viral DNA replication is necessary for DDR activation 

(Fig. 8). In this sub-Aim we will test the hypothesis that replicating viral DNA is sufficient to activate the DDR. 

Experimental Design. We will construct a plasmid containing the BKPyV non-coding control region (78), which 

includes the BKPyV origin of replication. This plasmid or a control plasmid lacking BKPyV origin of replication 

sequences will be transfected into RPTE cells that are transduced with a lentivirus expressing TAg (27). To 

identify cells that are transfected with the BKPyV origin plasmid, we will use fluorescent in situ hybridization 

(FISH) with a probe that recognizes the plasmid sequence. We will compare DDR activation between cells that 

have TAg alone and cells that contain both TAg and viral origin of DNA replication. Because of the relatively 

low transfection efficiency of RPTE cells, we will not be able to perform Western blotting on whole cell lysates 

 

Contact PD/PI: Jiang, Mengxi 

Research Strategy Page 41
	
to probe for DDR activation. Instead, we will use immunofluorescence staining for specific DDR activation 

markers (such as Chk1-pS317) to quantify on a single-cell level if there is elevated DDR activation in cells that 

contain both TAg and the origin sequence. 

Expected Outcomes. If our hypothesis is correct, we predict that there will be a much greater DDR activation 

when both TAg and viral origin are present compared with TAg expression alone. If we get this result, we will 

also introduce mutations in either TAg or the origin of replication to ensure that the activation is dependent on 

viral DNA replication. We will first clone the D501A mutant TAg into a lentivirus expression vector since we 

have shown that this mutation abrogates viral DNA replication. We will also create another mutant TAg that 

lacks a functional origin-binding domain (28). Finally, we will construct a plasmid that contains mutations in the 

origin of viral replication that render the viral DNA incapable of being replicated by TAg (79). We expect that 

RPTE cells transduced with either mutant form of TAg with the wild-type origin, or wild-type TAg with the 

mutant origin will display decreased DDR activation compared with the wild-type TAg and origin combinations. 

Potential Problems and Alternatives. We have extensive experience in FISH techniques (80), but if the 

sensitivity of FISH becomes a problem, we will engineer a fluorescent protein onto the origin-containing 

plasmid to allow identification of transfected cells. If we cannot detect DDR activation by the combination of 

wild-type TAg and viral origin of DNA replication, additional viral components may be involved. We will then 

create expression constructs to introduce other viral proteins including the virally-encoded small T antigen or 

the truncated T antigen (81), deliver them into RPTE cells, and assess their effects on DDR activation with or 

without viral origin. 

Aim 2.2 Are there ssDNA lesions and double-strand breaks (DSBs) on the viral DNA? Rationale: During 

normal cellular replication, the uncoupling of MCM helicase and DNA polymerase during DNA synthesis results 

in ssDNA accumulation, which activates ATR (82). ATM is mainly activated by DSBs, which can arise from 

collapsed replication forks during replication stress (83). In addition, DSB-mediated fork restart is one 

mechanism to repair stalled replication forks (84). Our preliminary data 

show that viral DNA replication drives the activation for both ATR and 

ATM, leading to the hypothesis that both ssDNA lesions and DSBs occur 

on replicating viral DNA, which serve as DDR activation triggers. 

Experimental Design. (i) To determine if there is extensive viral ssDNA 

accumulation, we will use a non-denaturing FISH technique using a nicktranslated viral probe to detect viral ssDNA on a single-cell level (83). The 

average FISH signal intensities will allow us to quantify the amount of 

ssDNA present on viral DNA (83). (ii) To determine whether there are 

DSBs on viral DNA, We will perform ChIP experiments in infected cells 

with a commercially available antibody against γH2AX, a protein known to 

mark sites of DSBs and recruit DNA repair proteins to these sites (85). 

The IP will be followed with real-time PCRs using primers against different 

regions on the viral chromosome. γH2AX level is markedly increased with 

BKPyV infection (Fig. 1A). We also observed that Mre11 (an early DSB 

sensor) and γH2AX are both recruited to TAg-containing nuclear foci 

during infection (Fig. 10). Therefore we reason that γH2AX-associated 

viral DNA will be a good initial marker for DSBs on viral DNA. In these 

experiments, DNA damaging reagents such as doxorubicin and the 

known damage sites on the host chromosome will be used as positive 

controls (86). Our preliminary data did show that γH2AX is associated 

with viral DNA (Fig. 11). (iii) We will also examine the accumulation of 

viral ssDNA lesions and DSBs in cells treated with hydroxyurea (HU), 

which is a ribonucleotide reductase inhibitor that can cause replication 

stress (83) and therefore may enrich these signals that are indicative of 

damage on viral DNA. 

Expected Outcomes. If our hypothesis is correct, we predict that both viral 

ssDNA and the association of γH2AX with viral DNA will be detected 

during normal infection, and that there will be an increase in viral ssDNA 

lesions and γH2AX-associated viral DNA in HU treated replication stress 

conditions. If we get these results, we will perform the following 

Figure 10. γH2AX and Mre11 are 

recruited to TAg foci. RPTE cells 

were infected with BKPyV infection at 

an MOI 0.5 IU/cells. Cells were 

immunostained for γH2AX, TAg, and 

Mre11 at 3 dpi. Bar, 10 μm.

Figure 11. γH2AX associates with 

BKPyV viral DNA. RPTE cells were 

infected with BKPyV at an MOI of 0.5 

IU/cell and cell lysates were subjected 

to ChIP analyses using antibodies 

against γH2AX or IgG at 3 dpi. The 

immunoprecipitated DNA were analyzed 

using real-time PCR with primers 

amplifying TAg or NCCR region on the 

viral genome and data were shown as 

percent of input controls. 

 

Contact PD/PI: Jiang, Mengxi 

Research Strategy Page 42
	
experiments: (i) Determine whether we can detect the opposite phenotype by supplementing infected cells with 

nucleotides to reduce viral replication stress during normal infection. (ii) Since the uncoupling of MCM helicase 

and host cell DNA polymerase α has been proposed to be the main mechanism for ATR activation (82), we will 

test whether this is also the case on viral DNA, i.e., the uncoupling between TAg helicase and DNA 

polymerase α results in the accumulation of ssDNA and activation of ATR. We will briefly treat the infected 

cells with aphidicolin, a DNA polymerase α inhibitor (82) and measure whether there is any increase in ssDNA 

lesions and DSBs on viral DNA. (iii) We will also measure DSB formation via γH2AX ChIP assays in ATR 

knockdown cells. If DSB formation is a result of replication fork destabilizing and collapsing, we will expect to 

see an increase of DSBs in the absence of ATR. If we do not observe such an increase, it suggests that the 

DSBs are derived from other sources, for example, oxidative damage caused by viral infection. 

Potential Problems and Alternatives. If the sensitivity of single-cell FISH is not high enough, we will modify our 

Southern procedure to perform a non-denaturing Southern blotting instead (78). Another caveat of the 

proposed study is that from a single DSB, γH2AX can also be spread several kilobases away from the damage 

site (87). Therefore it is possible that occupancy by γH2AX does not reflect the actual location of the DSB. To 

circumvent this problem and as an alternative method to map DNA damage, we will use ligation-mediated PCR 

(88) to directly map DSBs on the viral DNA. In this method, low-molecular weight DNA with broken ends from 

infected cells will be ligated with a biotinylated double-stranded oligonucleotide followed by restriction enzyme 

digest. This allows for the purification of these damaged DNA with streptavidin beads and the subsequent 

regular or real-time PCR to reveal sequence information and to perform quantitative analysis. 

If we do not detect either ssDNA lesions or DSBs on viral DNA, it may suggest that the DDR is activated 

by a different mechanism. One possibility is that TAg recruits DDR proteins to sites of viral replication and the 

prolonged association of these proteins with viral DNA or chromatin activates the DDR without any actual DNA 

lesion. This possibility has been shown previously by targeting DNA repair factors to the host chromatin (89). 

To test this idea, we will use a recently developed technique iPOND (isolate proteins on nascent DNA) (43, 

90), to investigate whether DDR proteins such as ATR, ATM, and MMR proteins are associated with replicating 

viral DNA. Infected cells will be labeled with biotinylated EdU, and proteins bound to the newly replicated DNA 

will be purified using streptavidin following click chemistry and be confirmed by Western blotting. To ensure 

that we are examining proteins associated with replicating viral DNA instead of cellular DNA, we will isolate the 

viral minichromosomes (33) prior to the purification. We have already performed EdU labeling experiments and 

have confirmed that the infected cells can be efficiently labeled (data not shown). 

Aim 3. To elucidate the molecular mechanism by which polyomavirus induces host genome instability. 

It is well accepted in the field that TAg is able to induce DNA damage (18, 19), but little is known about the 

exact mechanism of how the damage arises. The goal of this aim is to probe the mechanistic link between viral 

DNA replication and host DNA damage induced by TAg. It has previously been shown by one of the classic 

cell fusion experiments that fusion of an S-phase cell with a mitotic cell can result in a “shattered chromosome” 

phenotype (91). We hypothesize that TAg-induced host replication stress coupled with mitosis leads to host 

genome instability, which can be prevented by virus-replication induced DDR activation via G2/M arrest. 

Aim 3.1 Is polyomavirus-induced host DNA damage associated with mitosis? Rationale: According to our 

model (Fig. 1), TAg-induced aberrant host DNA replication stress followed by mitosis causes chromosome 

damage. This damage is not observed during normal infection because virus replication activates the DDR and 

G2/M checkpoint, which inactivates Cdc25C, a phosphatase necessary for mitotic entry (92). If our model is 

correct, we predict that if we force cells into mitosis during normal infection, we will detect DNA damage similar 

to that seen in DDR-deficient infected cells. 

Experimental Design. To bypass the G2/M checkpoint and induce the cells into mitosis, we will use two 

independent strategies (i) siRNA knockdown of Wee1, and (ii) overexpression of Cdc25C and cyclin B1 using 

lentivirus constructs. Wee1 is a kinase that inhibits mitotic entry through phosphorylation of Cdk1 (93). 

Overexpression of Cdc25C and cyclin B1 has previously been shown to induce mitosis (94). Timing is 

important in these experiments. We will first infect RPTE cells for two days to allow for S-phase induction by 

virus infection before we perform the siRNA knockdowns or lentivirus transductions. Metaphase analyses will 

be used following siRNA transfection or lentivirus transduction to examine the degree of chromosome damage. 

Expected Outcomes. In cells that are knocked down for Wee1 or overexpressed with Cdc25C and cyclin B1, 

we predict that the chromosome damage will be more severe in infected cells compared with uninfected cells. 

Among the infected cells, we predict that by driving the cells into mitosis we will observe an increase in 

abnormal chromosomes compared with normal infected cells. If we observe this, we will also directly measure 

 

Contact PD/PI: Jiang, Mengxi 

Research Strategy Page 43
	
 

Contact PD/PI: Jiang, Mengxi 

the mitotic status of the cells using a well-established mitotic index assay (Life Technologies). During mitotic 

entry, histone H3 is phosphorylated at the Ser10 position with chromatin condensation (95) and therefore can 

serve as a marker for mitosis. We will examine the following conditions for mitotic index: (i) wild-type BKPyV 

infection, (ii) TAg D501A mutant virus infection, and (iii) lentivirus-expressing TAg. We predict that the mitotic 

index will increase with the latter two conditions compared with normal virus infection, as the G2/M checkpoint 

is not activated in these cells. 

Potential Problems and Alternatives. If we do not detect an increase in chromosome damage from the above 

experiments, there could be two explanations. One possibility is that our experimental conditions do not

completely remove other checkpoints for mitosis, for example, the spindle assembly checkpoint (SAC). To test

this idea, we will also combine the previous experiments with SAC inactivation, for example, using a small 

inhibitor Gö6976 (96) to determine whether we can induce more damage in infected cells. Another possibility is 

that the chromosome damage in DDR-deficient cells is caused by DNA repair inhibition instead of a loss of 

checkpoint in infected cells. We will then inhibit the two major DNA repair pathways (SCR7 inhibitor to target 

NHEJ (97) and Rad51 siRNA knockdown to inhibit HR (98)) in infected cells and examine the chromosome 

damage under these conditions. 

Aim 3.2 Does large T antigen induce host cell replication stress? Rationale: There are increasing reports 

on oncogenes inducing replication stress thereby leading to DNA damage (99-101). In our system, TAg 

expression alone can cause DNA damage. Moreover, SV40 TAg-induced DNA damage can be reversed by 

exogenous supplementation of nucleotides (29). Together, these observations lead to the hypothesis that TAg 

induces host DNA replication stress. 

Experimental Design. To directly determine the impact of TAg on host DNA replication, we will use a novel 

DNA fiber assay (102) to measure how TAg affects host DNA replication initiation and elongation in lentivirusTAg transduced cells. This is a quantitative approach that labels nascent DNA in vivo by sequential 

incorporation of two halogenated nucleotides. Individual DNA fibers are stretched onto a microscope slide, and 

the labeled DNA replication tracts can be visualized by antibody staining and fluorescence microscopy. 

Replication origin firing, replication elongation, and replication fork terminations can all be quantitatively 

measured based on different labeling patterns. laboratory is experienced in this 

technique and she will provide expertise for these analyses (please see attached letter). We will also inhibit 

replication initiation in these cells using siRNA targeting the origin licensing factor Cdc6 (100) and determine 

whether it will prevent TAg-induced replication stress and DNA damage. 

Expected Outcomes. We expect to see increased origin firing and impaired replication fork progression in the 

presence of TAg. We also expect that these defects will be abolished through Cdc6 knockdown. If we observe 

this, we will introduce several mutations in TAg and assess which function of TAg is required for the replication 

stress phenotype: (i) A pRb-binding mutant since TAg may induce replication stress through inactivation of 

pRb, thus allowing E2F to drive cells into S phase (36, 72). (ii) An origin-binding domain mutant and a helicase 

mutant, as it is possible that TAg binds to and unwinds non-specific or pseudo-origins of replication sequences 

present on host DNA, thereby triggering aberrant replication (103). This idea has been proposed for the human 

papillomavirus E1 helicase (104). (iii) An RPA-binding mutant, since it has been shown that TAg binds to RPA 

and this interaction may prevent normal function of RPA during cellular replication (20, 105). 

Potential Problems and Alternatives. The DNA fiber assay only involves a short period of halogenated 

nucleotide labeling. If we do not detect any difference between lentivirus-TAg transduced cells and control 

cells, we will create an inducible-TAg system by cloning TAg under a tetracycline-controlled promoter (106) to 

better control the relative timing between TAg expression and DNA labeling. If we do not detect any significant 

replication stress defect induced by TAg, we will then test the hypothesis that TAg directly causes DNA 

damage either by melting host DNA or through a potential nuclease activity. As a first step to test this idea, we 

will perform a ChIP-sequencing experiment to determine whether there are specific host DNA sequences that 

are associated with TAg. 

C4) OVERALL SUMMARY AND CONCLUSIONS. We believe these studies will allow us to gain a more indepth understanding of how polyomaviruses usurp the host DDR to promote viral replication and how the DDR 

impacts polyomavirus-induced genome instability. We have gathered sufficient preliminary results and 

developed the expertise to ensure that the studies proposed here are highly feasible to accomplish. This 

research will have significant impact on our understanding of oncogenic polyomavirus replication and will lay 

the groundwork for the future development of DDR inhibitors to selectively target polyomavirus-infected cells. 

Research Strategy Page 44
	
Literature Cited 

1. Gardner SD, Field AM, Coleman DV, Hulme B. 1971. New human papovavirus (B.K.) isolated from 

urine after renal transplantation. Lancet 1:1253-1257. 

2. Padgett BL, Walker DL, ZuRhein GM, Eckroade RJ, Dessel BH. 1971. Cultivation of papova-like 

virus from human brain with progressive multifocal leucoencephalopathy. Lancet 1:1257-1260. 

3. Jiang M, Abend JR, Johnson SF, Imperiale MJ. 2009. The role of polyomaviruses in human disease. 

Virology 384:266-273. 

4. Nickeleit V, Singh HK, Goldsmith CS, Miller SE, Kenan DJ. 2013. BK virus-associated urinary 

bladder carcinoma in transplant recipients: productive or nonproductive polyomavirus infections in 

tumor cells? Hum Pathol 44:2870-2871. 

5. Bulut Y, Ozdemir E, Ozercan HI, Etem EO, Aker F, Toraman ZA, Seyrek A, Firdolas F. 2013. 

Potential relationship between BK virus and renal cell carcinoma. J Med Virol 85:1085-1089. 

6. van Aalderen MC, Yapici U, van der Pol JA, de Reijke TM, van Donselaar-van der Pant KA, 

Florquin S, Bemelman FJ, Ten Berge IJ. 2013. Polyomavirus BK in the pathogenesis of bladder 

cancer. Neth J Med 71:26-28. 

7. Alexiev BA, Randhawa P, Vazquez Martul E, Zeng G, Luo C, Ramos E, Drachenberg CB, 

Papadimitriou JC. 2013. BK virus-associated urinary bladder carcinoma in transplant recipients: report 

of 2 cases, review of the literature, and proposed pathogenetic model. Hum Pathol 44:908-917. 

8. Pino L, Rijo E, Nohales G, Frances A, Ubre A, Arango O. 2013. Bladder transitional cell carcinoma 

and BK virus in a young kidney transplant recipient. Transpl Infect Dis 15:E25-27. 

9. Emerson LL, Carney HM, Layfield LJ, Sherbotie JR. 2008. Collecting duct carcinoma arising in 

association with BK nephropathy post-transplantation in a pediatric patient. A case report with 

immunohistochemical and in situ hybridization study. Pediatr Transplant 12:600-605. 

10. Geetha D, Tong BC, Racusen L, Markowitz JS, Westra WH. 2002. Bladder carcinoma in a transplant 

recipient: evidence to implicate the BK human polyomavirus as a causal transforming agent. 

Transplantation 73:1933-1936. 

11. Narayanan M, Szymanski J, Slavcheva E, Rao A, Kelly A, Jones K, Jaffers G. 2007. BK virus 

associated renal cell carcinoma: case presentation with optimized PCR and other diagnostic tests. Am 

J Transplant 7:1666-1671. 

12. Ferenczy MW, Marshall LJ, Nelson CD, Atwood WJ, Nath A, Khalili K, Major EO. 2012. Molecular 

biology, epidemiology, and pathogenesis of progressive multifocal leukoencephalopathy, the JC virusinduced demyelinating disease of the human brain. Clin Microbiol Rev 25:471-506. 

13. DeCaprio JA, Garcea RL. 2013. A cornucopia of human polyomaviruses. Nat Rev Microbiol 11:264-

276. 

14. Houben R, Shuda M, Weinkam R, Schrama D, Feng H, Chang Y, Moore PS, Becker JC. 2010. 

Merkel cell polyomavirus-infected Merkel cell carcinoma cells require expression of viral T antigens. J 

Virol 84:7064-7072. 

15. Dalianis T, Hirsch HH. 2013. Human polyomaviruses in disease and cancer. Virology 437:63-72. 

16. Feng H, Shuda M, Chang Y, Moore PS. 2008. Clonal integration of a polyomavirus in human Merkel 

cell carcinoma. Science 319:1096-1100. 

17. Fanning E, Zhao K. 2009. SV40 DNA replication: from the A gene to a nanomachine. Virology 

384:352-359. 

18. Boichuk S, Hu L, Hein J, Gjoerup OV. 2010. Multiple DNA damage signaling and repair pathways 

deregulated by simian virus 40 large T antigen. J Virol 84:8007-8020. 

19. Li J, Wang X, Diaz J, Tsang SH, Buck CB, You J. 2013. Merkel cell polyomavirus large T antigen 

disrupts host genomic integrity and inhibits cellular proliferation. J Virol 87:9173-9188. 

20. Banerjee P, DeJesus R, Gjoerup O, Schaffhausen BS. 2013. Viral interference with DNA repair by 

targeting of the single-stranded DNA binding protein RPA. PLoS Pathog 9:e1003725. 

21. Trojanek J, Croul S, Ho T, Wang JY, Darbinyan A, Nowicki M, Del Valle L, Skorski T, Khalili K, 

Reiss K. 2006. T-antigen of the human polyomavirus JC attenuates faithful DNA repair by forcing 

nuclear interaction between IRS-1 and Rad51. J Cell Physiol 206:35-46. 

22. Jiang M, Zhao L, Gamez M, Imperiale MJ. 2012. Roles of ATM and ATR-mediated DNA damage 

responses during lytic BK polyomavirus infection. PLoS Pathog 8:e1002898. 

23. Low J, Humes HD, Szczypka M, Imperiale M. 2004. BKV and SV40 infection of human kidney tubular 

epithelial cells in vitro. Virology 323:182-188. 

 

Contact PD/PI: Jiang, Mengxi 

References Cited Page 45
	
24. Jiang M, Entezami P, Gamez M, Stamminger T, Imperiale MJ. 2011. Functional reorganization of 

promyelocytic leukemia nuclear bodies during BK virus infection. MBio 2:e00281-00210. 

25. Jiang M, Abend JR, Tsai B, Imperiale MJ. 2009. Early events during BK virus entry and disassembly. 

J Virol 83:1350-1358. 

26. Randhawa PS, Finkelstein S, Scantlebury V, Shapiro R, Vivas C, Jordan M, Picken MM, Demetris 

AJ. 1999. Human polyoma virus-associated interstitial nephritis in the allograft kidney. Transplantation 

67:103-109. 

27. Verhalen B, Justice JL, Imperiale MJ, Jiang M. 2015. Viral DNA Replication-Dependent DNA 

Damage Response Activation during BK Polyomavirus Infection. J Virol 89:5032-5039. 

28. Orba Y, Suzuki T, Makino Y, Kubota K, Tanaka S, Kimura T, Sawa H. 2010. Large T antigen 

promotes JC virus replication in G2-arrested cells by inducing ATM- and ATR-mediated G2 checkpoint 

signaling. J Biol Chem 285:1544-1554. 

29. Hu L, Filippakis H, Huang H, Yen TJ, Gjoerup OV. 2013. Replication stress and mitotic dysfunction in 

cells expressing simian virus 40 large T antigen. J Virol 87:13179-13192. 

30. Mao Z, Bozzella M, Seluanov A, Gorbunova V. 2008. Comparison of nonhomologous end joining and 

homologous recombination in human cells. DNA Repair (Amst) 7:1765-1771. 

31. Tsang SH, Wang X, Li J, Buck CB, You J. 2014. Host DNA damage response factors localize to 

merkel cell polyomavirus DNA replication sites to support efficient viral DNA replication. J Virol 

88:3285-3297. 

32. Topalis D, Andrei G, Snoeck R. 2013. The large tumor antigen: a "Swiss Army knife" protein 

possessing the functions required for the polyomavirus life cycle. Antiviral Res 97:122-136. 

33. Zlotkin T, Kaufmann G, Jiang Y, Lee MY, Uitto L, Syvaoja J, Dornreiter I, Fanning E, Nethanel T. 

1996. DNA polymerase epsilon may be dispensable for SV40- but not cellular-DNA replication. EMBO J 

15:2298-2305. 

34. Moyer SE, Lewis PW, Botchan MR. 2006. Isolation of the Cdc45/Mcm2-7/GINS (CMG) complex, a 

candidate for the eukaryotic DNA replication fork helicase. Proc Natl Acad Sci U S A 103:10236-10241. 

35. Zhao X, Madden-Fuentes RJ, Lou BX, Pipas JM, Gerhardt J, Rigell CJ, Fanning E. 2008. Ataxia 

telangiectasia-mutated damage-signaling kinase- and proteasome-dependent destruction of Mre11-

Rad50-Nbs1 subunits in Simian virus 40-infected primate cells. J Virol 82:5316-5328. 

36. Chellappan S, Kraus VB, Kroger B, Munger K, Howley PM, Phelps WC, Nevins JR. 1992. 

Adenovirus E1A, simian virus 40 tumor antigen, and human papillomavirus E7 protein share the 

capacity to disrupt the interaction between transcription factor E2F and the retinoblastoma gene 

product. Proc Natl Acad Sci U S A 89:4549-4553. 

37. Harris KF, Christensen JB, Radany EH, Imperiale MJ. 1998. Novel mechanisms of E2F induction by 

BK virus large-T antigen: requirement of both the pRb-binding and the J domains. Mol Cell Biol 

18:1746-1756. 

38. Bargonetti J, Reynisdottir I, Friedman PN, Prives C. 1992. Site-specific binding of wild-type p53 to 

cellular DNA is inhibited by SV40 T antigen and mutant p53. Genes Dev 6:1886-1898. 

39. Jiang D, Srinivasan A, Lozano G, Robbins PD. 1993. SV40 T antigen abrogates p53-mediated 

transcriptional activity. Oncogene 8:2805-2812. 

40. Sachsenmeier KF, Pipas JM. 2001. Inhibition of Rb and p53 is insufficient for SV40 T-antigen 

transformation. Virology 283:40-48. 

41. Cimprich KA, Cortez D. 2008. ATR: an essential regulator of genome integrity. Nat Rev Mol Cell Biol 

9:616-627. 

42. Jackson SP. 2002. Sensing and repairing DNA double-strand breaks. Carcinogenesis 23:687-696. 

43. Sirbu BM, Couch FB, Feigerle JT, Bhaskara S, Hiebert SW, Cortez D. 2011. Analysis of protein 

dynamics at active, stalled, and collapsed replication forks. Genes Dev 25:1320-1327. 

44. Dickey JS, Redon CE, Nakamura AJ, Baird BJ, Sedelnikova OA, Bonner WM. 2009. H2AX: 

functional roles and potential applications. Chromosoma 118:683-692. 

45. Podhorecka M, Skladanowski A, Bozko P. 2010. H2AX Phosphorylation: Its Role in DNA Damage 

Response and Cancer Therapy. J Nucleic Acids 2010. 

46. Zachos G, Rainey MD, Gillespie DA. 2003. Chk1-deficient tumour cells are viable but exhibit multiple 

checkpoint and survival defects. EMBO J 22:713-723. 

47. Maya-Mendoza A, Petermann E, Gillespie DA, Caldecott KW, Jackson DA. 2007. Chk1 regulates 

the density of active replication origins during the vertebrate S phase. EMBO J 26:2719-2731. 

 

Contact PD/PI: Jiang, Mengxi 

References Cited Page 46
	
48. Hollingworth R, Grand RJ. 2015. Modulation of DNA Damage and Repair Pathways by Human 

Tumour Viruses. Viruses 7:2542-2591. 

49. Chaurushiya MS, Weitzman MD. 2009. Viral manipulation of DNA repair and cell cycle checkpoints. 

DNA Repair (Amst) 8:1166-1176. 

50. Sowd GA, Li NY, Fanning E. 2013. ATM and ATR activities maintain replication fork integrity during 

SV40 chromatin replication. PLoS Pathog 9:e1003283. 

51. Fenech M, Kirsch-Volders M, Natarajan AT, Surralles J, Crott JW, Parry J, Norppa H, Eastmond 

DA, Tucker JD, Thomas P. 2011. Molecular mechanisms of micronucleus, nucleoplasmic bridge and 

nuclear bud formation in mammalian and human cells. Mutagenesis 26:125-132. 

52. Drummond JT, Li GM, Longley MJ, Modrich P. 1995. Isolation of an hMSH2-p160 heterodimer that 

restores DNA mismatch repair to tumor cells. Science 268:1909-1912. 

53. Matton N, Simonetti J, Williams K. 2000. Identification of mismatch repair protein complexes in HeLa 

nuclear extracts and their interaction with heteroduplex DNA. J Biol Chem 275:17808-17813. 

54. Edelbrock MA, Kaliyaperumal S, Williams KJ. 2013. Structural, molecular and cellular functions of 

MSH2 and MSH6 during DNA mismatch repair, damage signaling and other noncanonical activities. 

Mutat Res 743-744:53-66. 

55. Jiao J, Simmons DT. 2003. Nonspecific double-stranded DNA binding activity of simian virus 40 large 

T antigen is involved in melting and unwinding of the origin. J Virol 77:12720-12728. 

56. Buck CB, Pastrana DV, Lowy DR, Schiller JT. 2005. Generation of HPV pseudovirions using 

transfection and their use in neutralization assays. Methods Mol Med 119:445-462. 

57. Broekema NM, Imperiale MJ. 2012. Efficient propagation of archetype BK and JC polyomaviruses. 

Virology 422:235-241. 

58. Pabla N, Ma Z, McIlhatton MA, Fishel R, Dong Z. 2011. hMSH2 recruits ATR to DNA damage sites 

for activation during DNA damage-induced apoptosis. J Biol Chem 286:10411-10418. 

59. Yoshioka K, Yoshioka Y, Hsieh P. 2006. ATR kinase activation mediated by MutSalpha and 

MutLalpha in response to cytotoxic O6-methylguanine adducts. Mol Cell 22:501-510. 

60. Wang Y, Qin J. 2003. MSH2 and ATR form a signaling module and regulate two branches of the 

damage response to DNA methylation. Proc Natl Acad Sci U S A 100:15387-15392. 

61. Liu Y, Fang Y, Shao H, Lindsey-Boltz L, Sancar A, Modrich P. 2010. Interactions of human 

mismatch repair proteins MutSalpha and MutLalpha with proteins of the ATR-Chk1 pathway. J Biol 

Chem 285:5974-5982. 

62. Dandjinou AT, Larrivee M, Wellinger RE, Wellinger RJ. 2006. Two-dimensional agarose gel analysis 

of DNA replication intermediates. Methods Mol Biol 313:193-208. 

63. Abraham RT. 2001. Cell cycle checkpoint signaling through the ATM and ATR kinases. Genes Dev 

15:2177-2196. 

64. Kunkel TA, Erie DA. 2005. DNA mismatch repair. Annu Rev Biochem 74:681-710. 

65. Bronner CE, Baker SM, Morrison PT, Warren G, Smith LG, Lescoe MK, Kane M, Earabino C, 

Lipford J, Lindblom A, et al. 1994. Mutation in the DNA mismatch repair gene homologue hMLH1 is 

associated with hereditary non-polyposis colon cancer. Nature 368:258-261. 

66. Liu B, Parsons R, Papadopoulos N, Nicolaides NC, Lynch HT, Watson P, Jass JR, Dunlop M, 

Wyllie A, Peltomaki P, de la Chapelle A, Hamilton SR, Vogelstein B, Kinzler KW. 1996. Analysis of 

mismatch repair genes in hereditary non-polyposis colorectal cancer patients. Nat Med 2:169-174. 

67. Prolla TA, Christie DM, Liskay RM. 1994. Dual requirement in yeast DNA mismatch repair for MLH1 

and PMS1, two homologs of the bacterial mutL gene. Mol Cell Biol 14:407-415. 

68. Shahi A, Lee JH, Kang Y, Lee SH, Hyun JW, Chang IY, Jun JY, You HJ. 2011. Mismatch-repair 

protein MSH6 is associated with Ku70 and regulates DNA double-strand break repair. Nucleic Acids 

Res 39:2130-2143. 

69. Li GM. 1999. The role of mismatch repair in DNA damage-induced apoptosis. Oncol Res 11:393-400. 

70. York SJ, Modrich P. 2006. Mismatch repair-dependent iterative excision at irreparable O6-

methylguanine lesions in human nuclear extracts. J Biol Chem 281:22674-22683. 

71. Abend JR, Low JA, Imperiale MJ. 2010. Global effects of BKV infection on gene expression in human 

primary kidney epithelial cells. Virology 397:73-79. 

72. Harris KF, Christensen JB, Imperiale MJ. 1996. BK virus large T antigen: interactions with the 

retinoblastoma family of tumor suppressor proteins and effects on cellular growth control. J Virol 

70:2378-2386. 

 

Contact PD/PI: Jiang, Mengxi 

References Cited Page 47
	
73. Sun Y, Xu Y, Roy K, Price BD. 2007. DNA damage-induced acetylation of lysine 3016 of ATM 

activates ATM kinase activity. Mol Cell Biol 27:8502-8509. 

74. Burdett V, Baitinger C, Viswanathan M, Lovett ST, Modrich P. 2001. In vivo requirement for RecJ, 

ExoVII, ExoI, and ExoX in methyl-directed mismatch repair. Proc Natl Acad Sci U S A 98:6765-6770. 

75. Tran PT, Simon JA, Liskay RM. 2001. Interactions of Exo1p with components of MutLalpha in 

Saccharomyces cerevisiae. Proc Natl Acad Sci U S A 98:9760-9765. 

76. Spell RM, Jinks-Robertson S. 2003. Role of mismatch repair in the fidelity of RAD51- and RAD59-

dependent recombination in Saccharomyces cerevisiae. Genetics 165:1733-1744. 

77. Nicholson A, Hendrix M, Jinks-Robertson S, Crouse GF. 2000. Regulation of mitotic homeologous 

recombination in yeast. Functions of mismatch repair and nucleotide excision repair genes. Genetics 

154:133-146. 

78. Broekema NM, Abend JR, Bennett SM, Butel JS, Vanchiere JA, Imperiale MJ. 2010. A system for 

the analysis of BKV non-coding control regions: application to clinical isolates from an HIV/AIDS 

patient. Virology 407:368-373. 

79. Kwun HJ, Guastafierro A, Shuda M, Meinke G, Bohm A, Moore PS, Chang Y. 2009. The minimum 

replication origin of merkel cell polyomavirus has a unique large T-antigen loading architecture and 

requires small T-antigen expression for optimal replication. J Virol 83:12118-12128. 

80. Jiang M, Entezami P, Gamez M, Stamminger T, Imperiale MJ. 2011. Functional Reorganization of 

Promyelocytic Leukemia Nuclear Bodies during BK Virus Infection. mBio. 

81. Abend JR, Joseph AE, Das D, Campbell-Cecen DB, Imperiale MJ. 2009. A truncated T antigen 

expressed from an alternatively spliced BK virus early mRNA. J Gen Virol 90:1238-1245. 

82. Byun TS, Pacek M, Yee MC, Walter JC, Cimprich KA. 2005. Functional uncoupling of MCM helicase 

and DNA polymerase activities activates the ATR-dependent checkpoint. Genes Dev 19:1040-1052. 

83. Couch FB, Bansbach CE, Driscoll R, Luzwick JW, Glick GG, Betous R, Carroll CM, Jung SY, Qin 

J, Cimprich KA, Cortez D. 2013. ATR phosphorylates SMARCAL1 to prevent replication fork collapse. 

Genes Dev 27:1610-1623. 

84. Branzei D, Foiani M. 2010. Maintaining genome stability at the replication fork. Nat Rev Mol Cell Biol 

11:208-219. 

85. Lobrich M, Shibata A, Beucher A, Fisher A, Ensminger M, Goodarzi AA, Barton O, Jeggo PA. 

2010. gammaH2AX foci analysis for monitoring DNA double-strand break repair: strengths, limitations 

and optimization. Cell Cycle 9:662-669. 

86. Seo J, Kim SC, Lee HS, Kim JK, Shon HJ, Salleh NL, Desai KV, Lee JH, Kang ES, Kim JS, Choi 

JK. 2012. Genome-wide profiles of H2AX and gamma-H2AX differentiate endogenous and exogenous 

DNA damage hotspots in human cells. Nucleic Acids Res 40:5965-5974. 

87. Price BD, D'Andrea AD. 2013. Chromatin remodeling at DNA double-strand breaks. Cell 152:1344-

1354. 

88. Villalobos MJ, Betti CJ, Vaughan AT. 2006. Detection of DNA double-strand breaks and 

chromosome translocations using ligation-mediated PCR and inverse PCR. Methods Mol Biol 314:109-

121. 

89. Soutoglou E, Misteli T. 2008. Activation of the cellular DNA damage response in the absence of DNA 

lesions. Science 320:1507-1510. 

90. Sirbu BM, Couch FB, Cortez D. 2012. Monitoring the spatiotemporal dynamics of proteins at 

replication forks and in assembled chromatin using isolation of proteins on nascent DNA. Nat Protoc 

7:594-605. 

91. Johnson RT, Rao PN. 1970. Mammalian cell fusion: induction of premature chromosome 

condensation in interphase nuclei. Nature 226:717-722. 

92. Karlsson-Rosenthal C, Millar JB. 2006. Cdc25: mechanisms of checkpoint inhibition and recovery. 

Trends Cell Biol 16:285-292. 

93. Den Haese GJ, Walworth N, Carr AM, Gould KL. 1995. The Wee1 protein kinase regulates T14 

phosphorylation of fission yeast Cdc2. Mol Biol Cell 6:371-385. 

94. Karlsson C, Katich S, Hagting A, Hoffmann I, Pines J. 1999. Cdc25B and Cdc25C differ markedly in 

their properties as initiators of mitosis. J Cell Biol 146:573-584. 

95. Hirota T, Lipp JJ, Toh BH, Peters JM. 2005. Histone H3 serine 10 phosphorylation by Aurora B 

causes HP1 dissociation from heterochromatin. Nature 438:1176-1180. 

 

Contact PD/PI: Jiang, Mengxi 

References Cited Page 48
	
96. Stolz A, Vogel C, Schneider V, Ertych N, Kienitz A, Yu H, Bastians H. 2009. Pharmacologic 

abrogation of the mitotic spindle checkpoint by an indolocarbazole discovered by cellular screening 

efficiently kills cancer cells. Cancer Res 69:3874-3883. 

97. Srivastava M, Nambiar M, Sharma S, Karki SS, Goldsmith G, Hegde M, Kumar S, Pandey M, 

Singh RK, Ray P, Natarajan R, Kelkar M, De A, Choudhary B, Raghavan SC. 2012. An inhibitor of 

nonhomologous end-joining abrogates double-strand break repair and impedes cancer progression. 

Cell 151:1474-1487. 

98. Koch K, Wrona A, Dikomey E, Borgmann K. 2009. Impact of homologous recombination on 

individual cellular radiosensitivity. Radiother Oncol 90:265-272. 

99. Dominguez-Sola D, Ying CY, Grandori C, Ruggiero L, Chen B, Li M, Galloway DA, Gu W, Gautier 

J, Dalla-Favera R. 2007. Non-transcriptional control of DNA replication by c-Myc. Nature 448:445-451. 

100. Jones RM, Mortusewicz O, Afzal I, Lorvellec M, Garcia P, Helleday T, Petermann E. 2013. 

Increased replication initiation and conflicts with transcription underlie Cyclin E-induced replication 

stress. Oncogene 32:3744-3753. 

101. Bester AC, Roniger M, Oren YS, Im MM, Sarni D, Chaoat M, Bensimon A, Zamir G, Shewach DS, 

Kerem B. 2011. Nucleotide deficiency promotes genomic instability in early stages of cancer 

development. Cell 145:435-446. 

102. Schwab RA, Niedzwiedz W. 2011. Visualization of DNA replication in the vertebrate model system 

DT40 using the DNA fiber technique. J Vis Exp doi:10.3791/3255. 

103. Galli I, Iguchi-Ariga SM, Ariga H. 1993. Mammalian genomic sequences can substitute for the SV40 

AT stretch in sustaining replication of the SV40 origin of replication. FEBS Lett 318:335-340. 

104. Sakakibara N, Mitra R, McBride AA. 2011. The papillomavirus E1 helicase activates a cellular DNA 

damage response in viral replication foci. J Virol 85:8981-8995. 

105. Ning B, Feldkamp MD, Cortez D, Chazin WJ, Friedman KL, Fanning E. 2015. Simian virus Large T 

antigen interacts with the N-terminal domain of the 70 kD subunit of Replication Protein A in the same 

mode as multiple DNA damage response factors. PLoS One 10:e0116093. 

106. Urlinger S, Baron U, Thellmann M, Hasan MT, Bujard H, Hillen W. 2000. Exploring the sequence 

space for tetracycline-dependent transcriptional activators: novel mutations yield expanded range and 

sensitivity. Proc Natl Acad Sci U S A 97:7963-7968. 

 

Contact PD/PI: Jiang, Mengxi 

References Cited Page 49