SUMMARY STATEMENT

PROGRAM CONTACT: ( Privileged Communication ) Release Date: 11/09/2015

Application Number: 1 R01 AI123162-01

Principal Investigator 

JIANG, MENGXI PHD

Applicant Organization: UNIVERSITY OF ALABAMA AT BIRMINGHAM

Review Group: VIRB

Virology - B Study Section

Meeting Date: 10/29/2015 RFA/PA: PA13-302

Council: JAN 2016 PCC: M34A

Requested Start: 04/01/2016

Project Title: Intersection of polyomavirus infection and host cellular responses

SRG Action: Impact Score: Percentile:

Next Steps: Visit http://grants.nih.gov/grants/next_steps.htm

Human Subjects: 10-No human subjects involved

Animal Subjects: 10-No live vertebrate animals involved for competing appl.

Project Direct Costs Estimated

Year Requested Total Cost 

1 

2

3

4

5

TOTAL

ADMINISTRATIVE BUDGET NOTE: The budget shown is the requested budget and has not been 

adjusted to reflect any recommendations made by reviewers. If an award is planned, the costs will be 

calculated by Institute grants management staff based on the recommendations outlined below in the 

COMMITTEE BUDGET RECOMMENDATIONS section.

ADMINISTRATIVE NOTE, EARLY STAGE INVESTIGATOR, NEW INVESTIGATOR

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1 R01 AI123162-01 2 VIRB 

JIANG, M 

1R01AI123162-01 JIANG, MENGXI 

EARLY STAGE INVESTIGATOR

NEW INVESTIGATOR

SCIENTIFIC REVIEW OFFICER'S NOTES 

RESUME AND SUMMARY OF DISCUSSION: The application will examine how polyoma viruses 

control and destabilize normal host cellular processes to facilitate viral replication, and how these 

interactions may result in polyomavirus-induced oncogenesis. The investigator hypothesizes that “an 

activated host cellular DNA damage Response (DDR) is crucial in facilitating viral replication and 

maintaining host genome stability during polyomavirus infection”. More precisely, the investigators want 

to determine how host mismatch repair (MMR) proteins are required for polyoma virus replication and 

DDR activation, the viral DNA structures that are recognized by the host, and how T Ag affects host cell 

DNA replication. The proposed work is considered highly significant and likely to advance the field and 

uncover novel molecular mechanisms underlying polyomavirus replication and viral oncogenesis. 

Additional strengths include: preliminary data indicating that viral replication rather than T Ag may be 

the key trigger for DDR activation; cutting edge approaches, such as the primary human renal proximal 

tubule epithelial cell culture infection model; the accomplished investigative team; thorough 

consideration of alternative approaches. In sum, there is considerable enthusiasm in support of the 

investigator, the important topic, the highly compelling preliminary data, and the innovative 

experimental plan. 

DESCRIPTION (provided by applicant): 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. 

PUBLIC HEALTH RELEVANCE: 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. 
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JIANG, M 

CRITIQUE 1: 

Significance: 2 

Investigator(s): 2 

Innovation: 2 

Approach: 2 

Environment: 2 

Overall Impact: The enthusiasm for this application is based on the significance, innovation, and 

approach. The research proposed in this application should fill gaps in knowledge about the connection 

between polyomavirus-induced DNA damage response (DDR), viral replication, and host genome 

stability. This is significant, because a number of polyomavirus and other DNA viruses modulate the 

DDR pathway, with implications for viral replication and oncogenesis. Dr. Mengxi Jiang is a new 

investigator, holding an assistant professor position at UAB. She has made and published important 

findings about the human polyomavirus BKPyV induction of the DDR pathway, and in particular, she 

has new preliminary data that identify host mismatch repair proteins upregulated and involved in the 

DDR activation. She also has evidence that is paradigm-shifting that indicates that viral replication 

rather than T Ag may be the key trigger for DDR activation. The strengths of the proposal are the 

significance, because the investigator is in a strong position to make important findings that will be 

applicable to the DNA virus field. There are clearly designed experiments to test logical hypotheses. In 

addition to the preliminary data, feasibility of the approaches is evident because the investigator and 

co-investigator have experience in the techniques. Experimental design is thoroughly described, and 

expected outcomes are discussed. Even the possibility of results that disprove the hypotheses are 

discussed with alternative experiments and strong experimental design to test new hypotheses. There 

are a few minor weaknesses that do not significantly affect the overall impact and are outweighed by 

the significance, innovation, and hypothesis-driven comprehensive approach. 

1. Significance: 

Strengths 

 A tractable model system is being used to study a human pathogen; findings can likely be 

extended to other human polyomaviruses and potentially other viruses: other DNA viruses 

modulate the DDR pathway, promoting viral replication and/or oncogenesis. 

Builds on important findings newly implicating the host mismatch repair proteins in activating the 

DNA damage response during infection. 

May shift paradigms about the viral triggers important for DDR activation, which could inform 

future studies to prevent host DNA damage caused by polyomaviruses, with implications for 

oncogenesis 

Determination of how polyomavirus (and likely T Ag specifically) induces host genome instability 

(that is overcome if the virus replicates or if DDR is activated) might lead to approaches to 

selectively induce host DNA damage to help eliminated infected cells. 

Weaknesses 

None noted. 

2. Investigator(s): 

Strengths 
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JIANG, M 

Dr. Mengxi Jiang, investigator, is an Assistant Professor at the University of Alabama, 

Birmingham. She did her postdoctoral training with Dr. Michael Imperiale at the University of 

Michigan, where she focused on molecular aspects of host-BK polyomavirus interactions (viral 

entry and replication, and host nuclear architecture rearrangements). She discovered that 

polyomavirus replication and host genome stability rely on activated host DDR pathways, 

leading to the basis for this proposal. She continued this work in her own independent 

laboratory, making the key discoveries underpinning this proposal, i.e., that mismatch repair 

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

activation of the DDR. She has a strong publication record and is well positioned to carry out the 

proposed research.

 Her research focus is replicative stress in yeast, and chemotherapeutic drug action and 

cellular pathways regulating drug responses in human cell and mouse models. Apropos of the 

proposed project, she has extensive experience in analysis of DNA damage response 

pathways, including 2-D gel and DNA fiber assays. She is well qualified for the proposed role in 

this project.

Weaknesses 

None noted. 

3. Innovation: 

Strengths 

Conceptual innovation is high: paradigm-shifting model that polyoma activates the host DDR 

response leading to both productive replication and prevention of host DNA damage, especially 

with the novel concepts of connection between viral DNA replication, DDR activation and virusinduced host genome instability. Model that viral DNA replication triggers DDR is innovative. 

Some cutting-edge approaches are proposed, which alone are not innovative, but together 

comprise innovative application to polyomavirus and the host DNA damage response. These 

include 2-D gels and DNA fiber assays to examine replication intermediates, metaphase 

spreads, FISH and single-cell immunofluorescence staining, and iPOND (involving click 

chemistry and immunoblotting to identify proteins on nascent DNA). 

Use of the RPTE primary cell culture model that is biologically relevant because the cells have 

intact cell cycle checkpoint regulation (contrast to transformed cell lines). 

Weaknesses 

None noted 

4. Approach: 

Strengths 

Testable model is proposed that is addressed in three independent but related aims. 

Strong preliminary data indicating that BKPyV activates and requires the DDR for productive 

infection and that DDR is required for host DNA stability. 

Use of powerful SILAC and 2-D LC-MS/MS to identify host mismatch repair protein upregulation 

of MSH6 and MSH2 by BKPyV, and good confirming evidence of the importance of MSH6 and 

MSH2 in productive infection and DDR activation. 
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JIANG, M 

Good evidence of requirement of viral replication for DDR activation using both ectopic T Ag 

expression and viral infection. This is key evidence for the paradigm-shifting hypothesis that T 

Ag presence is not the sole (or even major) driver of DDR activation. 

Aims are clearly described, with strong rationale and testable hypotheses. 

Appropriate controls are included, and experiments are feasible. Expected outcomes are 

described. Outcomes that disprove the hypotheses are discussed, with additional experiments 

proposed to test the alternative outcomes. 

Aim 1 is a logical set of experiments that thoroughly examine the role of host mismatch repair 

proteins in BKPyV replication and DDR activation. 

Building on the preliminary findings that viral DNA replication is necessary for DDR activation, 

the investigator will test whether it is sufficient. She will use a strong genetic approach that 

should be informative, transfecting RPTE cells with a plasmid with a wt or nonreplicable BKPyV 

ori (or no-ori control). She will introduce various forms of T Ag using lentivirus constructs: either 

a wild-type T Ag or a mutant that abolishes viral replication (in hand) or one she will construct 

that will lack an ori-binding domain. 

The examination of specific DNA lesions on viral DNA (Aim 2.2) on a single-cell level is exciting 

and may be the first comprehensive look at such lesions for a DNA virus genome. 

Aim 3.1, examining whether forcing cells into mitosis produces host DNA damage, tests an 

intriguing hypothesis that T Ag induced replication stress plus mitosis causes chromosome 

damage; it uses approaches that are in hand and should be successful. 

Aim 3.2, is exciting because it tests directly whether T Ag induces host DNA replication stress 

using powerful cutting-edge techniques. The findings could be related to reports of other 

oncogenes causing replication stress. 

Weaknesses 

The reduction in viral titer is only ~ 1 log unit in some assays (Fig. 3C, 7C). This requires 

biological replicates in order to claim statistical significance; it’s not clear if the statistics shown 

are based on technical or biological replicates. 

The rationale for using the siRNA-resistant form of MSH6 or MSH2 in Aim 1.1 is not clear. 

5. Environment: 

Strengths 

The physical and scientific environment as described are outstanding. In addition, Dr. Jiang’s 

department chair provides a strong letter of support describing the institutional commitment to 

her success as an independent investigator. 

Weaknesses 

None noted. 

Protections for Human Subjects: 

Not Applicable (No Human Subjects) 

Vertebrate Animals: 

Not Applicable (No Vertebrate Animals) 
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JIANG, M 

Biohazards: 

Not Applicable (No Biohazards) 

Select Agents: 

Not Applicable (No Select Agents) 

Resource Sharing Plans: 

Not Applicable (No Relevant Resources) 

Budget and Period of Support: 

Recommend as Requested 

CRITIQUE 2: 

Significance: 2 

Investigator(s): 1 

Innovation: 2 

Approach: 3 

Environment: 1 

Overall Impact: This is a new application to determine the interplay between polyomavirus genome 

replication and the double strand DNA break (DDB) response. Dr. Jiang is a well trained new 

investigator and the application is based on her discovery that BKV genome replication and not simply 

TAg expression, is necessary to fully induce the DDB response. The established co-investigator is an 

expert in DNA damage, particularly replication stress. The overall hypothesis that drives the application 

is that “an activated cellular DNA damage response (DDR) is important for facilitating viral replication 

and maintaining host genome stability during polyomavirus infection.” There are many strengths to this 

proposal. These include the well qualified principal investigator and co-investigator, an interesting and 

innovative hypothesis and an extraordinarily well crafted, logical and compelling research plan that 

includes the following aims: (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; and (3) To elucidate the molecular mechanism by which 

polyomavirus induces host genome instability. The hypotheses driving individual aims are generally 

well supported by strong preliminary data. Concerns are that some of the proposed state-of-the art 

experimental approaches that are proposed lack feasibility data. These concerns, however, are 

mitigated by the fact that in most cases more standard alternative methods are proposed and they do 

not detract from the generally very high enthusiasm for this application. 

1. Significance: 

Strengths 

 Human polyomaviruses, including BK virus cause significant disease particularly in 

immunosuppressed patients. The incidence is likely to increase given the more frequent use of 

immunosuppressive drugs in organ transplant patients and for the treatment of other diseases. 
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JIANG, M 

These studies may yield data that are significant for other viruses, including other 

polyomaviruses, papillomaviruses and potentially other viruses with small double stranded DNA 

genomes. 

Weaknesses 

None noted 

2. Investigator(s): 

Strengths 

Mengxi Jiang is a well-trained new investigator. She presents an impressive set of published 

and unpublished preliminary data that support this application, and she is well suited to direct 

these studies. 

 is an established senior investigator with a strong record of accomplishments in the 

area of DNA damage. She has also developed and implemented a novel DNA fiber assay, 

which will be used in aim 3 to address the hypothesis that BKV TAg induces DNA replication 

stress. She is an asset to this proposal. 

Weaknesses 

None noted 

3. Innovation: 

Strengths 

While the overall concept that some DNA viruses induce double strand DNA breaks and 

harness some of these pathways to support their replication cycles is not novel, the 

investigator’s concept that the viral replication process induces the DNA damage response to 

stabilize stalled viral replication forks and prevent host genome destabilization is novel and 

potentially paradigm shifting. 

Technical approaches are cutting edge. 

Weaknesses

None noted 

4. Approach: 

Strengths 

The research plan is well conceived and follows a logical plan. 

The overarching hypothesis of the proposal is supported by strong preliminary data. 

Expected and unexpected outcomes are carefully considered and discussed, and appropriate 

follow-up experiments are proposed even those that would be performed if the results do not 

support their hypotheses. 

Appropriate alternative approaches are considered and proposed. 

Experiments will be performed in primary human renal proximal tubule epithelial (RPTE) cells. 

Weaknesses 
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JIANG, M 

Feasibility data for some of the proposed approaches (single-cell FISH, iPOND) are missing. 

This is a minor weakness as in most cases more standard alternative approaches are proposed. 

5. Environment: 

Strengths 

The research environment at UAB is excellent 

Weaknesses 

None noted. 

Protections for Human Subjects: 

Not Applicable (No Human Subjects) 

Vertebrate Animals: 

Not Applicable (No Vertebrate Animals) 

Biohazards: 

Not Applicable (No Biohazards) 

Select Agents: 

Not Applicable (No Select Agents) 

Resource Sharing Plans: 

Not Applicable (No Relevant Resources) 

Budget and Period of Support: 

Recommend as Requested 

CRITIQUE 3: 

Significance: 3 

Investigator(s): 4 

Innovation: 5 

Approach: 4 

Environment: 3 

Overall Impact: Polyomaviruses cause a variety of severe human diseases particularly in 

immunocompromised individuals. The numbers of individuals affected are relatively modest compared 

to other viral diseases. However, specific anti-viral treatments are lacking for this family of viruses. The 

proposed studies will examine the interactions between the BKPyV and the DDR. These studies are 

likely to be successful and may suggest potential targets for the development of antiviral therapies. 

More likely, these studies will reveal additional details regarding the mechanisms of activation of the 
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JIANG, M 

cellular DDR. The proposed analyses are clearly articulated and are likely to be successful. Technical 

issues have generally been well considered and appropriate collaborations are in place which makes it 

likely that useful information can be developed as a result of these studies. A greater focus of the role 

of the DDR in cellular transformation/cancer development using BKPyV as a probe of these processes 

may have increased the medical relevance of these studies. 

1. Significance: 

Strengths 

 BKPyV appears like an excellent probe to study the DDR. Understanding the relationships 

between the DDR and BKPyV may permit the development of novel antiviral agents. 

Weaknesses 

BKPyV is not a major human pathogen (when compared to other viral pathogens). 

2. Investigator(s): 

Strengths 

Dr. Jiang, Assistant Professor, UAB, is well qualified to perform the proposed studies. 

, will 

provide the required support for the proposed 2-D gel and DNA fiber assays of BK polyomavirus 

DNA replication intermediates. 

Weaknesses 

None noted. 

3. Innovation: 

Strengths 

The central hypothesis that polyomavirus modulates the host DDR to facilitate productive viral 

replication and to prevent host DNA damage is testable. 

The investigator has 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. 

In RPTE cells, 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. This suggests the DDR is induced, in part, by BKPyV replication in these cells 

(which is distinct from transformed cells where TAg alone is sufficient for the induction of the 

DDR). 

Weaknesses 

None noted. 

4. Approach: 

Strengths 

In Aim 1, the investigator plans to define the role of host mismatch repair (MMR) proteins in 

polyomavirus replication and polyomavirus-induced DDR activation. The analysis is well 

described and reasonable. The only caveat is the possibility that the majority of MMR proteins 
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JIANG, M 

do not play a major role in polyomavirus replication (with the exception MSH6 which has been 

shown to have a role). This seems unlikely and the approach is well justified. 

In Aim 2, the investigator plans to determine the viral DNA triggers that activate the DDR upon 

polyomavirus infection. These studies are designed to challenge the current paradigm that TAg 

is the main driver of DDR activation during polyomavirus infection. Although this may be true in 

RPTE cells, it is not clear if this is unique to this particular cell type, something the investigator 

should consider. It is probably worth repeating some the proposed analysis in another cell type 

which also demonstrates cell cycle checkpoint control. 

Weaknesses

In Aim 2, the hypothesis that both ssDNA lesions and DSBs occur on replicating viral DNA 

which serve as DDR activation triggers is proposed. Is it possible that BKPyV replication, either 

directly or indirectly, cause DNA lesions in the cellular DNA which are ultimately responsible for 

DDR activation? 

In Aim 3, the investigator plans to elucidate the molecular mechanism by which polyomavirus 

induces host genome instability. The model suggests TAg-induced aberrant host DNA 

replication stress followed by mitosis causes chromosome damage. If true, viral DNA triggers 

that activate the DDR upon polyomavirus infection may not be required for the DDR i.e. it may 

be caused by TAg affecting host DNA replication rather than BKPyV replication (although why 

BKPyV replication appears to enhance the DDR then becomes less obvious unless there are 

additional viral proteins/RNAs rather than viral DNA replication intermediates involved). This 

Aim is not described in sufficient detail to fully appreciate the expected goals and potential 

outcomes of the analysis. 

5. Environment: 

Strengths 

The environment is appropriate for these studies. 

Weaknesses 

None noted. 

Protections for Human Subjects: 

Not Applicable (No Human Subjects) 

Vertebrate Animals: 

Not Applicable (No Vertebrate Animals) 

Biohazards: 

Acceptable 

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. 
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JIANG, M 

Select Agents: 

Not Applicable (No Select Agents) 

Resource Sharing Plans: 

Unacceptable 

No resource sharing plan is presented. 

Budget and Period of Support: 

Recommend as Requested 

THE FOLLOWING SECTIONS WERE PREPARED BY THE SCIENTIFIC REVIEW OFFICER TO 

SUMMARIZE THE OUTCOME OF DISCUSSIONS OF THE REVIEW COMMITTEE, OR REVIEWERS' 

WRITTEN CRITIQUES, ON THE FOLLOWING ISSUES: 

COMMITTEE BUDGET RECOMMENDATIONS: The budget was recommended as requested. 

SCIENTIFIC REVIEW OFFICER’S NOTES: The VIRB Study Section has been recalibrated with the 

October 2015 meeting (January, 2016 Council Round). This process involves guiding the reviewers to 

address progressive score inflation and reset the percentile base to establish a benchmark for future 

VIRB meetings. 

The NIH special practice for new investigator R01 applications reviewed in the Center for Scientific 

Review study sections applies to this application. Resubmission (amended -A1) R01 applications from 

new investigators may be submitted on a special receipt date for review in the very next review cycle. 

See this notice in the NIH Guide for Grants and Contracts for more details: 

http://grants.nih.gov/grants/guide/notice-files/NOT-OD-11-057.html. 

You should contact the NIH program officer whose name is shown in the upper left hand corner of page 

one of this Summary Statement for information about whether this application may be fundable or 

whether you will need to submit an amended application. The program officer can also help you decide 

whether the changes and improvements necessary to address the weaknesses noted in the reviewers' 

critiques could be accomplished in the relatively short time available. You are also strongly advised to 

seek input from mentors, your Department chair, etc. 

If you choose to submit a resubmission application for the next review cycle under this policy for new 

investigators, your amended application must be received at NIH no later than Thursday, December 10, 

2015.

You may, of course, choose to take more time to resubmit your application. If so, you should prepare 

the resubmission for the normal dates for amended applications as specified in this table: 

http://grants1.nih.gov/grants/funding/submissionschedule.htm. 
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JIANG, M 

NIH has modified its policy regarding the receipt of resubmissions (amended applications). 

See Guide Notice NOT-OD-14-074 at http://grants.nih.gov/grants/guide/notice-files/NOT-OD14-074.html. The impact/priority score is calculated after discussion of an application by 

averaging the overall scores (1-9) given by all voting reviewers on the committee and 

multiplying by 10. The criterion scores are submitted prior to the meeting by the individual 

reviewers assigned to an application, and are not discussed specifically at the review meeting 

or calculated into the overall impact score. Some applications also receive a percentile 

ranking. For details on the review process, see 

http://grants.nih.gov/grants/peer_review_process.htm#scoring.