HIV Drug Resistance Program

National Cancer Institute

U.S. DEPARTMENT

OF HEALTH AND 

HUMAN SERVICES

National Institutes 

of Health

HIV Drug Resistance Program i

Table of Contents

HIV Drug Resistance Program, National Cancer Institute

Directory.............................................................................................................................. ii

Message from the Director of the HIV Drug Resistance Program..................................... 1

Retroviral Replication Laboratory ...................................................................................... 3

David Derse, Ph.D........................................................................................................ 4

Eric O. Freed, Ph.D....................................................................................................... 6

Wei-Shau Hu, Ph.D. ..................................................................................................... 8

Stephen H. Hughes, Ph.D........................................................................................... 10

Vineet N. KewalRamani, Ph.D................................................................................... 12

Stuart F.J. Le Grice, Ph.D. .......................................................................................... 14

Vinay K. Pathak, Ph.D................................................................................................ 16

Alan Rein, Ph.D. ......................................................................................................... 18

Stephen Oroszlan, Ph.D. ............................................................................................ 20

Clinical Program — Host-Virus Interaction Branch ....................................................... 21

Frank Maldarelli, M.D., Ph.D. ................................................................................... 22

Mary Kearney, Ph.D.................................................................................................... 24

Virology Core .............................................................................................................. 25

Training and Employment Opportunities in the HIV Drug Resistance Program........... 26
ii National Cancer Institute

Directory

Administration Office:

HIV Drug Resistance Program

National Cancer Institute

NCI-Frederick, P.O. Box B

Building 535

Frederick, MD 21702-1201

Phone: 301-846-5943 or -5487

Fax: 301-846-6863

E-mail: hivdrp@ncifcrf.gov 

Research Team:

Stephen H. Hughes, Ph.D., Director

Phone: 301-846-1619; Fax: 301-846-6966

E-mail: hughes@ncifcrf.gov

http://home.ncifcrf.gov/hivdrp/Hughes.html

David Derse, Ph.D.

Inquiries for the lab should be directed to 

Gisela Heidecker, Ph.D. 

Phone: 301-846-1440; Fax: 301-846-6863

E-mail: heidecke@ncifcrf.gov

http://home.ncifcrf.gov/hivdrp/Derse.html

Eric O. Freed, Ph.D.

Phone: 301-846-6223; Fax: 301-846-6777

E-mail: efreed@mail.nih.gov

http://home.ncifcrf.gov/hivdrp/Freed.html

Wei-Shau Hu, Ph.D.

Phone: 301-846-1250; Fax: 301-846-6013

E-mail: whu@ncifcrf.gov

http://home.ncifcrf.gov/hivdrp/Hu.html

Mary Kearney, Ph.D.

Phone: 301-846-6796; Fax: 301-846-6777

E-mail: kearneym@ncifcrf.gov

http://home.ncifcrf.gov/hivdrp/Kearney.html

Consultants:

John M. Coffin, Ph.D.

American Cancer Society Research Professor

Tufts University

Phone: 617-636-6528; Fax: 617-636-0337

E-mail: john.coffin@tufts.edu

http://www.tufts.edu/sackler/microbiology/faculty/coffin

John W. Mellors, M.D.

Chief, Division of Infectious Diseases

University of Pittsburgh

Phone: 412-383-7963; Fax: 412-383-7982

E-mail: jwm1@pitt.edu

http://www.dom.pitt.edu/id/faculty_info.aspx?fp=5058

Vineet N. KewalRamani, Ph.D.

Phone: 301-846-1249; Fax: 301-846-6777

E-mail: vineet@mail.nih.gov

http://home.ncifcrf.gov/hivdrp/KewalRamani.html

Stuart F.J. Le Grice, Ph.D.

Phone: 301-846-5256; Fax: 301-846-6013

E-mail: legrices@mail.nih.gov

http://home.ncifcrf.gov/hivdrp/Le_Grice.html

Frank Maldarelli, M.D., Ph.D.

Phone: 301-435-8019; Fax: 301-480-1735

E-mail: fmalli@mail.nih.gov

http://home.ncifcrf.gov/hivdrp/Maldarelli.html

Stephen Oroszlan, Ph.D.

Phone: 301-846-1355; Fax: 301-846-6013

E-mail: oroszlans@ncifcrf.gov

http://home.ncifcrf.gov/hivdrp/Oroszlan.html

Vinay K. Pathak, Ph.D.

Phone: 301-846-1710; Fax: 301-846-6013

E-mail: vpathak@ncifcrf.gov

http://home.ncifcrf.gov/hivdrp/Pathak.html

Alan Rein, Ph.D.

Phone: 301-846-1361; Fax: 301-846-6013

E-mail: rein@ncifcrf.gov

http://home.ncifcrf.gov/hivdrp/Rein.html
HIV Drug Resistance Program 1

Message from the Director of the HIV Drug Resistance Program

Approximately one million Americans and more than 30 million people 

worldwide are infected with HIV. 

Without effective therapy, virtually all 

will die of AIDS within 20 years. 

The real obstacle to successful 

therapy is the evolution of viruses 

that are resistant to the available 

antiviral drugs. At present, the only 

way to deal with this problem is with 

expensive, toxic, and strenuous 

multidrug regimens. Clearly, more 

research is needed to find new ways 

to deal with the resistance problem. 

The HIV Drug Resistance Program (DRP) was initiated in 1997 

with the mission of conducting and 

fostering multidisciplinary basic, 

translational, and clinical research 

focused on issues related to drugresistant HIV at all levels. The Program has built on existing strengths 

of the National Cancer Institute’s 

(NCI) basic and clinical research 

programs in retrovirology, drug 

discovery, and AIDS, exploiting 

structural biology, biochemistry, 

virology, virus-host interaction, 

evolution, and in vivo biology to 

accelerate drug discovery and better

understand mechanisms of drug 

action and resistance. Not only are 

these areas of critical importance to 

the resistance problem, they also 

raise many basic research challenges

whose solution will enhance our 

understanding of fundamental 

aspects of virology and cell biology.

Although the Program focuses on 

HIV, resistance development is also 

a major concern with other infectious agents as well as cancer, and

it is likely that our research will 

prove beneficial in these areas.

The DRP comprises the Office 

of the Director and research components that are divided into a basic

and a clinical arm. The basic arm

is the Retroviral Replication

Laboratory, composed of research

sections headed by eight Principal 

Investigators (PIs); the clinical arm is

the Host-Virus Interaction Branch. 

These subdivisions operate as a 

cohesive group, with considerable

interaction and collaboration among 

the researchers in the different 

components. Elements conducive to 

cohesion include biweekly senior 

staff meetings, often featuring chalk

talks by one of the PIs; frequent 

work-in-progress meetings, in which 

Fellows present their ongoing work; 

and other joint events such as an 

invited speaker seminar series and 

biweekly meetings to discuss the

clinical research with outside consultants Dr. John Coffin (Tufts 

University) and Dr. John Mellors 

(University of Pittsburgh). 

The DRP provides an important focus of HIV research within 

NCI and the National Institutes of

Health as a whole — not only 

because of the quality and interactiveness of the individual researchers, 

but also because of the organized 

events the Program sponsors. 

These include the annual Symposium on Antiviral Drug Resistance 

(SADR), a four-day conference that 

the DRP co-sponsors with the 

University of Pittsburgh. The SADR 

series brings together researchers 

from all over the world and has 

become a very popular meeting in

a very crowded schedule. Another 

major event is the annual Think 

Tank, a one-day meeting of NIH 

researchers interested in HIV, 

AIDS, and retrovirus research, 

which provides significant opportunities to learn about related work 

elsewhere on campus. Many 

valuable and productive intramural 

collaborations have been initiated 

http://home.ncifcrf.gov/hivdrp

Dr. Stephen H. Hughes
2 National Cancer Institute

at this meeting. Periodically, the DRP has broadened participation in the Think Tank Meeting by inviting senior 

graduate students involved in retrovirus research outside

the NIH to attend the Think Tank and to participate in an

informal symposium with members of the DRP the

following day. 

All of the elements within the DRP — high-quality 

research, interactions and collaborations, and formal 

events — provide considerable added value to the NCI. 

DRP investigators are also an integral part of the NCI’s 

Center of Excellence in HIV/AIDS & Cancer Virology 

(CEHCV), which was formed in 2006 to facilitate and 

rapidly communicate advances in the discovery, development, and delivery of antiviral and immunologic 

approaches for the prevention and treatment of HIV 

infection, AIDS-related malignancies, and cancerassociated viral diseases. Dr. Stuart Le Grice, a PI in the 

DRP, was appointed Head of the CEHCV. Through this Center of Excellence, we are working to enhance our interactions with both the intramural and extramural communities so that we can make the best use of the strengths and 

diversity within our Program and around us.

Stephen H. Hughes, Ph.D.

Director, HIV Drug Resistance Program

Center for Cancer Research

National Cancer Institute

National Institutes of Health 

We are deeply saddened by the passing of our friend and colleague 

Dave Derse. We will remember him as a gifted scientist and special 

friend. Our deepest condolences go out to his family.
HIV Drug Resistance Program 3

Retroviral Replication Laboratory

The Retroviral Replication Laboratory (RRL) focuses

on obtaining a detailed understanding of important

events in the retroviral life cycle with a strong emphasis

on the human retroviruses HIV and HTLV. Members

of the RRL study many aspects of the viral life cycle,

from the early steps in infection of the host cell, through

reverse transcription, to mechanisms of virus assembly

and release. There is an increasing emphasis on the

interactions of the host and the virus, and on the host

factors that either aid or inhibit viral replication. These

fundamental studies on the behavior of the virus and

its interactions with the host cell are complemented by

a strong commitment to understanding drug resistance.

Studies also include discovery, development, and 

mechanistic analysis of novel replication inhibitors, as

well as whole-organism studies, including the development of important animal models, and the development

and use of retroviral vectors.

The RRL is composed of eight Sections:

• The Retrovirus Gene Expression Section, headed by

Dr. David Derse, studies the replication of human

T-cell leukemia virus (HTLV). HTLVs are complex

pathogenic human retroviruses, but are only distantly

related to HIV-1. Both types of viruses evade the

immune system and are slow to cause disease; 

however, the underlying mechanisms are quite 

different.

• The Virus-Cell Interaction Section, directed by Dr. 

Eric O. Freed, is known for its work on the assembly 

and release of HIV-1 from infected cells and the 

host factors essential for efficient assembly and 

release. This Section has also been determining the 

mechanism of action of, and the development of 

resistance to, the Panacos maturation inhibitor PA-457. 

• The Viral Recombination Section, directed by Dr.

Wei-Shau Hu, focuses on mechanisms of recombination, RNA packaging, and virus assembly. 

• The Vector Design and Replication Section, directed by Dr. Stephen H. Hughes, has two principal 

areas of research interest: 1) structural and biochemical studies of HIV-1 reverse transcriptase 

(RT); and 2) retroviral vectors.

• The Model Development Section, directed by Dr. 

Vineet N. Kewal Ramani, is primarily interested in

understanding the role of host factors in HIV-1 

infection and in developing an RT-SHIV that can be 

used to study anti-RT drugs and drug responses in a 

macaque model.

• The RT Biochemistry Section, under the direction 

of Dr. Stuart F.J. Le Grice, focuses on the biochemistry of HIV RT and related retroviral RTs, 

on the interaction of these RTs with their nucleic 

acid substrates, and on the development of RNase 

H inhibitors.

• The Viral Mutation Section, headed by Dr. Vinay 

K. Pathak, focuses on mechanisms by which 

APOBEC3 host restriction factors inhibit HIV-1 

replication, interactions between HIV-1 Vif protein 

and APOBEC3 proteins, and how mutations in the 

connection subdomain and RNase H domain of 

HIV-1 RT confer resistance to antiviral RT inhibitors. 

• The Retrovirus Assembly Section, led by Dr. Alan 

Rein, has been focusing on the roles of elements of 

the Gag protein, nucleic acid, and host factors in 

virus assembly. 
4 National Cancer Institute

Dr. David Derse earned his Ph.D. in 1982 from the State University of New

York at Buffalo, studying inhibitors of herpesvirus and cellular DNA 

polymerases in the laboratory of Dr. Yung-Chi Cheng. Dr. Derse did his 

postdoctoral research on bovine leukemia virus gene regulation under the 

supervision of Dr. James W. Casey at the Louisiana State University Medical

Center. After joining the National Cancer Institute in 1986 as a Senior Staff

Fellow, Dr. Derse continued research on the Tax and Rex proteins encoded 

by deltaretroviruses. In 1991, he became a tenured Senior Investigator and 

extended his research into comparative biochemical analyses of lentivirus

Tat and Rev proteins. In 2004, Dr. Derse joined the HIV Drug Resistance

Program as Head of the Retrovirus Gene Expression Section. He is also

an Adjunct Professor in the Graduate Program in Genetics at George

Washington University in Washington, D.C. Dr. Derse currently serves on

the Editorial Board of Retrovirology and the Executive Committee of the

Center of Excellence in HIV/AIDS and Cancer Virology, Center for Cancer

Research. His current work focuses on the molecular mechanisms of

retrovirus infection, replication, and host cell restriction.

Head, Retrovirus Gene Expression Section, HIV 

Drug Resistance Program

Biography: 

A specialized type of retroviral transfer vector has been constructed to facilitate studies of cellto-cell infection for HIV-1 and HTLV-1. The upper part of the figure shows HIV-1 and HTLV-1

inLuc reporter vectors. In addition to the viral long terminal repeats and elements necessary for 

viral mRNA packaging and replication, the vectors contain an inverted reporter gene cassette. 

The cassette is composed of a CMV promoter, a luciferase gene containing a γ-globin intron 

oriented in the opposite direction, and a poly A signal. As shown in the lower part, when cotransfected with viral packaging plasmids, the spliced viral genomic mRNA is packaged into 

virus-like particles (VLPs) that infect target cells and generate a provirus capable of producing

luciferase. The reporter protein is expressed exclusively in target cells after reverse transcription and integration, making it possible to quantify virus infection in a coculture setting.

Contact Information:

HIV Drug Resistance Program

National Cancer Institute

NCI-Frederick, P.O. Box B

Building 535

Frederick, MD 21702-1201

Inquiries for the lab should be 

directed to Gisela Heidecker, Ph.D. 

Phone: 301-846-1440

Fax: 301-846-6863

E-mail: heidecke@ncifcrf.gov

http://home.ncifcrf.gov/hivdrp/Derse.html

Research Interests:

• Comparative retrovirology

• Mechanisms of HIV-1 and HTLV-1 

infection and replication

• Control of cell and virus gene 

expression

• Viral pathogenesis and persistence

David Derse, Ph.D.
HIV Drug Resistance Program 5

Although divergent retroviruses share a common mechanism for genome replication, they have evolved distinctly

different strategies for infectious transmission, evading

host immune responses, and resisting intrinsic cellular

defense mechanisms. These variations are likely to reveal 

important insights into cellular pathways that viruses 

hijack to ensure their propagation and persistence, and 

reveal how certain host-virus interactions are related to 

pathogenesis. In this light, it is useful to compare HIV 1 

with other retroviruses, in particular HTLV-1. Both of 

these human retroviruses target T cells for infection in vivo

but with distinctly different disease outcomes. Whereas 

HIV-1 causes AIDS in almost all infected individuals, 

HTLV-1, which infects approximately 20 million people 

worldwide, is associated with adult T-cell leukemia and a 

degenerative inflammatory disease of the central nervous 

system in about 5% of infected patients. We are comparing 

the molecular and cellular biology of HIV-1 and HTLV-1 

to understand fundamental mechanisms for cell-to-cell 

transmission of retroviruses, mechanism of action of 

APOBEC3 restriction factors, mechanisms of virus resistance to restriction, and mechanisms of virus protein trafficking essential for assembly of infectious virus particles.

To study mechanisms of cell-to-cell infection, we 

have developed special HIV-1 and HTLV-1 vectors and

quantitative infection methods that will complement 

existing imaging technologies. These new vectors and 

cell culture model systems now make it possible to 

examine infection in a coculture setting. Combined with

biochemical and microscopic image analysis, the new 

methods are helping to define how polarized virus 

assembly, release, transfer, and entry are coordinated with

adhesion-induced changes in cell morphology. 

We are using a comparative approach to better 

understand the mechanism of retrovirus restriction by 

APOBEC3 proteins. HIV-1 counteracts the antiviral 

activity of human A3G by producing an accessory protein, 

Vif, which hastens the degradation of A3G. In contrast, 

HTLV-1 encodes a peptide motif in the C-terminus of 

the HTLV-1 nucleocapsid domain that inhibits A3G 

packaging into nascent virions. Studies of HIV-1,

HTLV-1, and other retroviruses are helping to elucidate

how A3G is incorporated into virions, how it inhibits virus

replication, and how viruses resist its antiviral effects.

We are also examining mechanisms of Gag targeting

to specific membrane microdomains, envelope trafficking, 

regulation of Env activity, and the integration of Gag

and Env trafficking pathways involved in retrovirus 

assembly and release.

Selected Publications: 

1. Derse D, Hill SA, Princler G, Lloyd P, Heidecker G. 

Resistance of human T cell leukemia virus type 1 to 

APOBEC3G restriction is mediated by elements in 

nucleocapsid. Proc. Nat. Acad. Sci. USA 104: 2915-

2920, 2007.

2. Derse D, Crise B, Li Y, Princler G, Lum N, Stewart C, 

McGrath CF, Hughes SH, Munroe DJ, Wu X. Human 

T-cell leukemia virus type 1 integration target sites in 

the human genome: Comparison with those of other 

retroviruses. J. Virol. 81: 6731-6741, 2007.

3. Heidecker G, Lloyd PA, Soheilian F, Nagashima K, 

Derse D. The role of WWP1-Gag interaction and Gag 

ubiquitination in assembly and release of human T-cell 

leukemia virus type 1. J. Virol. 81: 9769-9777, 2007.

4. Mazurov D, Heidecker G, Derse D. The inner loop of

tetraspanins CD82 and CD81 mediates interactions with 

HTLV-1 Gag protein. J. Biol. Chem. 282: 3896-3903, 

2007. 

HTLV-1 Gag (magenta) is mobilized to sites of contact between HTLV-1-

transfected and nontransfected Jurkat T cells. Cells were stained with 

fluorescent-labeled antibodies that recognize HTLV-1 Gag (magenta), 

HTLV-1 Tax (green), and alpha-tubulin (blue). 

Research Summary:
6 National Cancer Institute

Contact Information:

HIV Drug Resistance Program

National Cancer Institute

NCI-Frederick, P.O. Box B

Building 535

Frederick, MD 21702-1201

Phone: 301-846-6223

Fax: 301-846-6777

E-mail: efreed@mail.nih.gov 

http://home.ncifcrf.gov/hivdrp/Freed.html

Research Interests:

• HIV and retroviral assembly and 

release

• Interactions between viral and 

cellular proteins

• Trafficking of retroviral proteins

• Envelope glycoprotein incorporation 

and function

HIV particle assembly and release. Research in the Freed lab focuses primarily on the late 

stages of the HIV/retroviral replication cycle, in particular on those steps highlighted in red.

Dr. Eric Freed received his Ph.D. in 1990 in the laboratories of Drs. Rex 

Risser and Howard Temin at the University of Wisconsin-Madison and did 

postdoctoral work with Dr. Temin at UW-Madison in 1991. His work in 

Madison focused on the function of the murine leukemia virus and HIV 

envelope glycoproteins in membrane fusion and virus entry. He joined the 

Laboratory of Molecular Microbiology at the National Institute of Allergy 

and Infectious Diseases (LMM/NIAID) in 1992, where he worked with 

Dr. Malcolm Martin on a variety of topics relating to virus assembly and 

entry/post-entry events in the HIV replication cycle. In 1997 he was 

appointed as a Tenure-Track Investigator in LMM/NIAID, and he was 

promoted to a tenured Senior Investigator position in 2002. Dr. Freed joined 

the National Cancer Institute in 2003 as Head of the Virus-Cell Interaction 

Section in the HIV Drug Resistance Program. Since 2004, he has served as 

an adjunct Associate Professor in the Department of Cell Biology and 

Molecular Genetics at the University of Maryland, College Park and a 

member of the University of Maryland Virology Program. Dr. Freed was an 

Organizer of the 2004 Cold Spring Harbor Retroviruses conference and the 

2006 ASCB Cell Biology of Retroviruses conference. In 2009 he was 

appointed as the first Editor-in-Chief of Viruses; he also currently serves on 

the Editorial Boards of the Journal of Virology, Virology, Open Virology Journal, 

Retrovirology, and Advances in Virology.

Head, Virus-Cell Interaction Section, HIV Drug 

Resistance Program

Biography: 

Eric O. Freed, Ph.D.
HIV Drug Resistance Program 7

The overall goal of the Virus-Cell Interaction Section is to 

understand basic mechanisms of retroviral replication at 

the molecular level, with an emphasis on the late stages of 

the HIV-1 replication cycle. Specifically, much of our 

current effort is aimed at understanding HIV-1 Gag 

trafficking, Env incorporation, virus assembly, budding, 

release, and maturation. 

We have a special interest in the complex relationship 

between viral proteins and cellular factors and pathways. 

We believe that the elucidation of fundamental aspects of 

the retrovirus replication cycle will suggest novel targets for

 the development of antiretroviral therapies. This concept 

finds validation in our recent work on the HIV-1 maturation inhibitor PA-457 (bevirimat), which is currently undergoing phase IIb clinical trials in HIV-1-infected patients.

HIV-1 virions are highly enriched in cholesterol 

relative to the host cell plasma membrane, and multiple 

lines of evidence suggest that HIV-1 assembly and entry 

occur in cholesterol-rich lipid raft microdomains. We 

recently demonstrated that the cholesterol-binding 

compound amphotericin B methyl ester (AME) potently 

blocks HIV-1 entry. The passaging of HIV-1 in the 

presence of AME led to viral escape from the compound 

and mutations conferring resistance mapped to the 

cytoplasmic tail of gp41. Remarkably, we found that the 

AME-resistance mutations induce the cleavage of the gp41 

cytoplasmic tail by the viral protease, leading to AME 

Blocking CA-SP1 cleavage with bevirimat (BVM) inhibits virion maturation 

and infectivity. Treatment with the HIV-1 maturation inhibitor PA-457 

(BVM) results in a defect in Gag processing that impairs normal core 

condensation and blocks virus replication.

resistance. These findings reveal a novel mechanism of 

viral resistance to an HIV-1 entry inhibitor and constitute 

the first example in which HIV-1 envelope glycoprotein 

function is activated by the viral protease.

Much effort in our lab has been directed at understanding the mechanisms and itineraries of Gag trafficking 

in HIV-1-infected cells and the host cell machinery involved 

in virus budding. We previously reported that the phospholipid PI(4,5)P2 is a key cellular cofactor in directing Gag to 

the plasma membrane. More recently, we found that two 

cellular protein families, the Golgi-localized, gamma-earcontaining, Arf-binding (GGA) and ADP ribosylation 

factor (Arf) proteins, play important roles in retroviral Gag 

trafficking and in modulating the release of virions from the 

cell surface. We have also recently established an imaging 

system that enables us to visualize Gag movement in living 

cells in the context of a fully infectious HIV-1 molecular 

clone. By using this system, we have observed that Gag 

moves rapidly from an internal site to the cell-cell junction, 

or synapse, formed between infected macrophages and 

uninfected T cells. These results suggest that HIV-1 virions 

are sequestered internally in infected macrophages but 

move to the synapse formed with susceptible target cells, 

allowing virus transmission to occur. 

Selected Publications: 

1. Adamson CS, Ablan SD, Boeras I, Goila-Gaur R, 

Soheilian F, Nagashima K, Li F, Salzwedel K, Sakalian 

M, Wild CT, Freed EO. In vitro resistance to the human 

immunodeficiency virus type 1 maturation inhibitor 

PA-457 (bevirimat). J. Virol. 80: 10957-10971, 2006. 

2. Waheed AA, Ablan SD, Roser JD, Sowder RC, 

Schaffner CP, Chertova E, Freed EO. HIV-1 escape 

from the entry-inhibiting effects of a cholesterolbinding compound via cleavage of gp41 by the viral 

protease. Proc. Natl. Acad. Sci. USA 104: 8467-8471, 

2007.

3. Gousset K, Ablan SD, Coren LV, Ono A, Soheilian F, 

Nagashima K, Ott DE, Freed EO. Real-time 

visualization of HIV-1 Gag trafficking in infected 

macrophages. PLoS Pathog. 4: e1000015, 2008.

4. Joshi A, Garg H, Nagashima K, Bonifacino JS, Freed 

EO. GGA and Arf proteins modulate retrovirus 

assembly and release. Mol. Cell 30: 227-238, 2008.

Research Summary:
8 National Cancer Institute

Dr. Wei-Shau Hu received her Ph.D. in Genetics from the University of 

California, Davis, in 1987. She studied the mechanisms of DNA recombination that lead to human α-thalassemia in Dr. James Shen’s laboratory. Under 

Dr. Howard Temin’s guidance, she studied the mechanisms of retroviral 

recombination as a Postdoctoral Fellow at the University of Wisconsin. In 

1991, Dr. Hu joined the faculty of West Virginia University as an Assistant 

Professor in the Department of Microbiology and Immunobiology and the 

Mary Babb Randolph Cancer Center. She was promoted to Associate 

Professor with tenure in 1998. In 1999, she joined the National Cancer 

Institute as Senior Investigator and Head of the Viral Recombination Section 

in the HIV Drug Resistance Program, Center for Cancer Research. Dr. Hu 

is an Organizer of the 2009 Cold Spring Harbor Retroviruses conference. She 

is also an Adjunct Professor at West Virginia University and serves on the 

Special Review Panel and Study Section of the National Institutes of Health 

extramural grant funding programs.

Head, Viral Recombination Section, HIV Drug 

Resistance Program

Biography: 

Wei-Shau Hu, Ph.D.

Contact Information:

HIV Drug Resistance Program

National Cancer Institute

NCI-Frederick, P.O. Box B

Building 535

Frederick, MD 21702-1201

Phone: 301-846-1250

Fax: 301-846-6013

E-mail: whu@ncifcrf.gov 

http://home.ncifcrf.gov/hivdrp/Hu.html

Research Interests:

• Mechanisms of reverse transcription 

and recombination

• RNA packaging and virus assembly

• HIV replication

• Interactions between distinct 

viruses

Multiple steps in viral replication can affect HIV-1 recombination. We study the mechanisms 

and factors that affect HIV recombination, including coinfection, heterozygous virion formation, and template-switching frequencies.
HIV Drug Resistance Program 9

We study the interactions among similar and different 

species of retroviruses — specifically, recombination and 

complementation via pseudotyping. Additionally, we 

study three aspects of viral infection that affect these 

interactions: infection by more than one virus (double 

infection); viral RNA packaging, including copackaging 

of two different RNAs (heterozygous virus); and virus 

assembly. Through these studies, our goal is to further 

our understanding of the plasticity of the retroviral 

genome and its replication mechanisms.

Recombination plays an important role in HIV 

replication and evolution. For example, many of the 

primary isolates of HIV are recombinants, and recombination can assort mutations to generate variants that 

escape host immune responses or are resistant to multidrug treatment protocols. Recombination of HIV-1 

occurs during reverse transcription using information 

in the two RNAs to produce a hybrid DNA. Therefore, 

phenotypically different recombinants are generated 

from viruses containing two copies of different RNAs. 

Only cells that are doubly infected can produce virions 

containing two different RNAs. To better understand 

interactions in HIV-1 populations, we study the 

dynamics of double infection and the frequency of 

recombination. 

In addition to recombination, retroviruses can interact with each other through complementation. Proteins 

and RNAs from two different viruses can interact in 

doubly infected cells. An estimated one million people 

are dually infected with HIV-1 and HIV-2, providing the 

basis for possible interactions between these two viruses 

in human populations. Although HIV-1/HIV-2 recombinants have not been reported thus far, given the large 

dual-infected human population and prior examples of 

novel lentiviruses generated by interspecies recombination, 

we cannot ignore the potential for the emergence of a 

hybrid virus. Our specific research aims are to investigate 

HIV-1 recombination and to explore genetic interactions 

between HIV-1 and HIV-2. In each aim, we are using 

experimental system studies to elucidate the mechanisms 

of recombination and will apply our knowledge to study 

patient samples. With this combined approach, we will 

elucidate the extent of HIV genetic recombination, thereby 

developing strategies to better control HIV-1 infection.

Selected Publications: 

1. Boyko V, Leavitt M, Gorelick R, Fu W, Nikolaitchik 

O, Pathak VK, Nagashima K, Hu W-S. Coassembly 

and complementation of Gag proteins from HIV-1 

and HIV-2, two distinct human pathogens. Mol. Cell

23: 281-287, 2006.

2. Chen J, Pathak VK, Peng W, Hu W-S. Capsid proteins 

from human immunodeficiency virus type 1 and simian 

immunodeficiency virus SIVmac can coassemble into 

mature cores of infectious viruses. J. Virol. 82: 8253-

8261, 2008.

3. Motomura K, Chen J, Hu W-S. Genetic recombination between human immunodeficiency virus type 1 

(HIV-1) and HIV-2, two distinct human lentiviruses.

J. Virol. 82: 1923-1933, 2008.

4. Chin MP, Lee S-K, Chen J, Nikolaitchik OA, Powell 

DA, Fivash MJ Jr, Hu W-S. Long-range recombination gradient between HIV-1 subtypes B and C 

variants caused by sequence differences in the 

dimerization initiation signal region. J. Mol. Biol. 377: 

1324-1333, 2008.

Functional complementation between proteins from different retroviruses. We study coassembly and complementation between viral 

proteins, including those of HIV-1 and HIV-2 Gag polyproteins. The 

figure shows interactions between HIV-1 and HIV-2 Gag detected by 

the bimolecular functional complementation assay.

Research Summary:
10 National Cancer Institute

Contact Information:

HIV Drug Resistance Program

National Cancer Institute

NCI-Frederick, P.O. Box B

Building 539

Frederick, MD 21702-1201

Phone: 301-846-1619

Fax: 301-846-6966

E-mail: hughes@ncifcrf.gov 

http://home.ncifcrf.gov/hivdrp/Hughes.

html

Research Interests:

• Retroviral replication

• Structure and function of HIV

 reverse transcriptase 

• Retroviral vectors 

Dr. Stephen H. Hughes received his Ph.D. from Harvard University under 

the direction of Dr. Mario Capecchi and did postdoctoral research under the 

direction of Drs. J. Michael Bishop and Harold Varmus at the University of 

California, San Francisco. From 1979 until 1984, Dr. Hughes was a Senior 

Staff Investigator at Cold Spring Harbor Laboratory. In 1984, he established 

the Gene Expression in Eukaryotes Section in the ABL–Basic Research 

Program at NCI-Frederick. Dr. Hughes became Deputy Director of the 

ABL–Basic Research Program in 1988 and Director of the Molecular Basis of 

Carcinogenesis Laboratory in 1995. In 1999, he joined the HIV Drug 

Resistance Program in the National Cancer Institute as Chief of the Retroviral 

Replication Laboratory. Dr. Hughes was appointed Director of the HIV Drug 

Resistance Program in 2006. He is an Organizer of the Annual Symposium 

on Antiviral Drug Resistance and has served as an Organizer of the 

Retroviruses and Viral Vectors Meetings at Cold Spring Harbor Laboratory 

and the Annual Meeting on Oncogenes. Dr. Hughes was named one of the 

most frequently cited AIDS researchers by Science Watch in 1996.

Director, HIV Drug Resistance Program; Chief, 

Retroviral Replication Laboratory; and Head, 

Vector Design and Replication Section, HIV Drug 

Resistance Program

Biography: 

Stephen H. Hughes, Ph.D.

The figure shows a ribbon diagram of the structure of HIV-1 reverse transcriptase (RT) in a 

complex with DNA. We are engaged in a long-term study of HIV-1 RT, with the expectation 

that this information will be useful in developing more effective anti-RT drugs. Our strategy 

involves the analysis of the structure and function of both wild-type and mutant HIV-1 RTs, 

including drug-resistant mutants.
HIV Drug Resistance Program 11 

Research Summary:

Selected Publications: 

1. Oh J, Chang KW, Hughes SH. Mutations in the U5 

sequences adjacent to the primer binding site do not 

affect tRNA cleavage by Rous sarcoma virus RNase H 

but do cause aberrant integrations in vivo. J. Virol. 80: 

451-459, 2006.

2. Boyer PL, Sarafianos SG, Arnold E, Hughes SH. 

Why do HIV-1 and HIV-2 use different pathways to 

develop AZT resistance? PLoS Pathog. 2: e10, 2006.

3. Boyer PL, Julias JG, Ambrose Z, Siddiqui MA, 

Marquez VE, Hughes SH. The nucleoside analogs 

4’C-methyl thymidine and 4'C-ethyl thymidine block 

DNA synthesis by wild-type HIV-1 RT and excision 

proficient NRTI resistant RT variants. J. Mol. Biol.

371: 873-882, 2007.

4. Das K, Bauman JD, Clark AD Jr, Frenkel YV, Lewi 

PJ, Shatkin AJ, Hughes SH, Arnold E. High-resolution 

structures of HIV-1 reverse transcriptase/TMC278 

complexes: Strategic flexibility explains potency against 

resistance mutations. Proc. Natl. Acad. Sci. USA 105: 

1466-1471, 2008.

Model showing the binding of ATP to AZT-resistant HIV-1 RT. The basis 

of the most common form of AZT resistance is ATP-mediated excision 

of AZTMP after it has been incorporated into viral DNA by RT. The 

mutations that give rise to AZT resistance help ATP bind appropriately 

for the excision reaction. Van der Waals surfaces are drawn for 

polymerase active site residues (magenta) and residues involved in 

ATP binding and AZT resistance (yellow). Mutated amino acids M41L, 

K70R, L210W, and T215Y are shown with black labels, and amino 

acids that could be involved with ATP binding but are not mutated (E44, 

K46) are shown with magenta labels. The wild-type amino acids at 

K219 and D67 were retained in the figure to show a potential salt 

bridge between the residues. The AZT resistance mutations at these 

residues will destroy this salt bridge and may increase the ability of the 

pyrophosphate donor to bind. K219 and D67 are shown as stick 

diagrams to avoid obscuring the pyrophosphate binding site.

effective against NRTI-resistant viruses. Drug resistance is

only one aspect of the behavior of RT. Our goals are to 

understand in detail how RT carries out reverse transcription in an infected cell and to correlate the wealth of 

structural and biochemical data on HIV-1 RT with the 

actual process of reverse transcription. 

For this reason, we perform complementary experiments with HIV-1-based vectors in cultured cells. We 

have a long-term commitment to developing and using 

retroviral vectors, which started with the development of 

avian retrovirus-based RCAS vectors. We are still using 

the RCAS system to study viral replication. However, 

given the importance of HIV-1 in human disease and the 

wealth of information about the structure and function of 

the HIV-1 proteins (including RT), we particularly want to 

understand HIV-1 replication. 

We have developed HIV-1-based vectors that carry 

most of the primary mutations that confer resistance to 

NRTIs and NNRTIs. These HIV-1-based vectors are used 

to study drug resistance and to evaluate the effectiveness of 

new anti-HIV-1 compounds. The HIV-1 vectors are also 

used to study important steps in the HIV 1 life cycle and to 

determine the effects of mutations on viral replication. 

HIV-1 is the causative agent of AIDS. Three viral 

enzymes — integrase, protease, and reverse transcriptase 

(RT) — have essential roles in the replication of HIV-1. 

We are engaged in a long-term effort to study HIV-1 RT, 

with the expectation that this information will be useful in 

the development of more effective anti-RT drugs. There 

are two classes of inhibitors of HIV-1 RT: nucleoside 

analogs (NRTIs) and nonnucleoside inhibitors (NNRTIs). 

While both have been used to successfully treat HIV-1 

infections, there are serious problems with drug toxicity as 

well as the development of resistance. 

A major focus of our work on HIV-1 RT is the mechanism(s) of RT inhibitor resistance. Our strategy has 

involved the biochemical and structural analysis of both 

wild-type and mutant HIV-1 RTs, including drug-resistant 

mutants. The structural studies are done by our long-term 

collaborator, Dr. Eddy Arnold (Rutgers University). A 

part of our collaboration with Dr. Arnold is intended to 

develop more effective NNRTIs. We are also collaborating 

with Dr. Victor Marquez (Laboratory of Medicinal 

Chemistry, NCI) to develop nucleoside analogs that are 
12 National Cancer Institute

Contact Information:

HIV Drug Resistance Program

National Cancer Institute

NCI-Frederick, P.O. Box B

Building 535

Frederick, MD 21702-1201

Phone: 301-846-1249

Fax: 301-846-6777

E-mail: vineet@mail.nih.gov 

http://home.ncifcrf.gov/hivdrp/KewalRamani.html

Research Interests:

• Virus-host interactions

• Cofactors in HIV infection

• Development of animal models for 

HIV infection

Head, Model Development Section, HIV Drug 

Resistance Program

Biography: 

Vineet N. KewalRamani, Ph.D.

Dr. Vineet N. KewalRamani first developed an interest in retroviruses as an 

undergraduate in the lab of Dr. Antonito T. Panganiban at the McArdle Cancer 

Center, University of Wisconsin at Madison. After choosing to pursue molecular biology as a career, he moved west to the University of Washington in 

Seattle and did his graduate work studying HIV molecular virology in the lab of 

Dr. Michael Emerman at the Fred Hutchinson Cancer Research Center. After 

completing his Ph.D. in 1996, he moved to New York to join the lab of Dr. 

Dan R. Littman to better understand HIV infection within an immunological 

context. While working in the Littman lab, Dr. KewalRamani joined in an 

effort to develop a murine model for HIV replication using transgenic technologies. These studies provided the foundation for his lab’s current studies of 

understanding the blocks to HIV replication in murine cells, identifying human 

cell cofactors involved in HIV replication, and translating these basic science 

findings to develop better cell culture and animal models for HIV infection 

studies. Dr. KewalRamani joined the National Cancer Institute (NCI) in 1999 

as Head of the Model Development Section in the HIV Drug Resistance 

Program. He currently serves on the Editorial Boards of the Journal of 

Virology, PLoS One, and Virology and the NIH Foundation Norman P. 

Salzman Virology Award and Symposium Committee and is Co-Chair of the 

Tenure-Track Investigator Community in the Center for Cancer Research, NCI. 

DC-SIGN captures HIV through interactions with virion Env trimers. HIV complexed by DCSIGN is efficiently released to CD4+ target cells resulting in their infection. We are mapping

the contacts that HIV Env makes with DC-SIGN for initial capture and examining the mechanisms that underlie subsequent release and transmission of captured virus. Dendritic cells 

present at mucosal surfaces, macrophage subpopulations, and activated B cells express 

DC-SIGN in vivo.
HIV Drug Resistance Program 13 

Selected Publications: 

1. Lee K, KewalRamani VN. In defense of the cell: 

TRIM5α interception of mammalian retroviruses. 

Proc. Natl. Acad. Sci. USA 101: 10496-10497, 2004.

2. Ambrose Z, Palmer S, Boltz VF, Kearney M, Larsen K, 

Polacino P, Flanary L, Oswald K, Piatak M, Smedley J,

 Shao W, Bischofberger N, Maldarelli F, Kimata JT, 

Mellors JW, Hu SL, Coffin JM, Lifson JD, KewalRamani

VN. Suppression of viremia and evolution of human 

immunodeficiency virus type 1 drug resistance in 

a macaque model for antiretroviral therapy. J. Virol. 81: 

12145-12155, 2007. 

3. Ambrose Z, Compton L, Piatak M Jr, Lu D, Alvord 

WG, Lubomirski MS, Hildreth JEK, Lifson JD, Miller 

CJ, KewalRamani VN. Incomplete protection against 

simian immunodeficiency virus vaginal transmission in 

rhesus macaques by a topical antiviral agent revealed by 

repeat challenges. J. Virol. 82: 6591-6599, 2008.

4. Hatziioannou T, Ambrose Z, Chung NPY, Piatak M Jr, 

Yuan F, Trubey CM, Coalter V, Kiser R, Schneider D, 

Smedley J, Pung R, Gathuka M, Estes JD, Veazey RS, 

KewalRamani VN, Lifson JD, Bieniasz PD. A macaque 

model of HIV-1 infection. Proc. Natl. Acad. Sci. USA

106: 4425-4429, 2009.

HIV-1 in the SIV genome to create a hybrid virus referred 

to as RT-SHIV. We showed that RT-SHIV infection of 

pigtail macaques leads to immune cell depletion and pathogenesis. Importantly, we also demonstrated that a drug 

cocktail commonly used to treat HIV-1-infected patients 

was highly effective in suppressing RT-SHIV in the pigtail 

macaques and preventing immune cell depletion. Furthermore, we observed that animals with prior transient exposure

to only one drug from the cocktail developed antiviral 

resistance more quickly when treated with the more potent 

three-drug combination. In animals that did not develop 

resistance, virus rebounded to pretherapy levels when ART 

was stopped, indicating persisting reservoirs of infected cells.

While our current infection model leverages a modified SIV encoding HIV-1 RT, infection models under 

development with intra- and extramural collaborators rely 

on HIV-1 containing minimal portions of SIV to increase 

tropism and virulence in macaques. Infection of macaques 

with closer facsimiles of HIV-1 will permit the use of 

powerful antiviral drug combinations to seek tissues where 

virus is able to persist and escape eradication.

We have taken a two-tiered approach to advance knowledge 

of HIV replication and pathogenesis. The first tier is 

rooted in the basic science of HIV-1 replication; the 

second is translational and applies basic science knowledge 

within animal models for HIV-1 infection to ask questions 

only possible within these systems. 

At a primary level, we are interested in defining the 

role of cellular factors in the transmission and early 

replication of HIV-1. The interaction of HIV-1 with the 

cell soon after infection is poorly defined. Understanding

the role of HIV-1 cofactors will aid in the development of

new antiviral therapies to combat HIV-1 and improved 

animal models in which to study HIV-1 replication. 

Projects in this area include identifying host factors that 

aid in HIV-1 uncoating after entry, defining the pathway 

of HIV-1 trafficking from cytoplasm to nucleus in 

nondividing cells, and examining the mechanisms through 

which the extracellular factor DC-SIGN captures HIV-1 

particles and facilitates their transfer to target cells. 

Second, we have developed a new pigtail macaque 

model in which to study the evolution of antiviral resistance 

and examine the presence of persisting reservoirs during 

antiretroviral therapy (ART). While pigtail macaques are 

not susceptible to infection with HIV-1, simian immunodeficiency virus (SIV), a related virus, efficiently infects 

these animals and causes an immunodeficiency and 

pathogenesis that are characteristic of AIDS. Because of

genetic differences, SIV is not responsive to many drugs 

used to treat HIV-1. We therefore spliced a portion of

Potent suppression of RT-SHIV during tenofovir (TDF), emtricitabine 

(FTC), and efavirenz (EFV) combination therapy, a common regimen 

for HIV-infected patients. RT-SHIV is derived from a pathogenic 

SIVmne isolate and encodes HIV-1 RT. EFV, a nonnucleoside HIV-1 

RT inhibitor, is otherwise ineffective against SIV RT molecules.

Research Summary:
14 National Cancer Institute

Dr. Stuart Le Grice received his Ph.D. from the Department of Biochemistry, 

University of Manchester, UK, in 1976, studying mechanisms of R-factormediated multi-drug resistance in Escherichia coli. After postdoctoral training 

in the United Kingdom, Germany, and the United States, he was appointed 

Senior Scientist in the Central Research Units of Hoffmann La Roche, 

Switzerland, where he worked from 1984 to 1990 evaluating HIV-1 and HIV-2 

enzymes as therapeutic targets. In 1990, he was recruited to the Division

of Infectious Diseases, Department of Medicine, Case Western Reserve 

University (CWRU), Cleveland, OH, receiving tenure in 1992, and promotion 

in 1995 to Professor of Medicine, Biochemistry, and Oncology. From 1994 

to 1999, he directed the NIH-accredited CWRU Center for AIDS Research. 

Dr. Le Grice joined the National Cancer Institute in 1999 as Chief of the 

Resistance Mechanisms Laboratory in the HIV Drug Resistance Program, 

Center for Cancer Research (CCR). In 2006, he was appointed Head of the 

Center of Excellence in HIV/AIDS & Cancer Virology, CCR and is also a 

Steering Committee Member of the Molecular Targets Discovery Program 

and the Chemistry and Structural Biology Faculty. In addition to serving on 

the Editorial Board of the Journal of Biological Chemistry, Dr. Le Grice has been 

both an ad hoc and permanent Study Section member of several NIH AIDS 

review panels, as well as an ad hoc reviewer for multiple international funding 

agencies. In 2007, Dr. Le Grice was designated an NCI Mentor of Merit.

Head, Center of Excellence in HIV/AIDS & Cancer 

Virology, Center for Cancer Research; and Head, 

RT Biochemistry Section, HIV Drug Resistance 

Program 

Biography: 

Stuart F.J. Le Grice, Ph.D.

Examining the 2-D structure of inhibitor-resistant variants of the HIV-1 Rev response element 

by selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE).

Contact Information:

HIV Drug Resistance Program

National Cancer Institute

NCI-Frederick, P.O. Box B

Building 535

Frederick, MD 21702-1201

Phone: 301-846-5256

Fax: 301-846-6013

E-mail: legrices@mail.nih.gov 

http://home.ncifcrf.gov/hivdrp/Le_Grice.html

Research Interests:

• Protein and nucleic acid footprinting 

• Bioconjugate evaluation of reverse 

transcription complexes 

• RNase H inhibitors 

• Unnatural amino acids as probes of 

protein function
HIV Drug Resistance Program 15 

Probing the orientational dynamics of HIV-1 RT on duplex and hybrid 

nucleic acid substrates via single-molecule spectroscopy. The fluorophores Cy3 and Cy5 are introduced at different sites of HIV-1 RT (a) and 

the nucleic acid substrate (b), respectively. The intensity of the FRET 

signal (c) is directly related to the proximity of Cy3 and Cy5. A detailed 

explanation of the experimental setup and use in determining enzyme 

orientation is given in Reference 2.

Novel drugs targeting virus entry and maturation have 

recently been added to the growing armory of antiviral 

agents aimed at stemming the continuing spread of HIV 

infection. In addition, host factors participating in key steps 

in HIV replication are under evaluation as targets for therapeutic intervention. Despite these promising approaches, 

the viral enzymes protease (PR), reverse transcriptase (RT), 

and integrase (IN) remain the primary targets of combination antiretroviral therapy, with more than 20 drugs in 

clinical use against these enzymes. 

Research in the RT Biochemistry Section (RTBS) 

combines biochemistry and biophysics with structural, 

molecular, and chemical biology to examine, at the 

molecular level, the manner in which retroviral RT and its

counterpart from Saccharomyces cerevisiae retrotransposons 

convert the single-stranded RNA genome into integrationcompetent double-stranded DNA. 

Nucleoside analogs imparting different properties 

into nucleic acid duplexes (eliminating hydrogen bonding 

or base stacking, decreasing duplex flexibility, neutralizing 

the phosphate backbone) are exploited to provide novel 

acumen into the interaction of the retroviral polymerase 

with cis-acting regulatory elements.

Nucleic acid strategies are complemented by the use 

of genetically engineered Escherichia coli to site-specifically 

introduce unnatural amino acids with a variety of biophysical properties into recombinant proteins. This 

approach has created novel HIV-1 RT mutants conferring 

resistance to inhibitors of both DNA polymerase and 

ribonuclease H (RNase H) function.

Chemical footprinting of RNA by automated selective 

2'-hydroxyl acylation analyzed by primer extension 

(SHAPE) is under development to examine the structure 

of regulatory elements on the viral RNA genome both in 

vitro and in vivo, while high-resolution FTICR-ESI mass 

spectrometry has been combined with NMR spectroscopy 

to examine the interaction of small-molecule ligands with 

the polypurine tract primer of plus-strand DNA synthesis. 

Finally, we have initiated single-molecule fluorescence 

studies to probe the orientation of RT on a variety of 

nucleic acid duplexes. Knowledge gained through these 

multidisciplinary approaches helps characterize potent and 

selective RNase H inhibitors identified by high-throughput 

screening. The RTBS enjoys the expertise of investigators 

and resources within the Intramural Research Program while 

collaborating with internationally recognized extramural 

investigators to promote new areas of investigation.

Selected Publications: 

1. Legiewicz M, Badorrek CS, Turner KB, Fabris D, 

Hamm TE, Rekosh D, Hammarskjöld M-L, Le Grice 

SFJ. Resistance to RevM10 inhibition reflects a conformational switch in the HIV-1 Rev response element. 

Proc. Natl. Acad. Sci. USA 105: 14365-14370, 2008.

2. Abbondanzieri EA, Bokinsky G, Rausch JW, Zhang J, 

Le Grice SFJ, Zhuang X. Dynamic binding orientations 

direct activity of HIV reverse transcriptase. Nature 453: 

184-189, 2008.

3. Wendeler M, Lee H-F, Bermingham A, Miller JT, 

Chertov O, Bona MK, Baichoo NS, Ehteshami M, 

Beutler J, O’Keefe BR, Götte M, Kvaratskhelia M,

Le Grice SFJ. Vinylogous ureas as a novel class of 

inhibitors of reverse transcriptase-associated ribonuclease H activity. ACS Chem. Biol. 3: 635-644, 2008.

4. Liu S, Abbondanzieri EA, Rausch JW, Le Grice SFJ, 

Zhuang X. Slide into action: Dynamic shuttling of HIV 

reverse transcriptase on nucleic acid substrates. Science

322: 1092-1097, 2008.

Research Summary:
16 National Cancer Institute

Contact Information:

HIV Drug Resistance Program

National Cancer Institute

NCI-Frederick, P.O. Box B

Building 535

Frederick, MD 21702-1201

Phone: 301-846-1710

Fax: 301-846-6013

E-mail: vpathak@ncifcrf.gov 

http://home.ncifcrf.gov/hivdrp/Pathak.html

Research Interests:

• Structure and function of APOBEC3 

host restriction factors

• Mechanisms of antiviral drug 

resistance

• Reverse transcriptase structure, 

fidelity, and template switching

Dr. Vinay K. Pathak received his B.A. in Biology from the University of 

California, Los Angeles, in 1979, and his M.S. in Comparative Pathology 

from the University of California, Davis (UCD), in 1983 for characterization of mouse mammary tumor virus proviral integration sites near the int1

and int2 loci in Dr. Robert Cardiff’s laboratory. He received his Ph.D. in 

Genetics for work on characterization of the eukaryotic protein synthesis 

initiation factors eIF-2α and eIF-2β in Dr. John W.B. Hershey’s laboratory 

(UCD) in 1988. From 1988 to 1991, he was a Postdoctoral Fellow under 

the guidance of Dr. Howard Temin at the University of Wisconsin, where 

he studied the in vivo fidelity and nature of mutations that arise during 

retroviral replication. In 1991, Dr. Pathak became an Assistant Professor 

in the Department of Biochemistry and the Mary Babb Randolph Cancer 

Center at West Virginia University. He was promoted to Associate 

Professor with tenure in 1998. He joined the National Cancer Institute in 

1999 as Senior Investigator and Head of the Viral Mutation Section in the 

HIV Drug Resistance Program. Dr. Pathak was appointed as Guest Editor 

for the HIV Drug Resistance special issue of Viruses, which will be 

published in October 2009.

Head, Viral Mutation Section, HIV Drug Resistance 

Program

Biography: 

Vinay K. Pathak, Ph.D.

In virus producer cells, APOBEC3 proteins are incorporated into HIV-1 virions in the 

absence of Vif. Vif binds to APOBEC3 proteins and targets them for proteasomal 

degradation. APOBEC3 proteins inhibit viral DNA synthesis, induce G-to-A hypermutation, 

and inhibit viral DNA integration. 
HIV Drug Resistance Program 17

Research Summary:

Selected Publications: 

1. Nikolenko GN, Delviks-Frankenberry KA, Palmer S,

Maldarelli F, Fivash MJ Jr, Coffin JM, Pathak VK. 

Mutations in the connection domain of HIV-1 reverse 

transcriptase increase AZT resistance. Proc. Natl. Acad. 

Sci. USA 104: 317-322, 2007.

2. Mbisa JL, Barr R, Thomas JA, Vandegraaff N, Dorweiler 

IJ, Svarovskaia ES, Brown WL, Mansky LM, Gorelick RJ, 

Harris RS, Engelman A, Pathak VK. Human immunodeficiency virus type 1 cDNAs produced in the presence 

of APOBEC3G exhibit defects in plus-strand DNA 

transfer and integration. J. Virol. 81: 7099-7110, 2007. 

3. Delviks-Frankenberry KA, Nikolenko GN, Boyer PL, 

Hughes SH, Coffin JM, Jere A, Pathak VK. HIV-1 reverse

transcriptase connection subdomain mutations reduce 

template RNA degradation and enhance AZT excision. 

Proc. Natl. Acad. Sci. USA 105: 10943-10948, 2008.

4. Russell RA, Smith J, Barr R, Bhattacharyya D, Pathak 

VK. Distinct domains within APOBEC3G and 

APOBEC3F interact with separate regions of human 

immunodeficiency virus type 1 Vif. J. Virol. 83: 

1992-2003, 2009.

phenotypic analyses of drug resistance. The improved 

genotypic and phenotypic assays might predict antiviral 

drug resistance more accurately and would lead directly to 

the development of new clinical protocols designed to 

improve drug choice in salvage therapy. Our specific 

research aims are to further define the role of the C-terminal 

connection and RNase H domains of RT in antiviral drug 

resistance in subtype B as well as non-B subtypes of HIV-1. 

Third, we are exploring the in vivo mechanisms of 

reverse transcription by using a novel quantitative strandspecific amplification (SSA) technology that we recently 

developed. We are using the SSA method to measure the 

rates of viral DNA synthesis in activated primary CD4+ T 

cells and macrophages, the target cells of HIV-1 infection. 

We are studying the mechanism of discontinuous plusstrand DNA synthesis and the influence of RT inhibitors 

on the kinetics of reverse transcription, providing insights 

into their mechanisms of inhibition. The SSA technology 

provides a novel approach to analyzing DNA replication 

processes and may facilitate the development of new 

antiretroviral drugs that target specific steps in HIV-1 

reverse transcription.

Our goal is to understand how HIV-1 replicates and how 

its interactions with the host cell influence its replication. 

We are active in three major research areas. First, we are 

defining the structure and function of APOBEC3G and 

3F proteins. We are identifying the determinants of 

interaction between HIV-1 Vif and APOBEC3 proteins 

through genetic and biochemical analysis. We are elucidating the mechanisms by which the APOBEC3 proteins 

inhibit HIV-1 DNA synthesis, integration, and viral replication. In addition, we are investigating the mechanisms 

by which APOBEC3 proteins are incorporated into virions 

and regulated in vivo. Our long-term goal is to identify 

small-molecule inhibitors that interfere with the interaction between HIV-1 Vif and APOBEC3 proteins, which 

could form the basis of a new class of antiretroviral drugs. 

Second, we are elucidating mechanisms by which 

HIV-1 reverse transcriptase (RT) acquires resistance to RT 

inhibitors. We have identified eight novel mutations in the 

C-terminal connection and RNase H domains of RT that 

substantially enhance NRTI and NNRTI resistance. 

Current genotypic and phenotypic studies only analyze the 

N-terminal domains of RT for drug resistance and do not 

include the C-terminal domains. However, our studies 

indicate that it is important to include the C-terminal 

connection and RNase H domains in future genotypic and

A mechanism by which C-terminal domain mutations increase NRTI resistance. NRTI is incorporated into DNA during RNA-template-directed DNA 

synthesis; RNase H degrades template RNA; the template and primer are 

then dissociated, leading to termination of reverse transcription. Mutations 

that reduce RNase H activity provide more time for NRTI excision to occur 

before template RNA degradation, resulting in increased NRTI resistance. 
18 National Cancer Institute

Contact Information:

HIV Drug Resistance Program

National Cancer Institute

NCI-Frederick, P.O. Box B

Building 535

Frederick, MD 21702-1201

Phone: 301-846-1361

Fax: 301-846-6013

E-mail: rein@ncifcrf.gov 

http://home.ncifcrf.gov/hivdrp/Rein.html

Research Interests:

• Molecular mechanisms of retroviral 

assembly and replication

• Interactions of retroviral proteins

with nucleic acids

• Interactions of retroviral components with host antiviral factors 

Dr. Alan Rein obtained his Ph.D. with Dr. Harry Rubin at the University of 

California at Berkeley and did postdoctoral research as an American Cancer 

Society Fellow under the direction of Dr. Sheldon Penman at the 

Massachusetts Institute of Technology. Dr. Rein has been associated with 

the National Cancer Institute (NCI) since 1976 and served as Head of the 

Retroviral Genetics Section in the ABL–Basic Research Program from 1984 

to 1999. In 1999, he joined the HIV Drug Resistance Program as Head of 

the Retrovirus Assembly Section and was appointed to the NCI Senior 

Biomedical Research Service. Dr. Rein was an Organizer of the 1995 Cold 

Spring Harbor Laboratory Meeting on Retroviruses and the 2001 

International Retroviral Nucleocapsid Symposium. He currently serves 

on the Editorial Boards of the Journal of Biological Chemistry, Virology, and

Journal of Virology. He is also a member of the Center of Excellence in 

HIV/AIDS & Cancer Virology and the Chemistry and Structural Biology 

Faculty in the Center for Cancer Research, NCI. His research has dealt 

with a number of aspects of the biology and molecular biology of murine 

and human retroviruses, including virus assembly and maturation, viral 

envelope function, translational suppression, and pathogenesis.

Head, Retrovirus Assembly Section, HIV Drug 

Resistance Program

Biography: 

Alan Rein, Ph.D.

Virus-like particles assembled from purified HIV-1 Gag protein that had been produced in 

bacteria and mixed with E. coli ribosomal RNA. Scale bar: 100 nm.
HIV Drug Resistance Program 19

Selected Publications: 

1. Fisher RJ, Fivash MJ, Stephen AG, Hagan NA,

Shenoy SR, Medaglia MV, Smith LR, Worthy KM,

Simpson JT, Shoemaker R, McNitt KL, Johnson DG,

Hixson CV, Gorelick RJ, Fabris D, Henderson LE,

Rein A. Complex interactions of HIV-1 nucleocapsid 

protein with oligonucleotides. Nucleic Acids Res. 34: 

472-484, 2006.

2. Rulli SJ, Hibbert CS, Mirro J, Pederson T, Biswal S, 

Rein A. Selective and nonselective packaging of cellular 

RNAs in retrovirus particles. J. Virol. 81: 6623-6631, 

2007.

3. Datta SA, Zhao Z, Clark PK, Tarasov S, Alexandratos 

JN, Campbell SJ, Kvaratskhelia M, Lebowitz J, Rein A. 

Interactions between HIV-1 Gag molecules in solution: 

An inositol phosphate-mediated switch. J. Mol. Biol.

365: 799-811, 2007.

4. Datta SA, Curtis JE, Ratcliff W, Clark PK, Crist RM, 

Lebowitz J, Krueger S, Rein A. Conformation of the 

HIV-1 Gag protein in solution. J. Mol. Biol. 365: 812-

824, 2007.

Inactivation of murine leukemia virus by mouse APOBEC3 and by human 

APOBEC3G. A murine leukemia virus-derived “reporter vector” encoding 

luciferase was produced in the presence of increasing amounts of either 

mouse APOBEC3 or human APOBEC3G expression vectors, and its 

infectivity was determined by assaying for luciferase following infection

of fresh cells.

We have found that RNA plays a crucial role in 

retrovirus particle structure, and are studying the nature of 

this contribution. Gag normally selects the viral genomic 

RNA for packaging into the assembling particle; the 

mechanism of this selection is unknown. We found that if 

this RNA is not present in the cell, Gag packages cellular 

mRNA molecules; most, but not all, mRNAs are packaged 

nonselectively. We are now analyzing how certain RNAs 

are enriched in virus particles. 

Another focus of our research is the interactions 

between retroviruses and antiviral factors produced by 

their hosts. Mouse cells encode an antiviral factor termed 

APOBEC3. We found that murine retroviruses are almost 

entirely resistant to its antiviral effects (Fig. 2 — compared 

with its human counterpart APOBEC3G). In collaboration with Dr. David Derse (HIV Drug Resistance Program), 

we are investigating the mechanism underlying this relative 

resistance of murine leukemia virus to APOBEC3, using

a number of experimental approaches. These studies 

should help elucidate the mechanisms of encapsidation

of, and virus inactivation by, APOBEC proteins.

Virus particle assembly is an essential step in the HIV 1 

replication cycle. This fact makes it a potential target for 

antiviral therapy. However, it has not been exploited 

therapeutically to date, presumably because the molecular 

mechanisms responsible for assembly are not understood 

in detail. 

Our research focuses on the molecular mechanisms 

underlying the assembly of infectious particles of HIV-1 

and other retroviruses. Expression of a single viral 

protein, termed Gag, is sufficient for the efficient assembly 

of retrovirus particles in mammalian cells. Virus assembly 

can be recapitulated in a fully defined system, containing 

only purified recombinant Gag protein and nucleic acid 

(Fig. 1). We have found that our recombinant Gag 

protein is in a folded, compact conformation in solution; 

in contrast, it is rod-shaped in the assembled virus particle. 

Therefore, our findings show for the first time that the 

Gag protein undergoes a major conformational change 

before or during virus assembly. This is a previously 

unsuspected event in virus replication, and represents a

 new potential target for antiviral therapy. Current efforts are 

directed toward identifying the “trigger” for this 

conformational change. 

We have also reported that the same site within Gag 

functions successively in two different steps in the 

replication cycle: first, in the initial assembly of the virus

particle, and second, in formation of the mature core 

following cleavage of Gag within the released particle. It

is remarkable that the same site is used twice in the viral 

replication cycle; this site would appear to be a particularly attractive target for therapeutic intervention.

Research Summary:
20 National Cancer Institute

Contact Information:

HIV Drug Resistance Program

National Cancer Institute

NCI-Frederick, P.O. Box B

Building 536

Frederick, MD 21702-1201

Phone: 301-846-1355

Fax: 301-846-6013

E-mail: oroszlans@ncifcrf.gov 

http://home.ncifcrf.gov/hivdrp/Oroszlan.html

Research Interests:

• Biochemistry and enzymology of 

retroviral replication

• Retroviral enzymes and structural 

proteins

• Structure and function of HIV 

protease

Scientist Emeritus, HIV Drug Resistance Program

Biography: 

Dr. Stephen Oroszlan obtained his Ph.D. in Pharmacology from Georgetown 

University. After his postdoctoral training at the National Cancer Institute 

(NCI) from 1961 to 1963, he conducted biochemical research on retroviruses 

at the Albert Einstein Medical Center, George Washington University School 

of Medicine, and Flow Laboratories. In 1976, he joined the Biological 

Carcinogenesis Program at NCI-Frederick as Head of the Immunochemistry 

Section, and served as Director of the Molecular Virology and Carcinogenesis 

Laboratory, ABL–Basic Research Program, from 1983 to 1995. Dr. Oroszlan 

was appointed Scientist Emeritus in 1995 and has been affiliated with the 

HIV Drug Resistance Program since 1999. His studies have provided 

invaluable information on the biosynthesis of retroviral enzymes and 

structural proteins. He has published more than 250 papers and was named 

one of the most highly cited researchers in microbiology by the Institute for 

Scientific Information. Dr. Oroszlan has been awarded three U.S. patents 

and one European patent on HIV protease and one U.S. patent on 

chloroquine as an inhibitor of infectious HIV production. His numerous 

honors include the award of a Doctor Honoris Causa Degree from the 

University of Debrecen Medical School in 1993, election to the Hungarian 

Academy of Sciences as a Foreign Associate in 1994, a Retroviral 

Retrospective Symposium to Honor Dr. Stephen Oroszlan held at Hood 

College in 1994, and the Mór Kaposi Research Foundation Award in 2000. 

At the Symposium on HIV Protease and Beyond: The Past, Present, and Future of HIV Structural 

Biology, held at NCI-Frederick in January 2009, Dr. Michael Gottesman (NIH Deputy Director for 

Intramural Research) presented an award to Dr. Stephen Oroszlan in recognition of his many 

contributions to our understanding of the structure of retroviruses, and for his seminal work on 

viral 

proteases. Dr. Oroszlan gave the opening talk at the symposium, describing the key events in 

retroviral protease research and the discovery of HIV protease.

Stephen Oroszlan, Ph.D.
HIV Drug Resistance Program 21

As the clinical arm of the HIV Drug Resistance 

Program, the Host-Virus Interaction Branch (HVIB) 

conducts fundamental studies on the nature of HIV 

drug resistance in vivo. The HVIB has developed 

sensitive and specific tools that are being used to 

analyze patient material to address questions of HIV 

population genetics, replication dynamics, and emergence of resistance in infected individuals. Ongoing 

studies are focused on several issues: understanding 

the origin and development of genetic variation in HIV 

populations, and using this information to infer the 

structure of the underlying population of HIV-infected 

cells; understanding the extent of replication of HIV—

and the potential for evolution of resistance — in 

patients whose virus load has become suppressed under 

successful therapy; and understanding the mechanism, 

kinetics, and consequences of appearance and disappearance of drug resistance mutations in infected 

patients before, during, and after treatment. 

The HVIB comprises the following elements:

• The In Vivo Biology Group, headed by Dr. Frank 

Maldarelli, directs the protocol development, 

regulatory affairs, patient recruitment, and sample 

collection effort in the NIH Clinical Center, in 

collaboration with the AIDS clinical research 

programs of the National Cancer Institute 

(HIV/AIDS Malignancy Branch, Dr. Robert 

Yarchoan), National Institute of Allergy and 

Infectious Diseases (Division of Clinical Research, 

Dr. H. Clifford Lane), and NIH Clinical Center 

(Critical Care Medicine Department, Dr. Henry 

Masur). The In Vivo Biology Group is studying 

patient samples derived from its own protocols 

(largely in collaboration with these NIH clinical 

groups) and obtained from numerous extramural 

collaborators. 

• The Virology Core, led by Dr. Mary Kearney,

is responsible for the development and implementation of new assays to detect and analyze HIV in 

patient samples. These assays are principally used 

by the HVIB and other sections of the HIV Drug 

Resistance Program. 

• Dr. John Coffin, American Cancer Society Research 

Professor of Molecular Biology and Microbiology at 

Tufts University and founding Director of the HIV 

Drug Resistance Program, now serves as advisor to 

the Director of the Center for Cancer Research and 

consultant to the clinical program, particularly in the 

areas of assay design and interpretation, study design, 

and data analysis.

• Collaboration with Dr. John Mellors from the 

University of Pittsburgh provides critical consultation 

and advice on all aspects of HIV clinical research, 

including protocol design and implementation.

 Dr. Mellors provides an invaluable interface with

the extramural HIV research community, including

the AIDS Clinical Trials Group and international

collaborative trials, making possible the Program’s

participation in major multicenter trials. The Mellors

laboratory also collaborates closely with the HVIB

on development of novel laboratory techniques for 

analysis of clinical material, and in basic studies 

of the mechanism of resistance to reverse transcriptase inhibitors. 

Clinical Program — Host-Virus Interaction Branch
22 National Cancer Institute

Head, In Vivo Biology Group, HIV Drug Resistance 

Program

Biography: 

Frank Maldarelli, M.D., Ph.D.

Contact Information:

HIV Drug Resistance Program

National Cancer Institute

National Institutes of Health

Building 10, Room 5A06

Bethesda, MD 20892

Phone: 301-435-8019

Fax: 301-480-1735

E-mail: fmalli@mail.nih.gov 

http://home.ncifcrf.gov/hivdrp/Maldarelli.

html

Research Interests:

• HIV-1 population genetics 

• Source of low-level viremia in

patients suppressed on antiretroviral therapy

• Clinical studies on HIV resistance

Dr. Frank Maldarelli received his Ph.D. from the City University of New 

York and his M.D. from Mount Sinai School of Medicine. After completing 

his residency in internal medicine at The Columbia-Presbyterian Hospital, 

New York, he joined the Laboratory of Molecular Microbiology at the 

National Institute of Allergy and Infectious Diseases (NIAID) as a Medical 

Staff Fellow. Dr. Maldarelli joined the National Cancer Institute (NCI) in 

1998 as Head of the In Vivo Biology Group in the HIV Drug Resistance 

Program (DRP). Representing the clinical research arm of the DRP, he has 

established extensive collaborations between the DRP in Frederick and both 

the NCI HIV and AIDS Malignancy Branch and the NIAID AIDS clinical 

research program in Bethesda. Dr. Maldarelli is an Attending Physician in the 

NIAID/CCMD HIV service, and is currently a faculty member of the 

International Society of Infectious Diseases HIV Training Program, NIH 

Infectious Disease Consult Service, and Center for Bio-Medical Communications Infectious Disease Board Review Course, and a lecturer in the 

Washington D.C. community outreach PACT Program.

Analysis of genetic diversity of HIV-1 in patients with HIV-1 infection. HIV-1 genetic diversity 

(expressed as percent average pairwise difference) was determined from alignments of 

single-genome sequences obtained from all samples from 27 HIV-1-infected patients and 

presented as a function of minimum duration of infection. Individual patients with multiple 

samples are color coded as indicated. Patients for whom only a single sample was available 

for analysis are coded in black. Early in infection (~days 1-200), diversity accumulated 

rapidly and approximated the mutation rate of HIV-1 reverse transcriptase (RT). In established 

HIV-1 infection, diversity accumulates slowly, and is likely the product of a balance of 

mutation and selection.
HIV Drug Resistance Program 23 

Research Summary:

The In Vivo Biology Group develops and implements 

clinical protocols to elucidate mechanisms underlying the

 emergence of resistance in vivo, the dynamics of infection 

under treatment, and the role of resistance mutations in the 

efficacy and failure of subsequent treatments. 

In the first of four projects conducted by the In Vivo 

Biology Group, our goal is to determine the dynamics of 

viral replication in HIV-infected patients on suppressive 

antiretroviral therapy. These studies have been extended in 

clinical trials performed at NIH and elsewhere to determine 

the levels of viremia when treatment regimens are either 

simplified or intensified. Initial results from these trials 

indicate that the source of HIV-1 viremia during suppressive antiretroviral therapy is not from active cycles of 

replication, but from reservoirs of long-lived cells. We 

are also exploring new strategies to decrease reservoirs of 

HIV-1 in infected individuals.

In a second research project, we are investigating the 

genetic structure of HIV populations in infected individuals. 

We are using the single-genome sequencing technology 

developed by the Virology Core to analyze and understand 

the accumulation of genetic variation in the gag/pol and env

genes of HIV-1 in a number of different patient groups, 

including chronically infected patients, both naïve and on 

therapy, as well as in primary and early HIV-1 infection. 

The goal of this study is to understand the nature of the 

forces (mutation, selection, drift, recombination) that mold 

the genetic diversity of virus populations before and after 

antiretroviral therapy is introduced.

Antiretroviral therapy intensification does not reduce HIV-1 viremia. HIV-1 

RNA levels are shown for all time points for all nine patients. Each set of 

colored symbols represents HIV-1 RNA values obtained for the indicated 

patient during each phase of the study. Open symbols represent measurements below the limit of detection (1 copy/ml). Levels of residual viremia 

during intensification were not statistically different from pre- and postintensification levels.

The third project is focused on the dynamics of the 

appearance and disappearance of drug resistance mutations 

in HIV-infected individuals, using assays developed in the 

Virology Core that are capable of detecting mutations with 

a frequency of >0.03%. In several collaborative studies, we 

are assessing the role of low-frequency mutations on subsequent treatment failure and analyzing the appearance and 

disappearance of NNRTI resistance mutations in women 

exposed to single-dose nevirapine to prevent mother-toinfant transmission. 

The goal of the fourth project is to improve our understanding of HIV-1 drug resistance in vivo. In a clinical 

protocol of patients with drug-resistant HIV-1, we are 

investigating the effects of short-duration, single-drug 

discontinuations. Using standard viral RNA level determinations and single-genome sequencing, we are investigating 

the contribution of individual antiretrovirals to partial suppression of virus, and identifying mutations associated with 

resistance to that drug. We are currently investigating the 

role of new RT connection domain mutations identified 

by Dr. Vinay Pathak of the HIV Drug Resistance Program.

Selected Publications: 

1. Maldarelli F, Palmer S, King MS, Wiegand A, Polis MA, 

Mican J, Kovacs JA, Davey RT, Rock-Kress D, Dewar 

R, Liu S, Metcalf JA, Rehm C, Brun SC, Hanna GJ, 

Kempf DJ, Coffin JM, Mellors JW. ART suppresses 

plasma HIV-1 RNA to a stable set point predicted by 

pretherapy viremia. PLoS Pathog. 3: e46, 2007.

2. Palmer S, Maldarelli F, Wiegand A, Bernstein B, Hanna 

GJ, Brun SC, Kempf DJ, Mellors JW, Coffin JM, King 

MS. Low-level viremia persists for at least 7 years in 

patients on suppressive antiretroviral therapy. Proc. 

Natl. Acad. Sci. USA 105: 3879-3884, 2008.

3. Wilkin TJ, McKinnon JE, Dirienzo AG, Mollan K, 

Fletcher CV, Margolis DM, Bastow B, Thal G,

Woodward W, Godfrey C, Wiegand A, Maldarelli F, 

Palmer S, Coffin JM, Mellors JW, Swindells S. Regimen 

simplification to atazanavir-ritonavir alone as maintenance antiretroviral therapy: Final 48-week clinical and 

virologic outcomes. J. Infect. Dis. 199: 866-871, 2009.

4. Dinoso JB, Kim SY, Wiegand AM, Palmer SE, Gange SJ, 

Cranmer L, O’Shea A, Callender M, Spivak A, Brennan T, 

Kearney M, Proschan MA, Mican JM, Rehm CA, Coffin 

JM, Mellors JW, Siliciano RF, Maldarelli F. Treatment 

intensification does not reduce residual HIV-1 viremia 

in patients on highly active antiretroviral therapy. 

Proc. Natl. Acad. Sci. USA, 9: 106-123, 2009.
24 National Cancer Institute

Contact Information:

HIV Drug Resistance Program

National Cancer Institute

NCI-Frederick, P.O. Box B

Building 535

Frederick, MD 21702-1201

Phone: 301-846-6796

Fax: 301-846-6777

E-mail: kearneym@ncifcrf.gov 

http://home.ncifcrf.gov/hivdrp/Kearney.html

Research Interests:

• Development of novel clinical HIV-1 

assays

• Generation of HIV-1 genetic diversity 

• Detection of minority HIV-1 drug 

resistance mutations

• Persistent viremia in patients on 

and off antiretroviral therapy

Biography: 

Mary Kearney, Ph.D.

Dr. Mary Kearney received her Ph.D. in Biology at Catholic University in 

2007 under the direction of Drs. John Coffin, Sarah Palmer, and Venigalla 

Rao. In 2008, she was awarded the Benedict T. DeCicco Award for 

Excellence in Graduate Research by the Biology Faculty of the Catholic 

University of America. Dr. Kearney joined the HIV Drug Resistance 

Program in 2001 as a Biologist in the Virology Core and was promoted

to Manager of the Virology Core in 2008. In her current position,

Dr. Kearney oversees a team that is developing novel analyses for the

virological studies undertaken within the HIV Drug Resistance Program,

as well as performing standard analyses of HIV in both clinical and basic 

studies. Her research is focused on developing methods for obtaining very 

large numbers of sequences from virus circulating in plasma, for measuring 

virus load with much greater sensitivity than can be reliably attained with 

currently available tests, and for detecting minor drug resistance mutations 

in vivo.

Studying the genetic relatedness of multiple single-virion amplicons by using a

limiting-dilution RT-PCR technique provides useful insight into the evolution of

genetic diversity of HIV-1 populations. The figure illustrates that chronically infected 

patients initiating treatment have highly diverse populations prior to therapy. Even 

though rapid viral load declines occur after initiation of treatment (shown by the 

arrow), phylogenetic analysis has revealed that HIV-1 population structure posttreatment is indistinguishable from pretherapy samples, even following greater than

100-fold decreases in plasma HIV-1 RNA levels. These results suggest a large 

replicating HIV-1 population in infected persons.
HIV Drug Resistance Program 25 

Selected Publications: 

1. Maldarelli F, Palmer S, King MS, Wiegand A, Polis MA, 

Mican J, Kovacs JA, Davey RT, Rock-Kress D, Dewar 

R, Liu S, Metcalf JA, Rehm C, Brun SC, Hanna GJ, 

Kempf DJ, Coffin JM, Mellors JW. ART suppresses 

plasma HIV-1 RNA to a stable set point predicted by 

pretherapy viremia. PLoS Pathog. 3: e46, 2007.

2. Kearney M, Palmer S, Maldarelli F, Shao W, Polis MA, 

Mican J, Rock-Kress D, Margolick JB, Coffin JM, 

Mellors JW. Frequent polymorphism at drug resistance 

sites in HIV-1 protease and reverse transcriptase. 

AIDS 22: 497-501, 2008.

3. Kearney M, Maldarelli F, Shao W, Margolick JB, Daar 

ES, Mellors JW, Rao V, Coffin JM, Palmer S. Human 

immunodeficiency virus type 1 population genetics and

adaptation in newly infected individuals. J. Virol. 83: 

2715-2727, 2009.

4. Dinoso JB, Kim SY, Wiegand AM, Palmer SE, Gange 

SJ, Cranmer L, O’Shea A, Callender M, Spivak A, 

Brennan T, Kearney M, Proschan MA, Mican JM, 

Rehm CA, Coffin JM, Mellors JW, Siliciano RF, 

Maldarelli F. Treatment intensification does not reduce 

residual HIV-1 viremia in patients on highly active 

antiretroviral therapy. Proc. Natl. Acad. Sci. USA, 

9: 106-123, 2009.

The Virology Core is primarily responsible for supporting 

the clinical research efforts of the HIV Drug Resistance 

Program by developing and applying new technologies 

to quantify low-level viremia, to detect low-frequency 

drug-resistant mutants, and to characterize the genetic 

diversity of HIV-1 in infected individuals. The Core has 

developed the following assays for application to 

projects within the Host-Virus Interaction Branch and 

other sections of the HIV Drug Resistance Program: 

• Single-Copy Assay (SCA) for Detection of HIV-1 

RNA in Plasma. Potent antiretroviral therapy is 

effective in suppressing, but not eradicating, HIV-1 

infection. Persistent viremia can be detected in 

patients on antiretroviral therapy despite suppression 

of plasma HIV-1 RNA to <50 copies/ml. The Core 

developed the SCA for detection of HIV-1 RNA, 

which has a dynamic range of 106 to 0.3 copies/ml. 

This technology is now being applied to characterize 

the prevalence, level, and change over time of 

persistent viremia. 

• Allele-Specific PCR (ASP) for Detection of 

Low-Frequency Mutations. Low-frequency drugresistant mutants are hypothesized to contribute to 

antiretroviral treatment failure. The Core developed 

the ASP assay for quantifying drug-resistant HIV-1 

variants at reverse transcriptase amino acid codon 

positions 103, 181, 184, and 190 at frequencies as 

low as 0.1%. The Core is applying the ASP assay 

to plasma samples from treatment-naïve and 

experienced patients to assess the frequency of 

mutant alleles and their relation to virologic 

response to antiretroviral treatment. 

• Single-Genome Sequencing (SGS) for Genetic 

Analysis of HIV-1 Populations. Characterization of 

the genetic diversity of HIV-1, including the prevalence of rare drug-resistant variants, in patients with 

acute infection or chronic untreated infection and in 

patients initiating antiretroviral therapy can provide 

important insights into the effective population size 

of HIV-1, and the likelihood of emergence of drugresistant variants. Standard population-based DNA 

sequencing methods cannot reliably detect HIV-1 

variants that comprise <20% of the virus population. 

Furthermore, these methods cannot distinguish 

whether alleles are present on the same genome or 

different genomes. The Core developed the SGS 

assay for detecting sequences derived from individual 

viral genomes in plasma. Using the SGS assay, the 

Core has studied HIV-1 diversity and divergence in 

recently infected patients followed for up to 5 years 

after infection, in patients who were chronically 

infected with HIV-1 for 5 years and longer, and in 

HIV-1-infected patients initiating therapy. 

Single-genome sequencing assay developed by the Virology Core for the 

genetic analysis of HIV-1 populations.

Virology Core
26 National Cancer Institute

postdoctoral openings by Principal Investigators in

 the HIV Drug Resistance Program, is available at 

http://www.training.nih.gov/postdoctoral/.

For postbaccalaureates, college undergraduates,

and high-school students interested in pursuing biomedical careers, a variety of programs provide excellent 

research training opportunities, ranging from summer 

laboratory internships to 1- to 2-year fellowships. 

Details about all of these programs may be found at the 

NIH Research and Training Opportunities website: 

http://www.training.nih.gov/student/.

In addition to postdoctoral and student research 

training opportunities, the HIV Drug Resistance Program 

periodically recruits for technical positions. For further 

information about all of the recruitment activities in

the HIV Drug Resistance Program, please visit 

The HIV Drug Resistance Program is committed to http://home.ncifcrf.gov/hivdrp/recruitment.html. 

providing excellent research training opportunities for 

postdoctoral and postbaccalaureate graduates, college 

undergraduates, and high-school students. The Program’s 

successful mentoring of young scientists has resulted in 

former trainees obtaining major positions in academia, 

industry, and government research programs.

Postdoctoral research training is supported primarily through two fellowship programs: the Cancer 

Research Training Award (CRTA) provides funding for 

postdoctoral fellows who are U.S. citizens or permanent 

residents, whereas the NIH Visiting Fellow Program, 

administered by the Division of International Services, provides postdoctoral awards to foreign nationals on

J1 visas. To be eligible to apply for either program, 

candidates must hold a doctoral degree or equivalent in 

the health sciences and have no more than 5 years of 

relevant research experience since receipt of their most 

recent doctoral degree. These awards are generally 

granted for 2 years, with 1-year extensions up to 5 years 

based on performance and availability of funds. More 

detailed information about both types of postdoctoral training programs, including a description of current 

The National Cancer Institute, NIH, DHHS, is an Equal Employment Opportunity and Affirmative Action

employer that values and fosters diversity throughout the entire organization.

Training and Employment Opportunities in the HIV Drug Resistance Program

NIH Publication No. 08-6530

Printed October 2009