Breadcrumb

Stephen H. Hughes, Ph.D.

Stephen H. Hughes, Ph.D.

  • Center for Cancer Research
  • National Cancer Institute
HIV Dynamics and Replication Program

RESEARCH SUMMARY

Dr. Hughes is internationally renowned for his work on the roles of reverse transcriptase (RT) and integrase (IN).  He is interested in how HIV becomes resistant to RT and IN inhibitors, in developing drugs that are more effective against the known resistance mutations, and in how the clonal expansion of HIV-infected cells contributes to the formation and persistence of the reservoir that has made it impossible to cure HIV infections with the available drugs.  In his role as NIH Scientist Emeritus, Dr. Hughes interacts with scientists inside and outside of the HIV-DRP, providing advice and mentoring.

Areas of Expertise

HIV Replication
Retroviruses
Antivirals
Reverse Transcription
Integration

Publications

Selected Key Publications

Specific HIV integration sites are linked to clonal expansion and persistence of infected cells

Maldarelli F, Wu X, Su L, Simonetti FR, Shao W, Hill S, Spindler J, Ferris AL, Mellors JW, Kearney MF, Coffin JM, Hughes SH
Science. 345: 179-183, 2014. [ Journal Article ]

Lens epithelium-derived growth factor fusion proteins redirect HIV-1 DNA integration

Ferris AL, Wu X, Hughes CM, Stewart C, Smith SJ, Milne TA, Wang GG, Shun MC, Allis CD, Engelman A, Hughes SH
Proc Natl Acad Sci U S A. 107: 3135-3140, 2010. [ Journal Article ]

Selective excision of AZTMP by drug-resistant human immunodeficiency virus reverse transcriptase

Boyer PL, Sarafianos SG, Arnold E, Hughes SH
J Virol. 75: 4832-4842, 2001.

A system for tissue-specific gene targeting: transgenic mice susceptible to subgroup A avian leukosis virus-based retroviral vectors

Federspiel MJ, Bates P, Young JA, Varmus HE, Hughes SH
Proc Natl Acad Sci U S A. 91: 11241-11245, 1994. [ Journal Article ]

Crystal structure of human immunodeficiency virus type 1 reverse transcriptase complexed with double-stranded DNA at 3.0 Å resolution shows bent DNA

Jacobo-Molina A, Ding J, Nanni RG, Clark AD Jr, Lu X, Tantillo C, Williams RL, Kamer G, Ferris AL, Clark P, Hizi A, Hughes SH, Arnold E
Proc Natl Acad Sci U S A. 90: 6320-6324, 1993.

News

Large HIV Integration Site Datasets Provide Insights into the Stability of Clones of Infected cells and the Persistence of the Reservoir

Although combination antiretroviral therapy (ART) blocks HIV replication, it is not curative because infected CD4+ T cells that carry intact, infectious proviruses persist. Understanding the behavior of clones of infected T cells is important for understanding the stability of the reservoir; however, the stabilities of clones of infected T cells in persons on long-term ART are not well defined. In a paper that was recently published in PLoS Pathogens (HIV infected CD4+ T cell clones are more stable than uninfected clones during long-term antiretroviral therapy. Guo S, Luke BT, Henry AR, Darko S, Brandt LD, Su L, Sun D, Wells D, Joseph KW, Demirov D, Halvas EK, Douek DC, Wu X, Mellors JW, Hughes SH. PLoS Pathog. 2022 Aug 31;18(8):e1010726. doi: 10.1371/journal.ppat.1010726. eCollection 2022 Aug. PMID: 36044447), Dr. Hughes and his colleagues and collaborators determined the relative stabilities of clones of infected and uninfected CD4+ T cells over time intervals of one to four years in three individuals who had been on ART for 9-19 years. Although the number of individuals in this study was relatively small, more than 19,000 integration sites were identified from each of the donors. The large integration site datasets made it possible to accurately compare the sizes of the T cell clones over time.

There are several important conclusions. First, the largest clones of uninfected T cells were larger than the largest clones of infected T cells. There are at least two explanations for this result. First, only a small fraction of the CD4+ T cells are infected. CD4+ T cells can be infected either before or after they have expanded to form a clone. If T cells are infected before they begin to expand, the cells that are infected are unlikely, based on simple probability, to be one of the rare T cells that is destined to become one of the largest T cell clones. Alternatively, if the initial infection happens in a T cell that is part of a clone that has already begun to expand, only a small fraction of the cells in the clone will be infected. Either way, the largest clones of infected cells will be smaller in size than the largest uninfected clones.

Second, clones of infected CD4+ T cells are more stable than clones of uninfected CD4+ T cells of a similar size. Because successful ART blocks new HIV infections, all of the infected clones must have derived from T cells that were infected before ART was initiated. Thus, all the infected clones in those on successful long-term ART are “old”. In order for a clone to survive long enough to become old, it would have had to be relatively stable. In contrast, the clones of infected T cells could have arisen either before or after ART was initiated (which means that the uninfected clones are a mixture of young and old).

Finally, because it is the clones that carry infectious proviruses that contribute to the reservoir, the behavior and stability of these clones are particularly important. Thus far, we only have data on the stability of three large clones of cells that carry infectious proviruses and more data are needed. One of the T cell clones that carried an infectious provirus decreased in size over time. However, in another donor, a large clone that carried an infectious provirus increased in size, becoming, at the last time point, the largest clone of infected cells in that donor. In a donor who had been on therapy for 19 years, a clone that carried an infectious provirus first increased in size and then decreased over about a four-year period. The data show that, although all three of the clones must all have persisted for at least 9 years, “old” clones that carry intact infectious proviruses can still change in size. Importantly, clones of T cell that carry infectious proviruses can still increase in size, perhaps in response to antigenic or homeostatic stimulation, many years after the cell that gave rise to the clone was first infected.

Alumni

Michael Abram, Ph.D.
2005-2010
Postdoctoral Fellow
Aamir Akram, M.D.
2013-2015
Postbaccalaureate Fellow
Eugene Barsov, Ph.D.
1999-2001
Research Fellow
Paul Boyer, Ph.D.
1994-2021
Staff Scientist
Kevin Chang, Ph.D.
2001-2007
Postdoctoral Fellow
Carolyn Crisp, B.S.
2012-2013
Postbaccalaureate Fellow
Caroline Davis, Ph.D.
2007-2012
Postdoctoral Fellow
Mark Driscoll, Ph.D.
1999-2000
Research Fellow
Linda Dunn
1999-2021
Boiologist
Hong-Qiang Gao, Ph.D.
1999-2000
Research Fellow
Marie-Pierre Golinelli, Ph.D.
1999-2001
Staff Scientist
Gunnar Gunnarsson, Ph.D.
2005-2007
Postdoctoral Fellow
Barry Johnson, Ph.D.
2007-2013
Postdoctoral Fellow
Fatima Jones, Ph.D.
2003-2004
Postdoctoral Fellow
John Julias, Ph.D.
1999-2006
Research Fellow
Stanislaw Kaczmarczyk, Ph.D.
2001-2008
Research Fellow
Mary Jane McWilliams, B.A.
1999-2015
Research Biologist
Jangsuk Oh, Ph.D.
2000-2007
Research Fellow
Elena Peletskaya, Ph.D.
1999-2010
Research Fellow, Guest Scientist
Valerian Pinto, Ph.D.
1999-2001
Research Fellow
Carolyn Rinke, Ph.D.
2002
Predoctoral Fellow
Steven Smith, Ph.D.
2008-2021
Research Biologist
Amber Steele, Ph.D.
2002-2003
Postdoctoral Fellow
B. Christie Vu, Ph.D.
2004-2010
Postdoctoral Fellow
Janani Varadarajan, Ph.D.
2007-2013
Postdoctoral Fellow
Daria Wells, M.S.
2014-2021
Bioinformatics Analyst (Contr)
Rafal Wierzchoslawski, Ph.D.
2005-2008
Research Fellow
Edward Chia-Kuei Wu, Ph.D.
2000-2007
Research Fellow

Covers

Inside cover graphic of ACS Infectious Disease Volume 7 Issue 6 June 2021

HIV-1 Integrase Inhibitors with Modifications That Affect Their Potencies against Drug Resistant Integrase Mutants

Published Date

Abstract:

Integrase strand transfer inhibitors (INSTIs) block the integration step of the retroviral lifecycle and are first-line drugs used for the treatment of HIV-1/AIDS.  INSTIs have a polycyclic core with heteroatom triads, chelate the metal ions at the active site, and have a halobenzyl group that interacts with viral DNA attached to the core by a flexible linker.  The most broadly effective INSTIs inhibit both wild-type (WT) integrase (IN) and a variety of well-known mutants.  However, because there are mutations that reduce the potency of all of the available INSTIs, new and better compounds are needed.  Models based on recent structures of HIV-1 and red-capped mangabey SIV INs suggest modifications in the INSTI structures that could enhance interactions with the 3'-terminal adenosine of the viral DNA, which could improve performance against INSTI resistant mutants.  We designed and tested a series of INSTIs having modifications to their naphthyridine scaffold.  One of the new compounds retained good potency against an expanded panel of HIV-1 IN mutants that we tested.  Our results suggest the possibility of designing inhibitors that combine the best features of the existing compounds, which could provide additional efficacy against known HIV-1 IN mutants.

On the cover:

INSTIs bound to HIV-1 IN.  Compound 5j (magenta) was docked onto the structures of 4d (yellow) and BIC (green) bound to the active site of HIV-1 IN; the surface of IN is shown in white.  The surface envelope of the unprocessed 3′ end of the vDNA is shown as brown mesh.  Ordered water molecules bound to the active site of IN in the absence of a bound INSTI are shown in cyan.

Cover graphic of Journal of Medicinal Chemistry Volume 60 Issue 17 September 14, 2017

Structure-Guided Optimization of HIV Integrase Strand Transfer Inhibitors

Published Date

Abstract:

Integrase mutations can reduce the effectiveness of the first-generation FDA-approved integrase strand transfer inhibitors (INSTIs), raltegravir (RAL) and elvitegravir (EVG).  The second-generation agent, dolutegravir (DTG), has enjoyed considerable clinical success; however, resistance-causing mutations that diminish the efficacy of DTG have appeared.  Our current findings support and extend the substrate envelope concept that broadly effective INSTIs can be designed by filling the envelope defined by the DNA substrates.  Previously, we explored 1-hydroxy-2-oxo-1,2-dihydro-1,8-naphthyridine-3-carboxamides as an INSTI scaffold, making a limited set of derivatives, and concluded that broadly effective INSTIs can be developed using this scaffold.  Herein, we report an extended investigation of 6-substituents as well the first examples of 7-substituted analogues of this scaffold.  While 7-substituents are not well-tolerated, we have identified novel substituents at the 6-position that are highly effective, with the best compound (6p) retaining better efficacy against a broad panel of known INSTI resistant mutants than any analogues we have previously described.

On the cover:

Shown is an HIV integrase strand transfer inhibitor possessing a good mutant profile, bound at the catalytic site of the prototype foamy virus intasome including metal cofactors and viral DNA.

Cover design by Joseph Myer

Cover graphic of ACS Chemical Biology Volume 11 Issue 4 April 1, 2016

HIV‑1 Integrase Strand Transfer Inhibitors with Reduced Susceptibility to Drug Resistant Mutant Integrases

Published Date

Abstract:

HIV integrase (IN) strand transfer inhibitors (INSTIs) are among the newest anti-AIDS drugs; however, mutant forms of IN can confer resistance.  We developed noncytotoxic naphthyridine-containing INSTIs that retain low nanomolar IC50 values against HIV-1 variants harboring all of the major INSTI-resistant mutations.  We found by analyzing crystal structures of inhibitors bound to the IN from the prototype foamy virus (PFV) that the most successful inhibitors show striking mimicry of the bound viral DNA prior to 3'-processing and the bound host DNA prior to strand transfer.  Using this concept of "bi-substrate mimicry," we developed a new broadly effective inhibitor that not only mimics aspects of both the bound target and viral DNA but also more completely fills the space they would normally occupy.  Maximizing shape complementarity and recapitulating structural components encompassing both of the IN DNA substrates could serve as a guiding principle for the development of new INSTIs.

On the cover:

Mutant forms of HIV-1 IN reduce the therapeutic effectiveness of integrase strand transfer inhibitors (INSTIs).  The cover figure shows the IN of prototype foamy virus complexed to a novel INSTI (gold) that retains potency against resistant mutants of HIV-1 IN.  Overlain are the host and viral DNA substrates (blue and green, respectively), showing substrate mimicry by the inhibitor.

Cover design by Joseph Myer