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Stephen H. Hughes
HIV Dynamics and Replication Program, Center for Cancer Research, National Cancer Institute, Frederick, Maryland, United States of America

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Article
Published: 17 August 2021 in Nature Communications
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Between 10 and 20 million people worldwide are infected with the human T-cell lymphotropic virus type 1 (HTLV-1). Despite causing life-threatening pathologies there is no therapeutic regimen for this deltaretrovirus. Here, we screened a library of integrase strand transfer inhibitor (INSTI) candidates built around several chemical scaffolds to determine their effectiveness in limiting HTLV-1 infection. Naphthyridines with substituents in position 6 emerged as the most potent compounds against HTLV-1, with XZ450 having highest efficacy in vitro. Using single-particle cryo-electron microscopy we visualised XZ450 as well as the clinical HIV-1 INSTIs raltegravir and bictegravir bound to the active site of the deltaretroviral intasome. The structures reveal subtle differences in the coordination environment of the Mg2+ ion pair involved in the interaction with the INSTIs. Our results elucidate the binding of INSTIs to the HTLV-1 intasome and support their use for pre-exposure prophylaxis and possibly future treatment of HTLV-1 infection.

ACS Style

Michal S. Barski; Teresa Vanzo; Xue Zhi Zhao; Steven J. Smith; Allison Ballandras-Colas; Nora B. Cronin; Valerie E. Pye; Stephen H. Hughes; Terrence R. Burke; Peter Cherepanov; Goedele N. Maertens. Structural basis for the inhibition of HTLV-1 integration inferred from cryo-EM deltaretroviral intasome structures. Nature Communications 2021, 12, 1 -10.

AMA Style

Michal S. Barski, Teresa Vanzo, Xue Zhi Zhao, Steven J. Smith, Allison Ballandras-Colas, Nora B. Cronin, Valerie E. Pye, Stephen H. Hughes, Terrence R. Burke, Peter Cherepanov, Goedele N. Maertens. Structural basis for the inhibition of HTLV-1 integration inferred from cryo-EM deltaretroviral intasome structures. Nature Communications. 2021; 12 (1):1-10.

Chicago/Turabian Style

Michal S. Barski; Teresa Vanzo; Xue Zhi Zhao; Steven J. Smith; Allison Ballandras-Colas; Nora B. Cronin; Valerie E. Pye; Stephen H. Hughes; Terrence R. Burke; Peter Cherepanov; Goedele N. Maertens. 2021. "Structural basis for the inhibition of HTLV-1 integration inferred from cryo-EM deltaretroviral intasome structures." Nature Communications 12, no. 1: 1-10.

Reply
Published: 03 August 2021 in Proceedings of the National Academy of Sciences
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ACS Style

Liguo Zhang; Alexsia Richards; M. Inmaculada Barrasa; Stephen H. Hughes; Richard A. Young; Rudolf Jaenisch. Response to Parry et al.: Strong evidence for genomic integration of SARS-CoV-2 sequences and expression in patient tissues. Proceedings of the National Academy of Sciences 2021, 118, 1 .

AMA Style

Liguo Zhang, Alexsia Richards, M. Inmaculada Barrasa, Stephen H. Hughes, Richard A. Young, Rudolf Jaenisch. Response to Parry et al.: Strong evidence for genomic integration of SARS-CoV-2 sequences and expression in patient tissues. Proceedings of the National Academy of Sciences. 2021; 118 (33):1.

Chicago/Turabian Style

Liguo Zhang; Alexsia Richards; M. Inmaculada Barrasa; Stephen H. Hughes; Richard A. Young; Rudolf Jaenisch. 2021. "Response to Parry et al.: Strong evidence for genomic integration of SARS-CoV-2 sequences and expression in patient tissues." Proceedings of the National Academy of Sciences 118, no. 33: 1.

Oncology
Published: 03 August 2021 in PLoS Pathogens
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MmuPV1 is a useful model for studying papillomavirus-induced tumorigenesis. We used RNA-seq to look for chimeric RNAs that map to both MmuPV1 and host genomes. In tumor tissues, a higher proportion of total viral reads were virus-host chimeric junction reads (CJRs) (1.9‰ - 7‰) than in tumor-free tissues (0.6‰ - 1.3‰): most CJRs mapped to the viral E2/E4 region. Although most of the MmuPV1 integration sites were mapped to intergenic regions and introns throughout the mouse genome, integrations were seen more than once in several genes: Malat1, Krt1, Krt10, Fabp5, Pard3, and Grip1; these data were confirmed by rapid amplification of cDNA ends (RACE)-Single Molecule Real-Time (SMRT)-seq or targeted DNA-seq. Microhomology sequences were frequently seen at host-virus DNA junctions. MmuPV1 infection and integration affected the expression of host genes. We found that factors for DNA double-stranded break repair and microhomology-mediated end-joining (MMEJ), such as H2ax, Fen1, DNA polymerase Polθ, Cdk1, and Plk1, exhibited a step-wise increase and Mdc1 a decrease in expression in MmuPV1-infected tissues and MmuPV1 tumors relative to normal tissues. Increased expression of mitotic kinases CDK1 and PLK1 appears to be correlated with CtIP phosphorylation in MmuPV1 tumors, suggesting a role for MMEJ-mediated DNA joining in the MmuPV1 integration events that are associated with MmuPV1-induced progression of tumors.

ACS Style

Lulu Yu; Vladimir Majerciak; Xiang-Yang Xue; Aayushi Uberoi; Alexei Lobanov; Xiongfong Chen; Maggie Cam; Stephen H. Hughes; Paul F. Lambert; Zhi-Ming Zheng. Mouse papillomavirus type 1 (MmuPV1) DNA is frequently integrated in benign tumors by microhomology-mediated end-joining. PLoS Pathogens 2021, 17, 1 .

AMA Style

Lulu Yu, Vladimir Majerciak, Xiang-Yang Xue, Aayushi Uberoi, Alexei Lobanov, Xiongfong Chen, Maggie Cam, Stephen H. Hughes, Paul F. Lambert, Zhi-Ming Zheng. Mouse papillomavirus type 1 (MmuPV1) DNA is frequently integrated in benign tumors by microhomology-mediated end-joining. PLoS Pathogens. 2021; 17 (8):1.

Chicago/Turabian Style

Lulu Yu; Vladimir Majerciak; Xiang-Yang Xue; Aayushi Uberoi; Alexei Lobanov; Xiongfong Chen; Maggie Cam; Stephen H. Hughes; Paul F. Lambert; Zhi-Ming Zheng. 2021. "Mouse papillomavirus type 1 (MmuPV1) DNA is frequently integrated in benign tumors by microhomology-mediated end-joining." PLoS Pathogens 17, no. 8: 1.

Journal article
Published: 29 July 2021 in Viruses
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In most cases, proteolytic processing of the retroviral Pol portion of the Gag-Pol polyprotein precursor produces protease (PR), reverse transcriptase (RT), and integrase (IN). However, foamy viruses (FVs) express Pol separately from Gag and, when Pol is processed, only the IN domain is released. Here, we report a 2.9 Å resolution crystal structure of the mature PR-RT from prototype FV (PFV) that can carry out both proteolytic processing and reverse transcription but is in a configuration not competent for proteolytic or polymerase activity. PFV PR-RT is monomeric and the architecture of PFV PR is similar to one of the subunits of HIV-1 PR, which is a dimer. There is a C-terminal extension of PFV PR (101-145) that consists of two helices which are adjacent to the base of the RT palm subdomain, and anchors PR to RT. The polymerase domain of PFV RT consists of fingers, palm, thumb, and connection subdomains whose spatial arrangements are similar to the p51 subunit of HIV-1 RT. The RNase H and polymerase domains of PFV RT are connected by flexible linkers. Significant spatial and conformational (sub)domain rearrangements are therefore required for nucleic acid binding. The structure of PFV PR-RT provides insights into the conformational maturation of retroviral Pol polyproteins.

ACS Style

Jerry Harrison; Steve Tuske; Kalyan Das; Francesc Ruiz; Joseph Bauman; Paul Boyer; Jeffrey DeStefano; Stephen Hughes; Eddy Arnold. Crystal Structure of a Retroviral Polyprotein: Prototype Foamy Virus Protease-Reverse Transcriptase (PR-RT). Viruses 2021, 13, 1495 .

AMA Style

Jerry Harrison, Steve Tuske, Kalyan Das, Francesc Ruiz, Joseph Bauman, Paul Boyer, Jeffrey DeStefano, Stephen Hughes, Eddy Arnold. Crystal Structure of a Retroviral Polyprotein: Prototype Foamy Virus Protease-Reverse Transcriptase (PR-RT). Viruses. 2021; 13 (8):1495.

Chicago/Turabian Style

Jerry Harrison; Steve Tuske; Kalyan Das; Francesc Ruiz; Joseph Bauman; Paul Boyer; Jeffrey DeStefano; Stephen Hughes; Eddy Arnold. 2021. "Crystal Structure of a Retroviral Polyprotein: Prototype Foamy Virus Protease-Reverse Transcriptase (PR-RT)." Viruses 13, no. 8: 1495.

Journal article
Published: 25 June 2021 in Viruses
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Efforts to cure HIV-1 infection require better quantification of the HIV-1 reservoir, particularly the clones of cells harboring replication-competent (intact) proviruses, termed repliclones. The digital droplet PCR assays commonly used to quantify intact proviruses do not differentiate among specific repliclones, thus the dynamics of repliclones are not well defined. The major challenge in tracking repliclones is the relative rarity of the cells carrying specific intact proviruses. To date, detection and accurate quantification of repliclones requires in-depth integration site sequencing. Here, we describe a simplified workflow using integration site-specific qPCR (IS-qPCR) to determine the frequencies of the proviruses integrated in individual repliclones. We designed IS-qPCR to determine the frequencies of repliclones and clones of cells that carry defective proviruses in samples from three donors. Comparing the results of IS-qPCR with deep integration site sequencing data showed that the two methods yielded concordant estimates of clone frequencies (r = 0.838). IS-qPCR is a potentially valuable tool that can be applied to multiple samples and cell types over time to measure the dynamics of individual repliclones and the efficacy of treatments designed to eliminate them.

ACS Style

Leah Brandt; Shuang Guo; Kevin Joseph; Jana Jacobs; Asma Naqvi; John Coffin; Mary Kearney; Elias Halvas; Xiaolin Wu; Stephen Hughes; John Mellors. Tracking HIV-1-Infected Cell Clones Using Integration Site-Specific qPCR. Viruses 2021, 13, 1235 .

AMA Style

Leah Brandt, Shuang Guo, Kevin Joseph, Jana Jacobs, Asma Naqvi, John Coffin, Mary Kearney, Elias Halvas, Xiaolin Wu, Stephen Hughes, John Mellors. Tracking HIV-1-Infected Cell Clones Using Integration Site-Specific qPCR. Viruses. 2021; 13 (7):1235.

Chicago/Turabian Style

Leah Brandt; Shuang Guo; Kevin Joseph; Jana Jacobs; Asma Naqvi; John Coffin; Mary Kearney; Elias Halvas; Xiaolin Wu; Stephen Hughes; John Mellors. 2021. "Tracking HIV-1-Infected Cell Clones Using Integration Site-Specific qPCR." Viruses 13, no. 7: 1235.

Journal article
Published: 06 May 2021 in Proceedings of the National Academy of Sciences
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Prolonged detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA and recurrence of PCR-positive tests have been widely reported in patients after recovery from COVID-19, but some of these patients do not appear to shed infectious virus. We investigated the possibility that SARS-CoV-2 RNAs can be reverse-transcribed and integrated into the DNA of human cells in culture and that transcription of the integrated sequences might account for some of the positive PCR tests seen in patients. In support of this hypothesis, we found that DNA copies of SARS-CoV-2 sequences can be integrated into the genome of infected human cells. We found target site duplications flanking the viral sequences and consensus LINE1 endonuclease recognition sequences at the integration sites, consistent with a LINE1 retrotransposon-mediated, target-primed reverse transcription and retroposition mechanism. We also found, in some patient-derived tissues, evidence suggesting that a large fraction of the viral sequences is transcribed from integrated DNA copies of viral sequences, generating viral–host chimeric transcripts. The integration and transcription of viral sequences may thus contribute to the detection of viral RNA by PCR in patients after infection and clinical recovery. Because we have detected only subgenomic sequences derived mainly from the 3′ end of the viral genome integrated into the DNA of the host cell, infectious virus cannot be produced from the integrated subgenomic SARS-CoV-2 sequences.

ACS Style

Liguo Zhang; Alexsia Richards; M. Inmaculada Barrasa; Stephen H. Hughes; Richard A. Young; Rudolf Jaenisch. Reverse-transcribed SARS-CoV-2 RNA can integrate into the genome of cultured human cells and can be expressed in patient-derived tissues. Proceedings of the National Academy of Sciences 2021, 118, 1 .

AMA Style

Liguo Zhang, Alexsia Richards, M. Inmaculada Barrasa, Stephen H. Hughes, Richard A. Young, Rudolf Jaenisch. Reverse-transcribed SARS-CoV-2 RNA can integrate into the genome of cultured human cells and can be expressed in patient-derived tissues. Proceedings of the National Academy of Sciences. 2021; 118 (21):1.

Chicago/Turabian Style

Liguo Zhang; Alexsia Richards; M. Inmaculada Barrasa; Stephen H. Hughes; Richard A. Young; Rudolf Jaenisch. 2021. "Reverse-transcribed SARS-CoV-2 RNA can integrate into the genome of cultured human cells and can be expressed in patient-derived tissues." Proceedings of the National Academy of Sciences 118, no. 21: 1.

Journal article
Published: 27 April 2021 in mBio
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HIV-1 integrates its genome into the DNA of host cells. Consequently, HIV-1 genomes are copied with the host cell DNA during cellular division.

ACS Style

Michael J. Bale; Mary Grace Katusiime; Daria Wells; Xiaolin Wu; Jonathan Spindler; Elias K. Halvas; Joshua C. Cyktor; Ann Wiegand; Wei Shao; Mark F. Cotton; Stephen H. Hughes; John W. Mellors; John M. Coffin; Gert U. Van Zyl; Mary F. Kearney. Early Emergence and Long-Term Persistence of HIV-Infected T-Cell Clones in Children. mBio 2021, 12, 1 .

AMA Style

Michael J. Bale, Mary Grace Katusiime, Daria Wells, Xiaolin Wu, Jonathan Spindler, Elias K. Halvas, Joshua C. Cyktor, Ann Wiegand, Wei Shao, Mark F. Cotton, Stephen H. Hughes, John W. Mellors, John M. Coffin, Gert U. Van Zyl, Mary F. Kearney. Early Emergence and Long-Term Persistence of HIV-Infected T-Cell Clones in Children. mBio. 2021; 12 (2):1.

Chicago/Turabian Style

Michael J. Bale; Mary Grace Katusiime; Daria Wells; Xiaolin Wu; Jonathan Spindler; Elias K. Halvas; Joshua C. Cyktor; Ann Wiegand; Wei Shao; Mark F. Cotton; Stephen H. Hughes; John W. Mellors; John M. Coffin; Gert U. Van Zyl; Mary F. Kearney. 2021. "Early Emergence and Long-Term Persistence of HIV-Infected T-Cell Clones in Children." mBio 12, no. 2: 1.

Research article
Published: 07 April 2021 in PLOS Pathogens
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HIV persists during antiretroviral therapy (ART) as integrated proviruses in cells descended from a small fraction of the CD4+ T cells infected prior to the initiation of ART. To better understand what controls HIV persistence and the distribution of integration sites (IS), we compared about 15,000 and 54,000 IS from individuals pre-ART and on ART, respectively, with approximately 395,000 IS from PBMC infected in vitro. The distribution of IS in vivo is quite similar to the distribution in PBMC, but modified by selection against proviruses in expressed genes, by selection for proviruses integrated into one of 7 specific genes, and by clonal expansion. Clones in which a provirus integrated in an oncogene contributed to cell survival comprised only a small fraction of the clones persisting in on ART. Mechanisms that do not involve the provirus, or its location in the host genome, are more important in determining which clones expand and persist.

ACS Style

John M. Coffin; Michael J. Bale; Daria Wells; Shuang Guo; Brian Luke; Jennifer M. Zerbato; Michele D. Sobolewski; Twan Sia; Wei Shao; Xiaolin Wu; Frank Maldarelli; Mary F. Kearney; John W. Mellors; Stephen H. Hughes. Integration in oncogenes plays only a minor role in determining the in vivo distribution of HIV integration sites before or during suppressive antiretroviral therapy. PLOS Pathogens 2021, 17, e1009141 .

AMA Style

John M. Coffin, Michael J. Bale, Daria Wells, Shuang Guo, Brian Luke, Jennifer M. Zerbato, Michele D. Sobolewski, Twan Sia, Wei Shao, Xiaolin Wu, Frank Maldarelli, Mary F. Kearney, John W. Mellors, Stephen H. Hughes. Integration in oncogenes plays only a minor role in determining the in vivo distribution of HIV integration sites before or during suppressive antiretroviral therapy. PLOS Pathogens. 2021; 17 (4):e1009141.

Chicago/Turabian Style

John M. Coffin; Michael J. Bale; Daria Wells; Shuang Guo; Brian Luke; Jennifer M. Zerbato; Michele D. Sobolewski; Twan Sia; Wei Shao; Xiaolin Wu; Frank Maldarelli; Mary F. Kearney; John W. Mellors; Stephen H. Hughes. 2021. "Integration in oncogenes plays only a minor role in determining the in vivo distribution of HIV integration sites before or during suppressive antiretroviral therapy." PLOS Pathogens 17, no. 4: e1009141.

Research article
Published: 09 March 2021 in ACS Infectious Diseases
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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.

ACS Style

Steven J. Smith; Xue Zhi Zhao; Dario Oliveira Passos; Valerie E. Pye; Peter Cherepanov; Dmitry Lyumkis; Terrence R. Burke Jr.; Stephen H. Hughes. HIV-1 Integrase Inhibitors with Modifications That Affect Their Potencies against Drug Resistant Integrase Mutants. ACS Infectious Diseases 2021, 7, 1469 -1482.

AMA Style

Steven J. Smith, Xue Zhi Zhao, Dario Oliveira Passos, Valerie E. Pye, Peter Cherepanov, Dmitry Lyumkis, Terrence R. Burke Jr., Stephen H. Hughes. HIV-1 Integrase Inhibitors with Modifications That Affect Their Potencies against Drug Resistant Integrase Mutants. ACS Infectious Diseases. 2021; 7 (6):1469-1482.

Chicago/Turabian Style

Steven J. Smith; Xue Zhi Zhao; Dario Oliveira Passos; Valerie E. Pye; Peter Cherepanov; Dmitry Lyumkis; Terrence R. Burke Jr.; Stephen H. Hughes. 2021. "HIV-1 Integrase Inhibitors with Modifications That Affect Their Potencies against Drug Resistant Integrase Mutants." ACS Infectious Diseases 7, no. 6: 1469-1482.

Review
Published: 29 January 2021 in Viruses
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Integrase strand transfer inhibitors (INSTIs) are currently recommended for the first line treatment of human immunodeficiency virus type one (HIV-1) infection. The first-generation INSTIs are effective but can select for resistant viruses. Recent advances have led to several potent second-generation INSTIs that are effective against both wild-type (WT) HIV-1 integrase and many of the first-generation INSTI-resistant mutants. The emergence of resistance to these new second-generation INSTIs has been minimal, which has resulted in alternative treatment strategies for HIV-1 patients. Moreover, because of their high antiviral potencies and, in some cases, their bioavailability profiles, INSTIs will probably have prominent roles in pre-exposure prophylaxis (PrEP). Herein, we review the current state of the clinically relevant INSTIs and discuss the future outlook for this class of antiretrovirals.

ACS Style

Steven J. Smith; Xue Zhi Zhao; Dario Oliveira Passos; Dmitry Lyumkis; Terrence R. Burke; Stephen H. Hughes. Integrase Strand Transfer Inhibitors Are Effective Anti-HIV Drugs. Viruses 2021, 13, 205 .

AMA Style

Steven J. Smith, Xue Zhi Zhao, Dario Oliveira Passos, Dmitry Lyumkis, Terrence R. Burke, Stephen H. Hughes. Integrase Strand Transfer Inhibitors Are Effective Anti-HIV Drugs. Viruses. 2021; 13 (2):205.

Chicago/Turabian Style

Steven J. Smith; Xue Zhi Zhao; Dario Oliveira Passos; Dmitry Lyumkis; Terrence R. Burke; Stephen H. Hughes. 2021. "Integrase Strand Transfer Inhibitors Are Effective Anti-HIV Drugs." Viruses 13, no. 2: 205.

Preprint content
Published: 26 November 2020
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HIV persists during antiretroviral therapy (ART) as integrated proviruses in cells descended from a small fraction of the CD4+ T cells infected prior to the initiation of ART. To better understand what controls HIV persistence and the distribution of integration sites (IS), we compared about 16,000 and 54,000 IS from individuals pre-ART and on ART, respectively, with approximately 385,000 IS from PBMC infected in vitro. The distribution of IS in vivo is quite similar to the distribution in PBMC, modified by selection against proviruses in expressed genes, by selection for proviruses integrated into one of 6 specific genes, and by clonal expansion. The clones in which a provirus integrated in an oncogene contributed to the survival of the clone comprise only a small fraction of the clones that persist in HIV-infected individuals on ART. Mechanisms that do not involve the provirus, or its location in the host genome, are more important in determining which clones expand and persist.Author SummaryIn HIV-infected individuals, a small fraction of the infected cells persist and divide. This reservoir persists on ART and can rekindle the infection if ART is discontinued. Because the number of possible sites of HIV DNA integration is very large, each infected cell, and all of its descendants, can be identified by the site where the provirus is integrated (IS). To understand the selective forces that determine the fates of infected cells in vivo, we compared the distribution of HIV IS in freshly-infected cells to cells from HIV-infected donors sampled both before and during ART. We found that, as has been previously reported, integration favors highly-expressed genes. However, over time the fraction of cells with proviruses integrated in highly-expressed genes decreases, implying that they grow less well. There are exceptions to this broad negative selection. When a provirus is integrated in a specific region in one of six genes, the proviruses affect the expression of the target gene, promoting growth and/or survival of the cell. Although this effect is striking, it is only a minor component of the forces that promote the growth and survival of the population of infected cells during ART.

ACS Style

John M. Coffin; Michael J. Bale; Daria Wells; Shuang Guo; Brian Luke; Jennifer M. Zerbato; Michele D. Sobolewskii; Twan Sia; Wei Shao; Xiaolin Wu; Frank Maldarelli; Mary F. Kearney; John W. Mellors; Stephen H. Hughes. Integration in or Near Oncogenes Plays Only a Minor Role in Determining the in Vivo Distribution of HIV Integration Sites Before or During Suppressive Antiretroviral Therapy. 2020, 1 .

AMA Style

John M. Coffin, Michael J. Bale, Daria Wells, Shuang Guo, Brian Luke, Jennifer M. Zerbato, Michele D. Sobolewskii, Twan Sia, Wei Shao, Xiaolin Wu, Frank Maldarelli, Mary F. Kearney, John W. Mellors, Stephen H. Hughes. Integration in or Near Oncogenes Plays Only a Minor Role in Determining the in Vivo Distribution of HIV Integration Sites Before or During Suppressive Antiretroviral Therapy. . 2020; ():1.

Chicago/Turabian Style

John M. Coffin; Michael J. Bale; Daria Wells; Shuang Guo; Brian Luke; Jennifer M. Zerbato; Michele D. Sobolewskii; Twan Sia; Wei Shao; Xiaolin Wu; Frank Maldarelli; Mary F. Kearney; John W. Mellors; Stephen H. Hughes. 2020. "Integration in or Near Oncogenes Plays Only a Minor Role in Determining the in Vivo Distribution of HIV Integration Sites Before or During Suppressive Antiretroviral Therapy." , no. : 1.

Journal article
Published: 05 October 2020 in Journal of Clinical Investigation
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BACKGROUNDHIV-1 viremia that is not suppressed by combination antiretroviral therapy (ART) is generally attributed to incomplete medication adherence and/or drug resistance. We evaluated individuals referred by clinicians for nonsuppressible viremia (plasma HIV-1 RNA above 40 copies/mL) despite reported adherence to ART and the absence of drug resistance to the current ART regimen.METHODSSamples were collected from at least 2 time points from 8 donors who had nonsuppressible viremia for more than 6 months. Single templates of HIV-1 RNA obtained from plasma and viral outgrowth of cultured cells and from proviral DNA were amplified by PCR and sequenced for evidence of clones of cells that produced infectious viruses. Clones were confirmed by host-proviral integration site analysis.RESULTSHIV-1 genomic RNA with identical sequences were identified in plasma samples from all 8 donors. The identical viral RNA sequences did not change over time and did not evolve resistance to the ART regimen. In 4 of the donors, viral RNA sequences obtained from plasma matched those sequences from viral outgrowth cultures, indicating that the viruses were replication competent. Integration sites for infectious proviruses from those 4 donors were mapped to the introns of the MATR3, ZNF268, ZNF721/ABCA11P, and ABCA11P genes. The sizes of the clones were estimated to be from 50 million to 350 million cells.CONCLUSIONThese findings show that clones of HIV-1-infected cells producing virus can cause failure of ART to suppress viremia. The mechanisms involved in clonal expansion and persistence need to be defined to effectively target viremia and the HIV-1 reservoir.FUNDINGNational Cancer Institute, NIH; Howard Hughes Medical Research Fellows Program, Howard Hughes Medical Institute; Bill and Melinda Gates Foundation; Office of AIDS Research; American Cancer Society; National Cancer Institute through a Leidos subcontract; National Institute for Allergy and Infectious Diseases, NIH, to the I4C Martin Delaney Collaboratory; University of Rochester Center for AIDS Research and University of Rochester HIV/AIDS Clinical Trials Unit.

ACS Style

Elias K. Halvas; Kevin W. Joseph; Leah D. Brandt; Shuang Guo; Michele D. Sobolewski; Jana L. Jacobs; Camille Tumiotto; John K. Bui; Joshua C. Cyktor; Brandon F. Keele; Gene D. Morse; Michael J. Bale; Wei Shao; Mary F. Kearney; John M. Coffin; Jason W. Rausch; Xiaolin Wu; Stephen H. Hughes; John W. Mellors. HIV-1 viremia not suppressible by antiretroviral therapy can originate from large T cell clones producing infectious virus. Journal of Clinical Investigation 2020, 130, 5847 -5857.

AMA Style

Elias K. Halvas, Kevin W. Joseph, Leah D. Brandt, Shuang Guo, Michele D. Sobolewski, Jana L. Jacobs, Camille Tumiotto, John K. Bui, Joshua C. Cyktor, Brandon F. Keele, Gene D. Morse, Michael J. Bale, Wei Shao, Mary F. Kearney, John M. Coffin, Jason W. Rausch, Xiaolin Wu, Stephen H. Hughes, John W. Mellors. HIV-1 viremia not suppressible by antiretroviral therapy can originate from large T cell clones producing infectious virus. Journal of Clinical Investigation. 2020; 130 (11):5847-5857.

Chicago/Turabian Style

Elias K. Halvas; Kevin W. Joseph; Leah D. Brandt; Shuang Guo; Michele D. Sobolewski; Jana L. Jacobs; Camille Tumiotto; John K. Bui; Joshua C. Cyktor; Brandon F. Keele; Gene D. Morse; Michael J. Bale; Wei Shao; Mary F. Kearney; John M. Coffin; Jason W. Rausch; Xiaolin Wu; Stephen H. Hughes; John W. Mellors. 2020. "HIV-1 viremia not suppressible by antiretroviral therapy can originate from large T cell clones producing infectious virus." Journal of Clinical Investigation 130, no. 11: 5847-5857.

Journal article
Published: 20 August 2020 in Antimicrobial Agents and Chemotherapy
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The currently recommended first-line therapy for HIV-1-infected patients is an integrase (IN) strand transfer inhibitor (INSTI), either dolutegravir (DTG) or bictegravir (BIC), in combination with two nucleoside reverse transcriptase inhibitors (NRTIs). Both DTG and BIC potently inhibit most INSTI-resistant IN mutants selected by the INSTIs raltegravir (RAL) and elvitegravir (EVG). BIC has not been reported to select for resistance in treatment-naive patients, and DTG has selected for a small number of resistant viruses in treatment-naive patients.

ACS Style

Steven J. Smith; Xue Zhi Zhao; Dario Oliveira Passos; Dmitry Lyumkis; Terrence R. Burke; Stephen H. Hughes. HIV-1 Integrase Inhibitors That Are Active against Drug-Resistant Integrase Mutants. Antimicrobial Agents and Chemotherapy 2020, 64, 1 .

AMA Style

Steven J. Smith, Xue Zhi Zhao, Dario Oliveira Passos, Dmitry Lyumkis, Terrence R. Burke, Stephen H. Hughes. HIV-1 Integrase Inhibitors That Are Active against Drug-Resistant Integrase Mutants. Antimicrobial Agents and Chemotherapy. 2020; 64 (9):1.

Chicago/Turabian Style

Steven J. Smith; Xue Zhi Zhao; Dario Oliveira Passos; Dmitry Lyumkis; Terrence R. Burke; Stephen H. Hughes. 2020. "HIV-1 Integrase Inhibitors That Are Active against Drug-Resistant Integrase Mutants." Antimicrobial Agents and Chemotherapy 64, no. 9: 1.

Research article
Published: 02 August 2020 in Chemical Biology & Drug Design
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Non‐nucleoside reverse transcriptase inhibitors (NNRTIs) inhibit reverse transcription and block the replication of HIV‐1. Currently, NNRTIs are usually used as part of a three‐drug combination given to patients as antiretroviral therapy. These combinations involve other classes of anti‐HIV‐1 drugs, commonly nucleoside reverse transcriptase inhibitors (NRTIs). However, attempts are being made to develop two‐drug maintenance therapies, some of which involve an NNRTI and an integrase strand transfer inhibitor. This has led to a renewed interest in developing novel NNRTIs, with a major emphasis on designing compounds that can effectively inhibit the known NNRTI resistant mutants. We have generated and tested novel rilpivirine (RPV) analogs. The new compounds were designed to exploit a small opening in the upper right periphery of the NNRTI binding pocket. The best of the new compounds, 12 , was a more potent inhibitor of the NNRTI‐resistant mutants we tested than either doravirine or efavirenz but was inferior to RPV. We describe the limitations on the modifications that can be appended to the “upper right side” of the RPV core and the effects of substituting other cores for the central pyrimidine core of RPV and make suggestions about how this information can be used in NNRTI design.

ACS Style

Steven J. Smith; Gary Pauly; Katharine Hewlett; Joel Schneider; Stephen H. Hughes. Structure‐based non‐nucleoside inhibitor design: Developing inhibitors that are effective against resistant mutants. Chemical Biology & Drug Design 2020, 97, 4 -17.

AMA Style

Steven J. Smith, Gary Pauly, Katharine Hewlett, Joel Schneider, Stephen H. Hughes. Structure‐based non‐nucleoside inhibitor design: Developing inhibitors that are effective against resistant mutants. Chemical Biology & Drug Design. 2020; 97 (1):4-17.

Chicago/Turabian Style

Steven J. Smith; Gary Pauly; Katharine Hewlett; Joel Schneider; Stephen H. Hughes. 2020. "Structure‐based non‐nucleoside inhibitor design: Developing inhibitors that are effective against resistant mutants." Chemical Biology & Drug Design 97, no. 1: 4-17.

Preprint content
Published: 31 January 2020
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BACKGROUND HIV-1 viremia that is not suppressed by combination antiretroviral therapy (ART) is generally attributed to incomplete medication adherence and/or drug resistance. We evaluated individuals referred for non-suppressible viremia (plasma HIV-1 RNA above 40 copies/ml) who reported adherence to ART and did not show drug resistance to their current regimen. METHODS Samples were collected from at least two time points from eight donors who had non-suppressible viremia for more than six months on ART. Single templates of HIV-1 RNA obtained from plasma and viral outgrowth of cultured cells and from proviral DNA were PCR-amplified and sequenced for evidence of clones of cells that produced infectious viruses. Clones were identified by host-proviral integration site analysis. RESULTS HIV-1 genomic RNAs with identical sequences were identified in plasma samples from all eight donors. The identical viral RNA sequences did not change over time and lacked resistance to the ART regimen. In four of the donors, viral RNA sequences obtained from plasma matched those sequences from viral outgrowth cultures, indicating that the viruses were replication-competent. Integration sites for infectious proviruses from those four donors were mapped to introns of theMATR3,ZNF268,ZNF721/ABCA11P, andABCA11Pgenes. The sizes of the clones were from 50 million to 350 million cells. CONCLUSION Clones of HIV-1-infected cells producing virus can cause failure of ART to suppress viremia despite medication adherence and absence of drug resistance. The mechanisms involved in clonal expansion and persistence need to be defined to eliminate viremia and the HIV-1 reservoir.

ACS Style

Elias K. Halvas; Kevin W. Joseph; Leah D. Brandt; Shuang Guo; Michele D. Sobolewski; Jana L. Jacobs; Camille Tumiotto; John K. Bui; Joshua C. Cyktor; Brandon F. Keele; Gene D. Morse; Michael J. Bale; Mary F. Kearney; John M. Coffin; Jason W. Rausch; Xiaolin Wu; Stephen H. Hughes; John W. Mellors. HIV-1 Viremia Not Suppressible By Antiretroviral Therapy Can Originate from Large T-Cell Clones Producing Infectious Virus. 2020, 1 .

AMA Style

Elias K. Halvas, Kevin W. Joseph, Leah D. Brandt, Shuang Guo, Michele D. Sobolewski, Jana L. Jacobs, Camille Tumiotto, John K. Bui, Joshua C. Cyktor, Brandon F. Keele, Gene D. Morse, Michael J. Bale, Mary F. Kearney, John M. Coffin, Jason W. Rausch, Xiaolin Wu, Stephen H. Hughes, John W. Mellors. HIV-1 Viremia Not Suppressible By Antiretroviral Therapy Can Originate from Large T-Cell Clones Producing Infectious Virus. . 2020; ():1.

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Elias K. Halvas; Kevin W. Joseph; Leah D. Brandt; Shuang Guo; Michele D. Sobolewski; Jana L. Jacobs; Camille Tumiotto; John K. Bui; Joshua C. Cyktor; Brandon F. Keele; Gene D. Morse; Michael J. Bale; Mary F. Kearney; John M. Coffin; Jason W. Rausch; Xiaolin Wu; Stephen H. Hughes; John W. Mellors. 2020. "HIV-1 Viremia Not Suppressible By Antiretroviral Therapy Can Originate from Large T-Cell Clones Producing Infectious Virus." , no. : 1.

Journal article
Published: 25 January 2020 in Viruses
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Combination antiretroviral therapy (cART) controls but does not eradicate HIV infection; HIV persistence is the principal obstacle to curing infections. The proportion of defective proviruses increases during cART, but the dynamics of this process are not well understood, and a quantitative analysis of how the proviral landscape is reshaped after cART is initiated is critical to understanding how HIV persists. Here, we studied longitudinal samples from HIV infected individuals undergoing long term cART using multiplexed Droplet Digital PCR (ddPCR) approaches to quantify the proportion of deleted proviruses in lymphocytes. In most individuals undergoing cART, HIV proviruses that contain gag are lost more quickly than those that lack gag. Increases in the fraction of gag-deleted proviruses occurred only after 1–2 years of therapy, suggesting that the immune system, and/or toxicity of viral re-activation helps to gradually shape the proviral landscape. After 10–15 years on therapy, there were as many as 3.5–5 times more proviruses in which gag was deleted or highly defective than those containing intact gag. We developed a provirus-specific ddPCR approach to quantify individual clones. Investigation of a clone of cells containing a deleted HIV provirus integrated in the HORMAD2 gene revealed that the cells underwent a massive expansion shortly after cART was initiated until the clone, which was primarily in effector memory cells, dominated the population of proviruses for over 6 years. The expansion of this HIV-infected clone had substantial effects on the overall proviral population.

ACS Style

Elizabeth M. Anderson; Francesco R. Simonetti; Robert J. Gorelick; Shawn Hill; Monica A. Gouzoulis; Jennifer Bell; Catherine Rehm; Liliana Pérez; Eli Boritz; Xiaolin Wu; Daria Wells; Stephen H. Hughes; Venigalla Rao; John M. Coffin; Mary F. Kearney; Frank Maldarelli. Dynamic Shifts in the HIV Proviral Landscape During Long Term Combination Antiretroviral Therapy: Implications for Persistence and Control of HIV Infections. Viruses 2020, 12, 136 .

AMA Style

Elizabeth M. Anderson, Francesco R. Simonetti, Robert J. Gorelick, Shawn Hill, Monica A. Gouzoulis, Jennifer Bell, Catherine Rehm, Liliana Pérez, Eli Boritz, Xiaolin Wu, Daria Wells, Stephen H. Hughes, Venigalla Rao, John M. Coffin, Mary F. Kearney, Frank Maldarelli. Dynamic Shifts in the HIV Proviral Landscape During Long Term Combination Antiretroviral Therapy: Implications for Persistence and Control of HIV Infections. Viruses. 2020; 12 (2):136.

Chicago/Turabian Style

Elizabeth M. Anderson; Francesco R. Simonetti; Robert J. Gorelick; Shawn Hill; Monica A. Gouzoulis; Jennifer Bell; Catherine Rehm; Liliana Pérez; Eli Boritz; Xiaolin Wu; Daria Wells; Stephen H. Hughes; Venigalla Rao; John M. Coffin; Mary F. Kearney; Frank Maldarelli. 2020. "Dynamic Shifts in the HIV Proviral Landscape During Long Term Combination Antiretroviral Therapy: Implications for Persistence and Control of HIV Infections." Viruses 12, no. 2: 136.

Journal article
Published: 06 August 2019 in Blood Advances
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Key Points Reexpansion of CAR T cells led to further investigations which confirmed the clonal nature of this expansion.

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Nirali N. Shah; Haiying Qin; Bonnie Yates; Ling Su; Haneen Shalabi; Mark Raffeld; Mark A. Ahlman; MaryAlice Stetler-Stevenson; Constance Yuan; Shuang Guo; Siyuan Liu; Stephen H. Hughes; Terry J. Fry; Xiaolin Wu. Clonal expansion of CAR T cells harboring lentivector integration in the CBL gene following anti-CD22 CAR T-cell therapy. Blood Advances 2019, 3, 2317 -2322.

AMA Style

Nirali N. Shah, Haiying Qin, Bonnie Yates, Ling Su, Haneen Shalabi, Mark Raffeld, Mark A. Ahlman, MaryAlice Stetler-Stevenson, Constance Yuan, Shuang Guo, Siyuan Liu, Stephen H. Hughes, Terry J. Fry, Xiaolin Wu. Clonal expansion of CAR T cells harboring lentivector integration in the CBL gene following anti-CD22 CAR T-cell therapy. Blood Advances. 2019; 3 (15):2317-2322.

Chicago/Turabian Style

Nirali N. Shah; Haiying Qin; Bonnie Yates; Ling Su; Haneen Shalabi; Mark Raffeld; Mark A. Ahlman; MaryAlice Stetler-Stevenson; Constance Yuan; Shuang Guo; Siyuan Liu; Stephen H. Hughes; Terry J. Fry; Xiaolin Wu. 2019. "Clonal expansion of CAR T cells harboring lentivector integration in the CBL gene following anti-CD22 CAR T-cell therapy." Blood Advances 3, no. 15: 2317-2322.

Research article
Published: 10 July 2019 in PLOS Pathogens
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Clonal expansion of HIV infected cells plays an important role in the formation and persistence of the reservoir that allows the virus to persist, in DNA form, despite effective antiretroviral therapy. We used integration site analysis to ask if there is a similar clonal expansion of SIV infected cells in macaques. We show that the distribution of HIV and SIV integration sites in vitro is similar and that both viruses preferentially integrate in many of the same genes. We obtained approximately 8000 integration sites from blood samples taken from SIV-infected macaques prior to the initiation of ART, and from blood, spleen, and lymph node samples taken at necropsy. Seven clones were identified in the pre-ART samples; one persisted for a year on ART. An additional 100 clones were found only in on-ART samples; a number of these clones were found in more than one tissue. The timing and extent of clonal expansion of SIV-infected cells in macaques and HIV-infected cells in humans is quite similar. This suggests that SIV-infected macaques represent a useful model of the clonal expansion of HIV infected cells in humans that can be used to evaluate strategies intended to control or eradicate the viral reservoir. Although antiretroviral therapy (ART) effectively blocks HIV replication, infected people are not cured. As a part of its normal replication cycle, HIV inserts (integrates) a DNA copy of its genome into the genome of infected host cells, which allows the virus to persist as long as the infected cells survive. Not only can these infected cells survive, they can grow and divide, increasing the numbers of infected cells without viral replication. The ability of the infected cells to proliferate plays an important role in maintaining the numbers of infected cells (and the infection) in people on successful therapy. However, there are some important experiments that cannot easily be done with samples that can be obtained from HIV infected people. SIV infected macaques are often used as a model to do experiments that cannot be done in HIV infected people. We show here that the distribution of HIV and SIV integration sites is similar, and that, in infected macaques, the timing and extent of the proliferation of SIV infected cells is also quite similar to HIV infected cells in humans. This shows that the SIV/macaque system can be used to model the clonal expansion of HIV infected cells.

ACS Style

Andrea L. Ferris; David W. Wells; Shuang Guo; Gregory Q. Del Prete; Adrienne E. Swanstrom; John M. Coffin; Xiaolin Wu; Jeffrey D. Lifson; Stephen H. Hughes. Clonal expansion of SIV-infected cells in macaques on antiretroviral therapy is similar to that of HIV-infected cells in humans. PLOS Pathogens 2019, 15, e1007869 .

AMA Style

Andrea L. Ferris, David W. Wells, Shuang Guo, Gregory Q. Del Prete, Adrienne E. Swanstrom, John M. Coffin, Xiaolin Wu, Jeffrey D. Lifson, Stephen H. Hughes. Clonal expansion of SIV-infected cells in macaques on antiretroviral therapy is similar to that of HIV-infected cells in humans. PLOS Pathogens. 2019; 15 (7):e1007869.

Chicago/Turabian Style

Andrea L. Ferris; David W. Wells; Shuang Guo; Gregory Q. Del Prete; Adrienne E. Swanstrom; John M. Coffin; Xiaolin Wu; Jeffrey D. Lifson; Stephen H. Hughes. 2019. "Clonal expansion of SIV-infected cells in macaques on antiretroviral therapy is similar to that of HIV-infected cells in humans." PLOS Pathogens 15, no. 7: e1007869.

Journal article
Published: 20 June 2019 in JCI Insight
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In HIV-infected individuals on long-term antiretroviral therapy (ART), more than 40% of the infected cells are in clones. Although most HIV proviruses present in individuals on long-term ART are defective, including those in clonally expanded cells, there is increasing evidence that clones carrying replication-competent proviruses are common in patients on long-term ART and form part of the HIV reservoir that makes it impossible to cure HIV infection with current ART alone. Given the importance of clonal expansion in HIV persistence, we determined how soon after HIV acquisition infected clones can grow large enough to be detected (clones larger than ca. 1 × 105 cells). We studied 12 individuals sampled in early HIV infection (Fiebig stage III-V/VI) and 5 who were chronically infected. The recently infected individuals were started on ART at or near the time of diagnosis. We isolated more than 6,500 independent integration sites from peripheral blood mononuclear cells before ART was initiated and after 0.5-18 years of suppressive ART. Some infected clones could be detected approximately 4 weeks after HIV infection and some of these clones persisted for years. The results help to explain how the reservoir is established early and persists for years.

ACS Style

John M. Coffin; David W. Wells; Jennifer M. Zerbato; JoAnn D. Kuruc; Shuang Guo; Brian T. Luke; Joseph J. Eron; Michael Bale; Jonathan Spindler; Francesco Roberto Simonetti; Shawn Hill; Mary F. Kearney; Frank Maldarelli; Xiaolin Wu; John W. Mellors; Stephen H. Hughes. Clones of infected cells arise early in HIV-infected individuals. JCI Insight 2019, 4, 1 .

AMA Style

John M. Coffin, David W. Wells, Jennifer M. Zerbato, JoAnn D. Kuruc, Shuang Guo, Brian T. Luke, Joseph J. Eron, Michael Bale, Jonathan Spindler, Francesco Roberto Simonetti, Shawn Hill, Mary F. Kearney, Frank Maldarelli, Xiaolin Wu, John W. Mellors, Stephen H. Hughes. Clones of infected cells arise early in HIV-infected individuals. JCI Insight. 2019; 4 (12):1.

Chicago/Turabian Style

John M. Coffin; David W. Wells; Jennifer M. Zerbato; JoAnn D. Kuruc; Shuang Guo; Brian T. Luke; Joseph J. Eron; Michael Bale; Jonathan Spindler; Francesco Roberto Simonetti; Shawn Hill; Mary F. Kearney; Frank Maldarelli; Xiaolin Wu; John W. Mellors; Stephen H. Hughes. 2019. "Clones of infected cells arise early in HIV-infected individuals." JCI Insight 4, no. 12: 1.

Journal article
Published: 01 June 2019 in Journal of Virology
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Although antiretroviral therapy (ART) is highly successful, drug-resistant variants can arise that blunt the efficacy of ART. New inhibitors that are broadly effective against known drug-resistant variants are needed, although such compounds might select for novel resistance mutations that affect the sensitivity of the virus to other compounds. Compound 13 selects for resistance mutations that differ from traditional NNRTI resistance mutations. These mutations cause increased sensitivity to NRTIs, such as AZT.

ACS Style

Paul L. Boyer; Kevin Melody; Steven J. Smith; Linda L. Dunn; Chris Kline; Douglas K. Fischer; Richa Dwivedi; Pat Clark; Stephen H. Hughes; Zandrea Ambrose. Two Coselected Distal Mutations in HIV-1 Reverse Transcriptase (RT) Alter Susceptibility to Nonnucleoside RT Inhibitors and Nucleoside Analogs. Journal of Virology 2019, 93, e00224-19 .

AMA Style

Paul L. Boyer, Kevin Melody, Steven J. Smith, Linda L. Dunn, Chris Kline, Douglas K. Fischer, Richa Dwivedi, Pat Clark, Stephen H. Hughes, Zandrea Ambrose. Two Coselected Distal Mutations in HIV-1 Reverse Transcriptase (RT) Alter Susceptibility to Nonnucleoside RT Inhibitors and Nucleoside Analogs. Journal of Virology. 2019; 93 (11):e00224-19.

Chicago/Turabian Style

Paul L. Boyer; Kevin Melody; Steven J. Smith; Linda L. Dunn; Chris Kline; Douglas K. Fischer; Richa Dwivedi; Pat Clark; Stephen H. Hughes; Zandrea Ambrose. 2019. "Two Coselected Distal Mutations in HIV-1 Reverse Transcriptase (RT) Alter Susceptibility to Nonnucleoside RT Inhibitors and Nucleoside Analogs." Journal of Virology 93, no. 11: e00224-19.