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John M. Coffin
Department of Molecular Biology and Microbiology, Tufts University, 145 Harrison Avenue, Jaharis 409, Boston, MA 02111, USA

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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: 29 April 2021 in Viruses
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The latent HIV-1 reservoir is comprised of stably integrated and intact proviruses with limited to no viral transcription. It has been proposed that latent infection may be maintained by methylation of pro-viral DNA. Here, for the first time, we investigate the cytosine methylation of a replication competent provirus (AMBI-1) found in a T cell clone in a donor on antiretroviral therapy (ART). Methylation profiles of the AMBI-1 provirus were compared to other proviruses in the same donor and in samples from three other individuals on ART, including proviruses isolated from lymph node mononuclear cells (LNMCs) and peripheral blood mononuclear cells (PBMCs). We also evaluated the apparent methylation of cytosines outside of CpG (i.e., CpH) motifs. We found no evidence for methylation in AMBI-1 or any other provirus tested within the 5′ LTR promoter. In contrast, CpG methylation was observed in the env-tat-rev overlapping reading frame. In addition, we found evidence for differential provirus methylation in cells isolated from LNMCs vs. PBMCs in some individuals, possibly from the expansion of infected cell clones. Finally, we determined that apparent low-level methylation of CpH cytosines is consistent with occasional bisulfite reaction failures. In conclusion, our data do not support the proposition that latent HIV infection is associated with methylation of the HIV 5′ LTR promoter.

ACS Style

Valerie Boltz; Cristina Ceriani; Jason Rausch; Wei Shao; Michael Bale; Brandon Keele; Rebecca Hoh; Jeffrey Milush; Steve Deeks; Frank Maldarelli; Mary Kearney; John Coffin. CpG Methylation Profiles of HIV-1 Proviral DNA in Individuals on ART. Viruses 2021, 13, 799 .

AMA Style

Valerie Boltz, Cristina Ceriani, Jason Rausch, Wei Shao, Michael Bale, Brandon Keele, Rebecca Hoh, Jeffrey Milush, Steve Deeks, Frank Maldarelli, Mary Kearney, John Coffin. CpG Methylation Profiles of HIV-1 Proviral DNA in Individuals on ART. Viruses. 2021; 13 (5):799.

Chicago/Turabian Style

Valerie Boltz; Cristina Ceriani; Jason Rausch; Wei Shao; Michael Bale; Brandon Keele; Rebecca Hoh; Jeffrey Milush; Steve Deeks; Frank Maldarelli; Mary Kearney; John Coffin. 2021. "CpG Methylation Profiles of HIV-1 Proviral DNA in Individuals on ART." Viruses 13, no. 5: 799.

Journal article
Published: 15 January 2021 in Molecular Biology of the Cell
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The simultaneous discovery in 1970 of reverse transcriptase in virions of retroviruses by Howard Temin and David Baltimore was perhaps the most dramatic scientific moment of the second half of the 20th century. Ten years previously, Temin’s observation of cells transformed by Rous Sarcoma virus led him to the conclusion that retroviruses replicate through a DNA intermediate he called the provirus. This heretical hypothesis was greeted with derision by fellow scientists; Temin and Baltimore performed a simple experiment, rapidly reproduced, and convincing to all. Its result was a major paradigm shift—reversal of the central dogma of molecular biology. It immediately grabbed the attention of both the scientific and lay press. It also came at a key time for cancer research, at the start of the “War on Cancer.” As a theoretical base and fundamental molecular tool, it enabled a decade of (largely fruitless) search for human oncogenic retroviruses but laid the foundation for the discovery of HIV 13 years later, leading to the development of effective therapy. I had the good fortune, as a student in Temin’s lab, to witness these events. I am honored to be able to share my recollection on the occasion of their 50th anniversary.

ACS Style

John M. Coffin. 50th anniversary of the discovery of reverse transcriptase. Molecular Biology of the Cell 2021, 32, 91 -97.

AMA Style

John M. Coffin. 50th anniversary of the discovery of reverse transcriptase. Molecular Biology of the Cell. 2021; 32 (2):91-97.

Chicago/Turabian Style

John M. Coffin. 2021. "50th anniversary of the discovery of reverse transcriptase." Molecular Biology of the Cell 32, no. 2: 91-97.

Brief report
Published: 14 December 2020 in Proceedings of the National Academy of Sciences
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In vivo clonal expansion of HIV-infected T cells is an important mechanism of viral persistence. In some cases, clonal expansion is driven by HIV proviral DNA integrated into one of a handful of genes. To investigate this phenomenon in vitro, we infected primary CD4+ T cells with an HIV construct expressing GFP and, after nearly 2 mo of culture and multiple rounds of activation, analyzed the resulting integration site distribution. In each of three replicates from each of two donors, we detected large clusters of integration sites with multiple breakpoints, implying clonal selection. These clusters all mapped to a narrow region within the STAT3 gene. The presence of hybrid transcripts splicing HIV to STAT3 sequences supports a model of LTR-driven STAT3 overexpression as a driver of preferential growth. Thus, HIV integration patterns linked to selective T cell outgrowth can be reproduced in cell culture. The single report of an HIV provirus in a case of AIDS-associated B-cell lymphoma with an HIV provirus in the same part of STAT3 also has implications for HIV-induced malignancy.

ACS Style

John K. Yoon; Joseph R. Holloway; Daria W. Wells; Machika Kaku; David Jetton; Rebecca Brown; John M. Coffin. HIV proviral DNA integration can drive T cell growth ex vivo. Proceedings of the National Academy of Sciences 2020, 117, 32880 -32882.

AMA Style

John K. Yoon, Joseph R. Holloway, Daria W. Wells, Machika Kaku, David Jetton, Rebecca Brown, John M. Coffin. HIV proviral DNA integration can drive T cell growth ex vivo. Proceedings of the National Academy of Sciences. 2020; 117 (52):32880-32882.

Chicago/Turabian Style

John K. Yoon; Joseph R. Holloway; Daria W. Wells; Machika Kaku; David Jetton; Rebecca Brown; John M. Coffin. 2020. "HIV proviral DNA integration can drive T cell growth ex vivo." Proceedings of the National Academy of Sciences 117, no. 52: 32880-32882.

Journal article
Published: 01 November 2020 in AIDS Research and Human Retroviruses
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The prevalence of HIV-1 drug resistance is increasing worldwide and monitoring its emergence is important for the successful management of populations receiving combination antiretroviral therapy. It is likely that pre-existing drug resistance mutations linked on the same viral genomes are predictive of treatment failure. Because of the large numbers of sequences generated by Ultrasensitive Single-Genome Sequencing (uSGS) and other similar next-generation sequencing methods, it is difficult to assess each sequence individually for linked drug resistance mutations. Several software/programs exist to report the frequencies of individual mutations in large datasets, but they provide no information on linkage of resistance mutations. Here, we report the HIV-DRLink program, a research tool that provides resistance mutation frequencies as well as their genetic linkage by parsing and summarizing the Sierra output from the Stanford HIV Database (https://hivdb.stanford.edu/). The HIV-DRLink program should only be used on datasets generated by methods that eliminate artifacts due to PCR recombination, for example, standard Single-Genome Sequencing (SGS) or uSGS. HIV-DRLink is exclusively a research tool and is not intended to inform clinical decisions.

ACS Style

Wei Shao; Valerie F. Boltz; Junko Hattori; Michael J. Bale; Frank Maldarelli; John M. Coffin; Mary F. Kearney. Short Communication: HIV-DRLink: A Tool for Reporting Linked HIV-1 Drug Resistance Mutations in Large Single-Genome Data Sets Using the Stanford HIV Database. AIDS Research and Human Retroviruses 2020, 36, 942 -947.

AMA Style

Wei Shao, Valerie F. Boltz, Junko Hattori, Michael J. Bale, Frank Maldarelli, John M. Coffin, Mary F. Kearney. Short Communication: HIV-DRLink: A Tool for Reporting Linked HIV-1 Drug Resistance Mutations in Large Single-Genome Data Sets Using the Stanford HIV Database. AIDS Research and Human Retroviruses. 2020; 36 (11):942-947.

Chicago/Turabian Style

Wei Shao; Valerie F. Boltz; Junko Hattori; Michael J. Bale; Frank Maldarelli; John M. Coffin; Mary F. Kearney. 2020. "Short Communication: HIV-DRLink: A Tool for Reporting Linked HIV-1 Drug Resistance Mutations in Large Single-Genome Data Sets Using the Stanford HIV Database." AIDS Research and Human Retroviruses 36, no. 11: 942-947.

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.

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.

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; 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.

Preprint content
Published: 06 December 2019
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The prevalence of HIV-1 drug resistance is increasing worldwide and monitoring its emergence is important for the successful management of populations receiving combination antiretroviral therapy (cART). Using Ultrasensitive Single-Genome Sequencing (uSGS), a next-generation method that avoids PCR bias and PCR recombination, a recent report showed that pre-existing dual-class drug resistance mutations linked on the same viral genomes were predictive of treatment failure while unlinked mutations were not. Because of the large numbers of sequences generated by uSGS and other next-generation sequencing methods, it is difficult to assess each sequence individually for linked resistance mutations. Several software/programs exist to report the frequencies of individual mutations in large datasets but they provide no information on their linkage. Here, we report the HIV-DRLink program, a research tool that provides mutation frequencies in the total dataset as well as their linkage to other mutations conferring resistance to the same or different drug classes. The HIV-DRLink program should only be used on datasets generated by methods that eliminate artifacts due to PCR recombination, for example, standard Single-Genome Sequencing (SGS) or Ultrasensitive Single-Genome Sequencing (uSGS). HIV-DRLink is exclusively a research tool and is not intended to inform clinical decisions.

ACS Style

Wei Shao; Valerie F. Boltz; Junko Hattori; Michael J. Bale; Frank Maldarelli; John M. Coffin; Mary F. Kearney. HIV-DRLink: A tool for detecting linked HIV-1 drug resistance mutations in next generation sequencing data. 2019, 866715 .

AMA Style

Wei Shao, Valerie F. Boltz, Junko Hattori, Michael J. Bale, Frank Maldarelli, John M. Coffin, Mary F. Kearney. HIV-DRLink: A tool for detecting linked HIV-1 drug resistance mutations in next generation sequencing data. . 2019; ():866715.

Chicago/Turabian Style

Wei Shao; Valerie F. Boltz; Junko Hattori; Michael J. Bale; Frank Maldarelli; John M. Coffin; Mary F. Kearney. 2019. "HIV-DRLink: A tool for detecting linked HIV-1 drug resistance mutations in next generation sequencing data." , no. : 866715.

Journal article
Published: 27 November 2019 in Proceedings of the National Academy of Sciences
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Understanding HIV-1 persistence despite antiretroviral therapy (ART) is of paramount importance. Both single-genome sequencing (SGS) and integration site analysis (ISA) provide useful information regarding the structure of persistent HIV DNA populations; however, until recently, there was no way to link integration sites to their cognate proviral sequences. Here, we used multiple-displacement amplification (MDA) of cellular DNA diluted to a proviral endpoint to obtain full-length proviral sequences and their corresponding sites of integration. We applied this method to lymph node and peripheral blood mononuclear cells from 5 ART-treated donors to determine whether groups of identical subgenomic sequences in the 2 compartments are the result of clonal expansion of infected cells or a viral genetic bottleneck. We found that identical proviral sequences can result from both cellular expansion and viral genetic bottlenecks occurring prior to ART initiation and following ART failure. We identified an expanded T cell clone carrying an intact provirus that matched a variant previously detected by viral outgrowth assays and expanded clones with wild-type and drug-resistant defective proviruses. We also found 2 clones from 1 donor that carried identical proviruses except for nonoverlapping deletions, from which we could infer the sequence of the intact parental virus. Thus, MDA-SGS can be used for “viral reconstruction” to better understand intrapatient HIV-1 evolution and to determine the clonality and structure of proviruses within expanded clones, including those with drug-resistant mutations. Importantly, we demonstrate that identical sequences observed by standard SGS are not always sufficient to establish proviral clonality.

ACS Style

Sean C. Patro; Leah D. Brandt; Michael Bale; Elias K. Halvas; Kevin W. Joseph; Wei Shao; Xiaolin Wu; Shuang Guo; Ben Murrell; Ann Wiegand; Jonathan Spindler; Castle Raley; Christopher Hautman; Michele Sobolewski; Christine M. Fennessey; Wei-Shau Hu; Brian Luke; Jenna M. Hasson; Aurelie Niyongabo; Adam A. Capoferri; Brandon F. Keele; Jeff Milush; Rebecca Hoh; Steven Deeks; Frank Maldarelli; Stephen H. Hughes; John M. Coffin; Jason W. Rausch; John W. Mellors; Mary Kearney. Combined HIV-1 sequence and integration site analysis informs viral dynamics and allows reconstruction of replicating viral ancestors. Proceedings of the National Academy of Sciences 2019, 116, 25891 -25899.

AMA Style

Sean C. Patro, Leah D. Brandt, Michael Bale, Elias K. Halvas, Kevin W. Joseph, Wei Shao, Xiaolin Wu, Shuang Guo, Ben Murrell, Ann Wiegand, Jonathan Spindler, Castle Raley, Christopher Hautman, Michele Sobolewski, Christine M. Fennessey, Wei-Shau Hu, Brian Luke, Jenna M. Hasson, Aurelie Niyongabo, Adam A. Capoferri, Brandon F. Keele, Jeff Milush, Rebecca Hoh, Steven Deeks, Frank Maldarelli, Stephen H. Hughes, John M. Coffin, Jason W. Rausch, John W. Mellors, Mary Kearney. Combined HIV-1 sequence and integration site analysis informs viral dynamics and allows reconstruction of replicating viral ancestors. Proceedings of the National Academy of Sciences. 2019; 116 (51):25891-25899.

Chicago/Turabian Style

Sean C. Patro; Leah D. Brandt; Michael Bale; Elias K. Halvas; Kevin W. Joseph; Wei Shao; Xiaolin Wu; Shuang Guo; Ben Murrell; Ann Wiegand; Jonathan Spindler; Castle Raley; Christopher Hautman; Michele Sobolewski; Christine M. Fennessey; Wei-Shau Hu; Brian Luke; Jenna M. Hasson; Aurelie Niyongabo; Adam A. Capoferri; Brandon F. Keele; Jeff Milush; Rebecca Hoh; Steven Deeks; Frank Maldarelli; Stephen H. Hughes; John M. Coffin; Jason W. Rausch; John W. Mellors; Mary Kearney. 2019. "Combined HIV-1 sequence and integration site analysis informs viral dynamics and allows reconstruction of replicating viral ancestors." Proceedings of the National Academy of Sciences 116, no. 51: 25891-25899.

Journal article
Published: 03 October 2019 in JCI Insight
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We hypothesized that HIV-1 with dual-class but not single-class drug resistance mutations linked on the same viral genome, present in the virus population before initiation of antiretroviral therapy (ART), would be associated with failure of ART to suppress viremia. To test this hypothesis, we utilized an ultrasensitive single-genome sequencing assay that detects rare HIV-1 variants with linked drug resistance mutations (DRMs). A case (ART failure) control (nonfailure) study was designed to assess whether linkage of DRMs in pre-ART plasma samples was associated with treatment outcome in the nevirapine/tenofovir/emtricitabine arm of the AIDS Clinical Trials Group A5208/Optimal Combined Therapy After Nevirapine Exposure (OCTANE) Trial 1 among women who had received prior single-dose nevirapine. Ultrasensitive single-genome sequencing revealed a significant association between pre-ART HIV variants with DRMs to 2 drug classes linked on the same genome (dual class) and failure of combination ART with 3 drugs to suppress viremia. In contrast, linked, single-class DRMs were not associated with ART failure. We conclude that linked dual-class DRMs present before the initiation of ART are associated with ART failure, whereas linked single-class DRMs are not.

ACS Style

Valerie F. Boltz; Wei Shao; Michael J. Bale; Elias K. Halvas; Brian Luke; James A. McIntyre; Robert T. Schooley; Shahin Lockman; Judith S. Currier; Fred Sawe; Evelyn Hogg; Michael D. Hughes; Mary Kearney; John M. Coffin; John W. Mellors. Linked dual-class HIV resistance mutations are associated with treatment failure. JCI Insight 2019, 4, 1 .

AMA Style

Valerie F. Boltz, Wei Shao, Michael J. Bale, Elias K. Halvas, Brian Luke, James A. McIntyre, Robert T. Schooley, Shahin Lockman, Judith S. Currier, Fred Sawe, Evelyn Hogg, Michael D. Hughes, Mary Kearney, John M. Coffin, John W. Mellors. Linked dual-class HIV resistance mutations are associated with treatment failure. JCI Insight. 2019; 4 (19):1.

Chicago/Turabian Style

Valerie F. Boltz; Wei Shao; Michael J. Bale; Elias K. Halvas; Brian Luke; James A. McIntyre; Robert T. Schooley; Shahin Lockman; Judith S. Currier; Fred Sawe; Evelyn Hogg; Michael D. Hughes; Mary Kearney; John M. Coffin; John W. Mellors. 2019. "Linked dual-class HIV resistance mutations are associated with treatment failure." JCI Insight 4, no. 19: 1.

Journal article
Published: 23 September 2019 in Journal of Clinical Investigation
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To investigate the possibility that HIV-1 replication in lymph nodes sustains the reservoir during ART, we looked for evidence of viral replication in 5 donors after up to 13 years of viral suppression. We characterized proviral populations in lymph nodes and peripheral blood before and during ART, evaluated the levels of viral RNA expression in single lymph node and blood cells, and characterized the proviral integration sites in paired lymph node and blood samples. Proviruses with identical sequences, identical integration sites, and similar levels of RNA expression were found in lymph nodes and blood samples collected during ART, and no single sequence with significant divergence from the pretherapy population was present in either blood or lymph nodes. These findings show that all detectable persistent HIV-1 infection is consistent with maintenance in lymph nodes by clonal proliferation of cells infected before ART and not by ongoing viral replication during ART.

ACS Style

William McManus; Michael J. Bale; Jonathan Spindler; Ann Wiegand; Andrew Musick; Sean C. Patro; Michele D. Sobolewski; Victoria K. Musick; Elizabeth M. Anderson; Joshua C. Cyktor; Elias K. Halvas; Wei Shao; Daria Wells; Xiaolin Wu; Brandon F. Keele; Jeffrey M. Milush; Rebecca Hoh; John W. Mellors; Stephen H. Hughes; Steven G. Deeks; John M. Coffin; Mary Kearney. HIV-1 in lymph nodes is maintained by cellular proliferation during antiretroviral therapy. Journal of Clinical Investigation 2019, 129, 4629 -4642.

AMA Style

William McManus, Michael J. Bale, Jonathan Spindler, Ann Wiegand, Andrew Musick, Sean C. Patro, Michele D. Sobolewski, Victoria K. Musick, Elizabeth M. Anderson, Joshua C. Cyktor, Elias K. Halvas, Wei Shao, Daria Wells, Xiaolin Wu, Brandon F. Keele, Jeffrey M. Milush, Rebecca Hoh, John W. Mellors, Stephen H. Hughes, Steven G. Deeks, John M. Coffin, Mary Kearney. HIV-1 in lymph nodes is maintained by cellular proliferation during antiretroviral therapy. Journal of Clinical Investigation. 2019; 129 (11):4629-4642.

Chicago/Turabian Style

William McManus; Michael J. Bale; Jonathan Spindler; Ann Wiegand; Andrew Musick; Sean C. Patro; Michele D. Sobolewski; Victoria K. Musick; Elizabeth M. Anderson; Joshua C. Cyktor; Elias K. Halvas; Wei Shao; Daria Wells; Xiaolin Wu; Brandon F. Keele; Jeffrey M. Milush; Rebecca Hoh; John W. Mellors; Stephen H. Hughes; Steven G. Deeks; John M. Coffin; Mary Kearney. 2019. "HIV-1 in lymph nodes is maintained by cellular proliferation during antiretroviral therapy." Journal of Clinical Investigation 129, no. 11: 4629-4642.

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: 04 January 2019 in Proceedings of the National Academy of Sciences
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Human endogenous retrovirus-K (HERV-K) human mouse mammary tumor virus-like 2 (HML-2) is the most recently active endogenous retrovirus group in humans, and the only group with human-specific proviruses. HML-2 expression is associated with cancer and other diseases, but extensive searches have failed to reveal any replication-competent proviruses in humans. However, HML-2 proviruses are found throughout the catarrhine primates, and it is possible that they continue to infect some species today. To investigate this possibility, we searched for gorilla-specific HML-2 elements using both in silico data mining and targeted deep-sequencing approaches. We identified 150 gorilla-specific integrations, including 31 2-LTR proviruses. Many of these proviruses have identical LTRs, and are insertionally polymorphic, consistent with very recent integration. One identified provirus has full-length ORFs for all genes, and thus could potentially be replication-competent. We suggest that gorillas may still harbor infectious HML-2 virus and could serve as a model for understanding retrovirus evolution and pathogenesis in humans.

ACS Style

Joseph R. Holloway; Zachary Williams; Michael M. Freeman; Uriel Bulow; John M. Coffin. Gorillas have been infected with the HERV-K (HML-2) endogenous retrovirus much more recently than humans and chimpanzees. Proceedings of the National Academy of Sciences 2019, 116, 1337 -1346.

AMA Style

Joseph R. Holloway, Zachary Williams, Michael M. Freeman, Uriel Bulow, John M. Coffin. Gorillas have been infected with the HERV-K (HML-2) endogenous retrovirus much more recently than humans and chimpanzees. Proceedings of the National Academy of Sciences. 2019; 116 (4):1337-1346.

Chicago/Turabian Style

Joseph R. Holloway; Zachary Williams; Michael M. Freeman; Uriel Bulow; John M. Coffin. 2019. "Gorillas have been infected with the HERV-K (HML-2) endogenous retrovirus much more recently than humans and chimpanzees." Proceedings of the National Academy of Sciences 116, no. 4: 1337-1346.

Journal article
Published: 20 August 2018 in Retrovirology
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These data suggest that stage-specific transcription factors at least partly contribute to LTR promoter activity during transformation and that, in some cases, transcription factor binding site polymorphisms may be responsible for the differential HML-2 expression often seen between individuals.

ACS Style

Meagan Montesion; Zachary H. Williams; Ravi P. Subramanian; Charlotte Kuperwasser; John M. Coffin. Promoter expression of HERV-K (HML-2) provirus-derived sequences is related to LTR sequence variation and polymorphic transcription factor binding sites. Retrovirology 2018, 15, 57 .

AMA Style

Meagan Montesion, Zachary H. Williams, Ravi P. Subramanian, Charlotte Kuperwasser, John M. Coffin. Promoter expression of HERV-K (HML-2) provirus-derived sequences is related to LTR sequence variation and polymorphic transcription factor binding sites. Retrovirology. 2018; 15 (1):57.

Chicago/Turabian Style

Meagan Montesion; Zachary H. Williams; Ravi P. Subramanian; Charlotte Kuperwasser; John M. Coffin. 2018. "Promoter expression of HERV-K (HML-2) provirus-derived sequences is related to LTR sequence variation and polymorphic transcription factor binding sites." Retrovirology 15, no. 1: 57.

Journal article
Published: 01 January 2018 in Journal of Virology
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Increasing evidence suggests that repetitive elements may play a role in host gene regulation, particularly through the donation of alternative promoters, enhancers, splice sites, and termination signals. Elevated transcript expression of the endogenous retrovirus group HERV-K (HML-2) is seen in many human cancers, although the identities of the individual proviral loci contributing to this expression as well as their mechanisms of activation have been unclear. Using high-throughput next-generation sequencing techniques optimized for the capture of HML-2 expression, we characterized the HML-2 transcriptome and means of activation in an in vitro model of human mammary epithelial cell transformation. Our analysis showed significant expression originating from 15 HML-2 full-length proviruses, through four modes of transcription. The majority of expression was in the antisense orientation and from proviruses integrated within introns. We found two instances of long terminal repeat (LTR)-driven provirus transcription but no evidence to suggest that these active 5′ LTRs were influencing nearby host gene expression. Importantly, LTR-driven transcription was restricted to tumorigenic cells, suggesting that LTR promoter activity is dependent upon the transcriptional environment of a malignant cell. IMPORTANCE Here, we use an in vitro model of human mammary epithelial cell transformation to assess how malignancy-associated shifts in the transcriptional milieu of a cell may impact HML-2 activity. We found 15 proviruses to be significantly expressed through four different mechanisms, with the majority of transcripts being antisense copies of proviruses located within introns. We saw active 5′ LTR use in tumorigenic cells only, suggesting that the cellular environment of a cancer cell is a critical component for induction of LTR promoter activity. These findings have implications for future studies investigating HML-2 as a target for immunotherapy or as a biomarker for disease.

ACS Style

Meagan Montesion; Neeru Bhardwaj; Zachary H. Williams; Charlotte Kuperwasser; John M. Coffin. Mechanisms of HERV-K (HML-2) Transcription during Human Mammary Epithelial Cell Transformation. Journal of Virology 2018, 92, e01258-17 .

AMA Style

Meagan Montesion, Neeru Bhardwaj, Zachary H. Williams, Charlotte Kuperwasser, John M. Coffin. Mechanisms of HERV-K (HML-2) Transcription during Human Mammary Epithelial Cell Transformation. Journal of Virology. 2018; 92 (1):e01258-17.

Chicago/Turabian Style

Meagan Montesion; Neeru Bhardwaj; Zachary H. Williams; Charlotte Kuperwasser; John M. Coffin. 2018. "Mechanisms of HERV-K (HML-2) Transcription during Human Mammary Epithelial Cell Transformation." Journal of Virology 92, no. 1: e01258-17.

Clinical trial
Published: 11 September 2017 in Journal of Clinical Investigation
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It remains controversial whether current antiretroviral therapy (ART) fully suppresses the cycles of HIV replication and viral evolution in vivo. If replication persists in sanctuary sites such as the lymph nodes, a high priority should be placed on improving ART regimes to target these sites. To investigate the question of ongoing viral replication on current ART regimens, we analyzed HIV populations in longitudinal samples from 10 HIV-1-infected children who initiated ART when viral diversity was low. Eight children started ART at less than ten months of age and showed suppression of plasma viremia for seven to nine years. Two children had uncontrolled viremia for fifteen and thirty months, respectively, before viremia suppression, and served as positive controls for HIV replication and evolution. These latter 2 children showed clear evidence of virus evolution, whereas multiple methods of analysis bore no evidence of virus evolution in any of the 8 children with viremia suppression on ART. Phylogenetic trees simulated with the recently reported evolutionary rate of HIV-1 on ART of 6 × 10-4 substitutions/site/month bore no resemblance to the observed data. Taken together, these data refute the concept that ongoing HIV replication is common with ART and is the major barrier to curing HIV-1 infection.

ACS Style

Gert U. Van Zyl; Mary Grace Katusiime; Ann Wiegand; William McManus; Michael Bale; Elias K. Halvas; Brian Luke; Valerie F. Boltz; Jonathan Spindler; Barbara Laughton; Susan Engelbrecht; John M. Coffin; Mark F. Cotton; Wei Shao; John W. Mellors; Mary F. Kearney. No evidence of HIV replication in children on antiretroviral therapy. Journal of Clinical Investigation 2017, 127, 3827 -3834.

AMA Style

Gert U. Van Zyl, Mary Grace Katusiime, Ann Wiegand, William McManus, Michael Bale, Elias K. Halvas, Brian Luke, Valerie F. Boltz, Jonathan Spindler, Barbara Laughton, Susan Engelbrecht, John M. Coffin, Mark F. Cotton, Wei Shao, John W. Mellors, Mary F. Kearney. No evidence of HIV replication in children on antiretroviral therapy. Journal of Clinical Investigation. 2017; 127 (10):3827-3834.

Chicago/Turabian Style

Gert U. Van Zyl; Mary Grace Katusiime; Ann Wiegand; William McManus; Michael Bale; Elias K. Halvas; Brian Luke; Valerie F. Boltz; Jonathan Spindler; Barbara Laughton; Susan Engelbrecht; John M. Coffin; Mark F. Cotton; Wei Shao; John W. Mellors; Mary F. Kearney. 2017. "No evidence of HIV replication in children on antiretroviral therapy." Journal of Clinical Investigation 127, no. 10: 3827-3834.

Journal article
Published: 01 July 2017 in Open Forum Infectious Diseases
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Lorenzo-Redondo et al. recently analyzed HIV RNA sequences in plasma virus and proviral DNA sequences in lymph nodes (LN) and peripheral blood mononuclear cells (PBMC) from samples collected over a 6-month period from 3 individuals following initiation of antiretroviral therapy (ART) and concluded that ongoing HIV replication occurred in LN despite ART and that this replication maintained the HIV reservoir. We analyzed the same sequences and found that the dataset was very limited (median of 5 unique RNA or DNA sequences per sample) after accounting for polymerase chain reaction resampling and hypermutation and that the few remaining DNA sequences after 3 and 6 months on ART were not more diverse or divergent from those in pre-ART in any of the individuals studied. These findings, and others, lead us to conclude that the claims of ongoing replication on ART made by Lorenzo-Redondo et al. are not justified from the dataset analyzed in their publication.

ACS Style

Mary F Kearney; Ann Wiegand; Wei Shao; William McManus; Michael Bale; Brian Luke; Frank Maldarelli; John W Mellors; John M Coffin. Ongoing HIV Replication During ART Reconsidered. Open Forum Infectious Diseases 2017, 4, 1 .

AMA Style

Mary F Kearney, Ann Wiegand, Wei Shao, William McManus, Michael Bale, Brian Luke, Frank Maldarelli, John W Mellors, John M Coffin. Ongoing HIV Replication During ART Reconsidered. Open Forum Infectious Diseases. 2017; 4 (3):1.

Chicago/Turabian Style

Mary F Kearney; Ann Wiegand; Wei Shao; William McManus; Michael Bale; Brian Luke; Frank Maldarelli; John W Mellors; John M Coffin. 2017. "Ongoing HIV Replication During ART Reconsidered." Open Forum Infectious Diseases 4, no. 3: 1.

Journal article
Published: 17 April 2017 in Proceedings of the National Academy of Sciences
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Little is known about the fraction of human immunodeficiency virus type 1 (HIV-1) proviruses that express unspliced viral RNA in vivo or about the levels of HIV RNA expression within single infected cells. We developed a sensitive cell-associated HIV RNA and DNA single-genome sequencing (CARD-SGS) method to investigate fractional proviral expression of HIV RNA (1.3-kb fragment of p6, protease, and reverse transcriptase) and the levels of HIV RNA in single HIV-infected cells from blood samples obtained from individuals with viremia or individuals on long-term suppressive antiretroviral therapy (ART). Spiking experiments show that the CARD-SGS method can detect a single cell expressing HIV RNA. Applying CARD-SGS to blood mononuclear cells in six samples from four HIV-infected donors (one with viremia and not on ART and three with viremia suppressed on ART) revealed that an average of 7% of proviruses (range: 2–18%) expressed HIV RNA. Levels of expression varied from one to 62 HIV RNA molecules per cell (median of 1). CARD-SGS also revealed the frequent expression of identical HIV RNA sequences across multiple single cells and across multiple time points in donors on suppressive ART consistent with constitutive expression of HIV RNA in infected cell clones. Defective proviruses were found to express HIV RNA at levels similar to those proviruses that had no obvious defects. CARD-SGS is a useful tool to characterize fractional proviral expression in single infected cells that persist despite ART and to assess the impact of experimental interventions on proviral populations and their expression.

ACS Style

Ann Wiegand; Jonathan Spindler; Feiyu F. Hong; Wei Shao; Joshua C. Cyktor; Anthony R. Cillo; Elias K. Halvas; John M. Coffin; John W. Mellors; Mary F. Kearney. Single-cell analysis of HIV-1 transcriptional activity reveals expression of proviruses in expanded clones during ART. Proceedings of the National Academy of Sciences 2017, 114, E3659 -E3668.

AMA Style

Ann Wiegand, Jonathan Spindler, Feiyu F. Hong, Wei Shao, Joshua C. Cyktor, Anthony R. Cillo, Elias K. Halvas, John M. Coffin, John W. Mellors, Mary F. Kearney. Single-cell analysis of HIV-1 transcriptional activity reveals expression of proviruses in expanded clones during ART. Proceedings of the National Academy of Sciences. 2017; 114 (18):E3659-E3668.

Chicago/Turabian Style

Ann Wiegand; Jonathan Spindler; Feiyu F. Hong; Wei Shao; Joshua C. Cyktor; Anthony R. Cillo; Elias K. Halvas; John M. Coffin; John W. Mellors; Mary F. Kearney. 2017. "Single-cell analysis of HIV-1 transcriptional activity reveals expression of proviruses in expanded clones during ART." Proceedings of the National Academy of Sciences 114, no. 18: E3659-E3668.

Comment
Published: 13 April 2017 in eLife
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Primates have co-opted a viral gene to produce an envelope protein that prevents infection by the HERV-T virus and likely contributed to the extinction of this virus.

ACS Style

Julia H Wildschutte; John M Coffin. Pushing the envelope. eLife 2017, 6, e26397 .

AMA Style

Julia H Wildschutte, John M Coffin. Pushing the envelope. eLife. 2017; 6 ():e26397.

Chicago/Turabian Style

Julia H Wildschutte; John M Coffin. 2017. "Pushing the envelope." eLife 6, no. : e26397.