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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.
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 StyleLeah 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 StyleLeah 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.
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.
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 StyleJohn 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 StyleJohn 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.
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.
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 StyleJohn 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 StyleJohn 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.
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.
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 StyleElias 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 StyleElias 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.
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.
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 StyleElias 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 StyleElias 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.
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.
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 StyleJohn 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 StyleJohn 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.