Dynamics and Genetics of HIV Proviruses before and during Antiretroviral Therapy

Division Of Basic Sciences - Nci

Investigators

Linked publications & trials

Abstract

BACKGROUND: We and others showed that HIV-infected cells can clonally expand and persist despite ART, and that the proviral integration site may influence this phenomenon (Maldarelli et al., Science 345:179-183, 2014; Wagner et al., Science 345:570-573, 2014). A major focus of this project is to determine how commonly such clonally expanded, infected cells that persist during ART carry replication-competent proviruses and in which tissues and cell types they persist. To answer this question, we developed a novel method called the multiple-displacement amplification single-genome sequencing (MDA-SGS) assay, which allows us to analyze the proviruses in highly expanded clones (Patro et al., PNAS 116:25891-25899, 2019). The MDA-SGS assay will determine if HIV proviruses in expanding clones that persist during ART have intact sequences or contain lethal mutations or deletions. We will further determine if, when activated, such clones are capable of producing infectious virions. If we show that latent, intact proviruses in infected cells commonly undergo clonal expansion, it will mean that strategies intended to cure patients will have to not only block viral replication, but also cope with the proliferation of these latently infected cells. ____ACCOMPLISHMENTS: Last year, to investigate the HIV reservoir in children, we conducted studies to determine if expanded, infected cell clones were present in children born with HIV infection and if these cell clones persisted during ART (Bale, Katsuimme et al., mBio 12:e00568-21, 2021). We found that infected T cell clones arose early in children (less than 2 months old) and that they persisted for at least 9 years during treatment. These findings demonstrated that the HIV reservoir in children is maintained by clonal expansion of cells that were infected prior to initiating treatment. This year, we expanded these studies to address two additional questions pertaining to children born with HIV. First, we investigated the proviral structures in the largest infected T cell clones in children on ART for more than 6 years and found all of them to contain only solo LTRs. These findings are important because they suggest that homologous recombination of the HIV LTRs after integration may lead to the deletion of HIV genes, leaving only a single LTR. If correct, then it is reasonable to assume that some intact HIV genomes will be deleted over time through this mechanism and that this mechanism may be able to be exploited towards a cure for HIV. The results of this study are currently being assembled into a manuscript. Our second accomplishment in this area is a finding that infected naive cells can be identified in virtually all children born with HIV and that, at least in some, clones of infected naive cells are found. These findings change our understanding of T cell biology and of HIV persistence, since, the proliferation of infected naive cells can lead to a self-renewing pool of infected central and effector memory T cells. We are currently in the process of defining the proviral structures in the clones of infected naive cells in children. ____We previously established two new public database to store and annotate HIV proviral sequences and their integration sites into the host genome, especially those that persist in donors on ART. These databases (PSD; https://psd.cancer.gov and RID; https://rid.ncifcrf.gov/index.php ) are available for meta-analyses of proviruses that persist on ART to better understand the HIV reservoir and to inform strategies towards a cure. This year, we grew the PSD to 4,870 full-length HIV proviruses collected from donors on ART. The PSD is accessed about 25 times daily by investigators worldwide. We grew the RID to 6,300,189 HIV integration sites. The RID is accessed about 45 times daily from investigators worldwide. ____We developed two new bioinformatic pipelines to annotate and detect intact HIV proviruses that persist during ART (Wright et al., Retrovirology 18:16, 2021) (Wright, et al. Cells 10, 2021). Our pipelines are unique because they can be incorporated into existing pipelines to extract and align HIV sequence reads from next-generation sequencing experiments (Illumina and Oxford Nanopore). This year, we also improved the pipelines to detect sequence inversions in some HIV proviruses that persist during treatment. ____In collaboration with John Mellors (University of Pittsburgh), we investigated the genetic structure of proviruses that persist on ART using a new integration sites assay that captures HIV LTR, gag, env, and nef sequences. This assay is an improvement on the previous HIV integration sites assay which is not capable of capturing HIV genetic information. We found that a high fraction of HIV proviruses that persist on treatment have asymmetric LTRs, possibly preventing recombination of the LTRs and allowing these proviruses to persist for years on treatment (Joseph et al., J. Virol. e0012222, 2022). ____ To build on our collaboration with John Mellors, we developed a qPCR-based assay that measures the size of HIV infected cell clones in donors on ART and can be used to investigate the dynamics of these cell clones over the course of treatment (Brandt, et al. Viruses. 13, 2021). We found that most infected cell clones are highly stable over the course of treatment, including those with intact HIV proviruses. However, a few infected T cell clones with intact HIV proviruses grew larger over time and a few diminished in size over longitudinal samples. ____ In collaboration with John Coffin (Tufts University) and Steve Hughes (HIV Dynamics and Replication Program, NCI), we conducted the most thorough investigation to date of selection pressures for and against HIV proviruses that persist in individuals on long-term ART (Coffin, et al. PLoS Pathog. 17: e1009141, 2021). We found that integration events into 6 different genes can results in selection for persistence of HIV proviruses. Integration events into these genes, which are proto-oncogenes such as BACH2 and STAT5B, enhance the proliferation of the T cells or promote their survival. This finding demonstrates that there is a self-renewing pool of HIV infected cells during treatment and show that halting viral replication alone will not be sufficient to cure HIV infection.