Dynamics and Genetics of HIV Proviruses before and during Antiretroviral Therapy

Division Of Basic Sciences - Nci

Investigators

Linked publications, trials & patents

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 expanded cell 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: We began our investigations of the HIV reservoir in children more than 4 years ago, initially focusing on whether expanded, infected cell clones were present in children born with HIV infection and if these cell clones persisted during ART (Bale, Katusiime 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. We followed up on these efforts by investigating the proviral structures in the largest infected T cell clones in children on ART for more than 6 years, and found them all to be solo LTRs (Botha, et al., mBio, in press). 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 an inactive remnant of what might have been a replication competent provirus contributing to the HIV reservoir. These findings suggest that LTR recombination may significantly contribute to reservoir decay over time on ART, suggesting that a new curative strategy might be devised that exploits or accelerates LTR recombination. In a related, ongoing study, we have determined that naive T cells collected from children with HIV can harbor integrated proviruses, sometimes in clonal populations, and that in one instance, the harbored provirus was sequence-intact. These findings change our understanding of both T cell biology and HIV persistence, since the proliferation of infected naive cells may lead to a self-renewing pool of infected central and effector memory T cells. A manuscript describing these findings is in preparation. Another ongoing, multifaceted effort toward genetically characterizing provirus populations utilizes samples from donors who have high levels of viremia on ART despite full suppression of viral. Notable findings produced in this ongoing, in-depth analysis of these samples include the observation that sequence-intact proviruses are disproportionately found integrated into the intronic regions of genes encoding the KRAB family of zinc-finger proteins, and that of the 10 largest infected T cell clones identified in these samples, 6 are solo LTRs - supporting analysis of samples obtained from children with HIV. To facilitate these efforts, we have recently devised and published a new combination provirus sequencing and integration site detection assay that permits more comprehensive provirus characterization that was previously possible (Joseph et al., J. Virol. e0012222, 2022).