Dynamics and Genetics of HIV Proviruses before and during Antiretroviral Therapy

Division Of Basic Sciences - Nci

Investigators

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Abstract

BACKGROUND: Despite the ability of antiretroviral therapy (ART) to inhibit HIV replication in infected patients, preventing disease progression, the virus persists in a stable reservoir and rebounds to pretherapy levels if ART is interrupted. Lifelong therapy is expensive and risks the emergence of drug resistance and toxic side effects. Consequently, identification of strategies to eliminate the reservoir is a major priority of HIV research. 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). In several cases, expanded proviruses were shown to match viral variants present at detectable levels in persistent viremia during ART; furthermore, we showed that the virus particles produced by the clonally expanded cells were replication competent (Simonetti et al., PNAS 113:1883-1888, 2016; Halvas et al., J. Clin. Invest. 130:5847-5857, 2020). These are examples where the source of infectious virus in blood has been traced to clones of infected cells carrying a mostly latent provirus. 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. ____Although there is considerable patient-to-patient variation, the frequency of resting CD4+ T cells that harbor HIV proviruses detectable by PCR has been very roughly estimated to average about 1 cell in 1000; however, the number of latently infected cells carrying replication-competent proviruses has been reported to be much lower (Ho et al., Cell 155:540-551, 2013). The difference is due to the presence of a large number of defective proviruses. Ya-Chi Ho and colleagues described the proviruses in resting CD4+ T cells that were not induced to produce replication-competent virus after a single round of maximal T-cell activation (Ho et al., Cell 155:540-551, 2013). Almost half of these proviruses had large internal deletions that preclude replication, while another third were lethally hypermutated by the host restriction factor APOBEC3G. Other defects and further analyses brought the fraction of defective proviruses up to 98% (Bruner et al., Nat. Med. 22:1043-1049, 2016). Additionally, Ho et al. found that some of the intact proviruses were capable of producing infectious virions following a second round of activation (Cell 155:540-551, 2013), even though they had not been induced by the prior activation. This result leads to the question of how commonly the expanding clones described by Maldarelli et al. and Wagner et al. carry intact and replication-competent proviruses. 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: We used our MDA-SGS method to demonstrate that infected cell clones with replication-competent proviruses are common in patients on ART and a source of persistent, low-level viremia during treatment (Halvas et al., J. Clin. Invest. 130:5847-5857, 2020). This study demonstrates the importance of investigating potential curative strategies for their effect on clonal expansion of infected cells. This study also confirms that the reservoir for HIV is the proliferation of T cells infected prior to the initiation of ART. ____To determine if cell clones carrying intact HIV proviruses persist in children with viremia suppressed on ART, as in adults, we analyzed HIV populations in longitudinal samples from HIV-1-infected children who initiated ART shortly after birth (Katusiime et al., J. Virol. 94:e01519-19, 2020). We found sequence-identical, intact proviruses in two of the children, demonstrating that such clones likely persist in children born with HIV. These data suggest that the HIV reservoir in children is not different from the HIV reservoir in adults. ____To further investigate the HIV reservoir in children, we conducted studies to determine if expanded, infected cell clones are present in children born with HIV infection and if these cell clones persist on ART (Bale, Katusiime et al., mBio 12:e00568-21, 2021). We found that infected cell clones arise early in children (2 months old) and persist for at least 9 years during treatment. These findings demonstrate that the HIV reservoir in children is maintained by clonal expansion of cells that were infected prior to initiating treatment. ____We established a new public database to store and annotate HIV proviral sequences that persist in donors on ART (Shao et al., AIDS Res. Hum. Retroviruses 36:1-3, 2020). This database (PSD; https://psd.cancer.gov) is available for meta-analyses of proviruses that persist on ART to better understand the HIV reservoir and to inform strategies towards a cure. ____We developed a new bioinformatic pipeline to annotate and detect intact HIV proviruses that persist during ART (Wright et al., Retrovirology 18:16, 2021). Our pipeline is unique because it can be incorporated into existing pipelines to extract and align HIV sequence reads from next-generation sequencing experiments. ____In collaboration with Dr. Frank Maldarelli (HIV Dynamics and Replication Program), we investigated the genetic structure of proviruses that persist on ART using a qPCR assay that detected potentially intact proviruses and those that are deleted. We found the number of potentially intact proviruses to decrease over time in patients on ART (Anderson et al., J. Virol. 12:136, 2020). These results suggest that the reservoir may shrink over time despite the proliferation of cells infected with intact proviruses.