Abstract
ABSTRACTA major goal in HIV eradication research is characterizing the reservoir cells that harbor HIV in the presence of antiretroviral therapy (ART), which reseed viremia after treatment is stopped. In general, it is assumed that the reservoir consists of CD4+ T cells that express no viral proteins. However, recent findings suggest that this may be an overly simplistic view and that the cells that contribute to the reservoir may be a diverse population that includes both CD4+ and CD4− cells. In this study, we directly infected resting CD4+ T cells and used fluorescence-activated cell sorting (FACS) and fiber-optic array scanning technology (FAST) to identify and image cells expressing HIV Gag. We found that Gag expression from integrated proviruses occurred in resting cells that lacked surface CD4, likely resulting from Nef- and Env-mediated receptor internalization. We also extended our approach to detect cells expressing HIV proteins in patients suppressed on ART. We found evidence that rare Gag+ cells persist during ART and that these cells are often negative for CD4. We propose that these double-negative α/β T cells that express HIV protein may be a component of the long-lived reservoir. IMPORTANCE A reservoir of infected cells persists in HIV-infected patients during antiretroviral therapy (ART) that leads to rebound of virus if treatment is stopped. In this study, we used flow cytometry and cell imaging to characterize protein expression in HIV-infected resting cells. HIV Gag protein can be directly detected in infected resting cells and occurs with simultaneous loss of CD4, consistent with the expression of additional viral proteins, such as Env and Nef. Gag+ CD4− cells can also be detected in suppressed patients, suggesting that a subset of infected cells express proteins during ART. Understanding the regulation of viral protein expression during ART will be key to designing effective strategies to eradicate HIV reservoirs.
Highlights
T cells that express no viral proteins
We reasoned that enrichment of integrated HIV DNA in Gagϩ cells sorted by fluorescence-activated cell sorting (FACS) would signify protein expression from integrated HIV DNA in infected cells
The Gag signal were an artifact of bound virions, we would expect Gagϩ cells to be enriched for HIV RNA but not integrated HIV DNA
Summary
T cells that express no viral proteins. recent findings suggest that this may be an overly simplistic view and that the cells that contribute to the reservoir may be a diverse population that includes both CD4؉ and CD4؊ cells. We extended our approach to detect cells expressing HIV proteins in patients suppressed on ART. Areservoir of infected cells persists in HIV-infected patients during antiretroviral therapy (ART) that leads to rebound of virus if treatment is stopped. HIV Gag protein can be directly detected in infected resting cells and occurs with simultaneous loss of CD4, consistent with the expression of additional viral proteins, such as Env and Nef. Gag؉ CD4؊ cells can be detected in suppressed patients, suggesting that a subset of infected cells express proteins during ART. Consistent with in vivo data from Kaiser et al [16], we found that gag unspliced RNA (usRNA) is the predominant viral transcript in resting CD4 T cells infected in vitro, whereas tat and rev msRNA is present at much lower levels [17]. Reverse transcriptase PCR (RT-PCR) assays that target the gag HIV RNA detect read-through transcripts from up-
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