HIV Infected T Cells Can Proliferate in vivo Without Inducing Expression of the Integrated Provirus.

Andrew Musick,Mary F Kearney,Francesco R Simonetti,Stephen H Hughes,Michael J Bale,Adam Capoferri,John M Coffin,Brandon F Keele,Wei Shao,John W Mellors,Daniel Crespo-Vélez,Jonathan Spindler,Carolyn Reid,Eli Boritz,Frank Maldarelli,Sean C Patro,Ann Wiegand,Michele D Sobolewski,Liliana Pérez

doi:10.3389/fmicb.2019.02204

Abstract

BackgroundHIV-1 proviruses can persist during ART in clonally-expanded populations of CD4+ T cells. To date, few examples of an expanded clones containing replication-competent proviruses exist, although it is suspected to be common. One such clone, denoted AMBI-1 (Maldarelli et al., 2014), was also a source of persistent viremia on ART, begging the question of how the AMBI-1 clone can survive despite infection with a replication-competent, actively-expressing provirus. We hypothesized that only a small fraction of cells within the AMBI-1 clone are activated to produce virus particles during cell division while the majority remain latent despite division, ensuring their survival. To address this question, we determined the fraction of HIV-1 proviruses within the AMBI-1 clone that expresses unspliced cell-associated RNA during ART and compared this fraction to 33 other infected T cell clones within the same individual.ResultsIn total, 34 different clones carrying either intact or defective proviruses in “Patient 1” from Maldarelli et al. (2014) were assessed. We found that 2.3% of cells within the AMBI-1 clone contained unspliced HIV-1 RNA. Highest levels of HIV-1 RNA were found in the effector memory (EM) T cell subset. The fraction of cells within clones that contained HIV-1 RNA was not different in clones with intact (median 2.3%) versus defective (median 3.5%) proviruses (p = 0.2). However, higher fractions and levels of RNA were found in cells with proviruses containing multiple drug resistance mutations, including those contributing to rebound viremia.ConclusionThese findings show that the vast majority of HIV-1 proviruses within expanded T cell clones, including intact proviruses, may be transcriptionally silent at any given time, implying that infected T cells may be able to be activated to proliferate without inducing the expression of the integrated provirus or, alternatelively, may be able to proliferate without cellular activation. The results of this study suggest that the long, presumed correlation between the level of cellular and proviral activation may not be accurate and, therefore, requires further investigation.

Highlights

HIV-1 replication is likely efficiently halted with antiretroviral therapy (ART), which prevents disease progression, but ART does not cure the infection (Persaud et al, 2007; Josefsson et al, 2013; Kearney et al, 2014; Maldarelli et al, 2014; Wagner et al, 2014; Palma et al, 2016; Vancoillie et al, 2017; Mok et al, 2018)
We identified a total of 34 different wild-type infected cell clones and possible clones, and used CARD-single-genome sequencing (SGS) (Wiegand et al, 2017) to determine the fraction of peripheral blood mononuclear cells (PBMC) within each clone, including the AMBI-1 clone, that had detectable amounts of ca-HIV RNA
To investigate HIV populations in this low-level viremia, a neighbor joining (NJ) phylogenetic distance tree was constructed using data obtained by P6-PR-RT SGS from plasma samples taken at three timepoints immediately before and after the ART regimen change (Figure 1B)

Summary

Introduction

HIV-1 replication is likely efficiently halted with antiretroviral therapy (ART), which prevents disease progression, but ART does not cure the infection (Persaud et al, 2007; Josefsson et al, 2013; Kearney et al, 2014; Maldarelli et al, 2014; Wagner et al, 2014; Palma et al, 2016; Vancoillie et al, 2017; Mok et al, 2018). The fractions of transcriptionally-silent proviruses versus transcriptionally-active proviruses remained unknown within populations of clonally-expanded infected cells, each of which contains the identical provirus at the identical site of integration, including those that carry intact proviruses (Simonetti et al, 2016; Einkauf et al, 2019) It is not known which CD4+ T cell subsets expand and support the expression of HIV-1 proviruses that persist on ART, effector memory (EM) cells have been suggested (Hiener et al, 2017; Pardons et al, 2019).

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Results

Conclusion