Chromosomal integration of genome-intact HIV-1 sequences into the host genome creates a reservoir of virally infected cells that persists throughout life, necessitating indefinite antiretroviral suppression therapy. During effective antiviral treatment, the majority of these proviruses remain transcriptionally silent, but mechanisms responsible for viral latency are insufficiently clear. Here, we used matched integration site and proviral sequencing (MIP-Seq), an experimental approach involving multiple displacement amplification of individual proviral species, followed by near-full-length HIV-1 next-generation sequencing and corresponding chromosomal integration site analysis to selectively map the chromosomal positions of intact and defective proviruses in 3 HIV-1-infected individuals undergoing long-term antiretroviral therapy. Simultaneously, chromatin accessibility and gene expression in autologous CD4+ T cells were analyzed by assays for transposase-accessible chromatin using sequencing (ATAC-Seq) and RNA-Seq. We observed that in comparison to proviruses with defective sequences, intact HIV-1 proviruses were enriched for non-genic chromosomal positions and more frequently showed an opposite orientation relative to host genes. In addition, intact HIV-1 proviruses were preferentially integrated in either relative proximity to or increased distance from active transcriptional start sites and to accessible chromatin regions. These studies strongly suggest selection of intact proviruses with features of deeper viral latency during prolonged antiretroviral therapy, and may be informative for targeting the genome-intact viral reservoir.

HIV-1 causes a chronic, incurable disease due to its persistence in CD4⁺ T cells that contain replication-competent provirus, but exhibit little or no active viral gene expression and effectively resist combination antiretroviral therapy (cART). These latently infected T cells represent an extremely small proportion of all circulating CD4⁺ T cells but possess a remarkable long-term stability and typically persist throughout life, for reasons that are not fully understood. Here we performed massive single-genome, near-full-length next-generation sequencing of HIV-1 DNA derived from unfractionated peripheral blood mononuclear cells, ex vivo-isolated CD4⁺ T cells, and subsets of functionally polarized memory CD4⁺ T cells. This approach identified multiple sets of independent, near-full-length proviral sequences from cART-treated individuals that were completely identical, consistent with clonal expansion of CD4⁺ T cells harboring intact HIV-1. Intact, near-full-genome HIV-1 DNA sequences that were derived from such clonally expanded CD4⁺ T cells constituted 62% of all analyzed genome-intact sequences in memory CD4 T cells, were preferentially observed in Th1-polarized cells, were longitudinally detected over a duration of up to 5 years, and were fully replication- and infection-competent. Together, these data suggest that clonal proliferation of Th1-polarized CD4⁺ T cells encoding for intact HIV-1 represents a driving force for stabilizing the pool of latently infected CD4⁺ T cells.

Signal transduction in Mycobacterium tuberculosis is mediated primarily by the Ser/Thr protein kinases and the two-component systems. The Ser/Thr kinase PknH has been shown to regulate growth of M. tuberculosis in a mouse model and in response to NO stress in vitro. Comparison of a pknH deletion mutant (ΔpknH) with its parental M. tuberculosis H37Rv strain using iTRAQ enabled us to quantify >700 mycobacterial proteins. Among these, members of the hypoxia- and NO-inducible dormancy (DosR) regulon were disregulated in the ΔpknH mutant. Using kinase assays, protein-protein interactions, and mass spectrometry analysis, we demonstrated that the two-component response regulator DosR is a substrate of PknH. PknH phosphorylation of DosR mapped to Thr¹⁹⁸ and Thr²⁰⁵ on the key regulatory helix α10 involved in activation and dimerization of DosR. PknH Thr phosphorylation and DosS Asp phosphorylation of DosR cooperatively enhanced DosR binding to cognate DNA sequences. Transcriptional analysis comparing ΔpknH and parental M. tuberculosis revealed that induction of the DosR regulon was subdued in the ΔpknH mutant in response to NO. Together, these results indicate that PknH phosphorylation of DosR is required for full induction of the DosR regulon and demonstrate convergence of the two major signal transduction systems for the first time in M. tuberculosis.

Therapeutic strategies aimed at achieving antiretroviral therapy (ART)-free HIV remission in infected individuals are under active investigation. Considering the vast majority of HIV-infected individuals experience plasma viral rebound upon cessation of therapy, clinical trials evaluating the efficacy of curative strategies would likely require inclusion of ART interruption. However, it is unclear what impact short-term analytical treatment interruption (ATI) and subsequent reinitiation of ART have on immunologic and virologic parameters of HIV-infected individuals. Here, we show a significant increase of HIV burden in the CD4⁺ T cells of infected individuals during ATI that was correlated with the level of plasma viral rebound. However, the size of the HIV reservoirs as well as immune parameters, including markers of exhaustion and activation, returned to pre-ATI levels 6–12 months after the study participants resumed ART. Of note, the proportions of near full-length, genome-intact and structurally defective HIV proviral DNA sequences were similar prior to ATI and following reinitiation of ART. In addition, there was no evidence of emergence of antiretroviral drug resistance mutations within intact HIV proviral DNA sequences following reinitiation of ART. These data demonstrate that short-term ATI does not necessarily lead to expansion of the persistent HIV reservoir nor irreparable damages to the immune system in the peripheral blood, warranting the inclusion of ATI in future clinical trials evaluating curative strategies. While we have made considerable advancements in the treatment of HIV, most infected individuals require life-long treatment to suppress plasma viremia, underscoring the need for the development of additional therapeutic strategies that would allow durable virologic remission in the absence of antiretroviral therapy (ART). While a definitive cure has not yet been identified, the field is moving in a promising direction, and with continued efforts we may arrive at a clinically acceptable alternative to ART. Clinical validation of new treatment options likely requires patients to stop therapy while monitoring for viral rebound, but the effect of treatment interruption and its precise impact on immunologic and virologic parameters in HIV-infected individuals has not been fully delineated. In this work, we measured a significant increase of HIV burden in the CD4⁺ T cells of infected individuals who underwent ATI with subsequent plasma viral rebound. However, the size of the HIV reservoirs as well as immune parameters returned to pre-ATI levels 6–12 months after the participants resumed ART. These data suggest ATI does not lead to expansion of the persistent HIV reservoir nor irreversible damages to the immune system in the peripheral blood.

Little is known about the genotypic make-up of HIV-1 DNA genomes during the earliest stages of HIV-1 infection. Here, we use near-full-length, single genome next-generation sequencing to longitudinally genotype and quantify subtype C HIV-1 DNA in four women identified during acute HIV-1 infection in Durban, South Africa, through twice-weekly screening of high-risk participants. In contrast to chronically HIV-1-infected patients, we found that at the earliest phases of infection in these four participants, the majority of viral DNA genomes are intact, lack APOBEC-3G/F-associated hypermutations, have limited genome truncations, and over one year show little indication of cytotoxic T cell-driven immune selections. Viral sequence divergence during acute infection is predominantly fueled by single-base substitutions and is limited by treatment initiation during the earliest stages of disease. Our observations provide rare longitudinal insights of HIV-1 DNA sequence profiles during the first year of infection to inform future HIV cure research.

HIV posttreatment controllers (PTCs) represent a natural model of sustained HIV remission, but they are rare and little is known about their viral reservoir. We obtained 1,450 proviral sequences after near-full-length amplification for 10 PTCs and 16 posttreatment noncontrollers (NCs). Before treatment interruption, the median intact and total reservoir size in PTCs was 7-fold lower than in NCs, but the proportion of intact, defective, and total clonally expanded proviral genomes was not significantly different between the 2 groups. Quantification of total but not intact proviral genome copies predicted sustained HIV remission as 81% of NCs, but none of the PTCs had a total proviral genome greater than 4 copies per million peripheral blood mononuclear cells (PBMCs). The results highlight the restricted intact and defective HIV reservoir in PTCs and suggest that total proviral genome burden could act as the first biomarker for identifying PTCs. Total and defective but not intact proviral copy numbers correlated with levels of cell-associated HIV RNA, activated NK cell percentages, and both HIV-specific CD4⁺ and CD8⁺ responses. These results support the concept that defective HIV genomes can lead to viral antigen production and interact with both the innate and adaptive immune systems.