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HIV persistence in the brain is a barrier to cure, and potentially contributes to HIV-associated neurocognitive disorders. Whether HIV transcription persists in the brain despite viral suppression with antiretroviral therapy (ART) and is subject to the same blocks to transcription seen in other tissues and blood, is unclear. Here, we quantified the level of HIV transcripts in frontal cortex tissue from virally suppressed or non-virally suppressed people with HIV (PWH). HIV transcriptional profiling of frontal cortex brain tissue (and PBMCs where available) from virally suppressed (n = 11) and non-virally suppressed PWH (n = 13) was performed using digital polymerase chain reaction assays (dPCR). CD68+ myeloid cells or CD3+ T cells expressing HIV p24 protein present in frontal cortex tissue was detected using multiplex immunofluorescence imaging. Frontal cortex brain tissue from PWH had HIV TAR (n = 23/24) and Long-LTR (n = 20/24) transcripts. Completion of HIV transcription was evident in brain tissue from 12/13 non-virally suppressed PWH and from 5/11 virally suppressed PWH, with HIV p24+CD68+ cells detected in these individuals. While a block to proximal elongation was present in frontal cortex tissue from both PWH groups, this block was more extensive in virally suppressed PWH. These findings suggest that the brain is a transcriptionally active HIV reservoir in a subset of virally suppressed PWH., Competing Interests: SRL has received investigator-initiated grant funding from Gilead, Merck and ViiV Healthcare. She has participated as a paid member of scientific advisory boards to Abivax, Immunocore, Efsam, Abbvie and Gilead., (Copyright: © 2024 Jamal Eddine et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Objectives: Some drugs that augment cell-intrinsic defenses or modulate cell death/survival pathways have been reported to selectively kill cells infected with HIV or Simian Immunodeficiency Virus (SIV), but comparative studies are lacking. We hypothesized that these drugs may differ in their ability to kill cells infected with intact and defective proviruses., Design: To investigate this hypothesis, drugs were tested ex vivo on peripheral blood mononuclear cells (PBMC) from nine antiretroviral therapy (ART)-suppressed individuals., Methods: We tested drugs currently in clinical use or human trials, including auranofin (p53 modulator), interferon alpha2A, interferon gamma, acitretin (RIG-I inducer), GS-9620/vesatolimod (TLR7 agonist), nivolumab (PD-1 blocker), obatoclax (Bcl-2 inhibitor), birinapant [inhibitor of apoptosis proteins (IAP) inhibitor], bortezomib (proteasome inhibitor), and INK128/sapanisertib [mammalian target of rapamycin mTOR] [c]1/2 inhibitor). After 6 days of treatment, we measured cell counts/viabilities and quantified levels of total, intact, and defective HIV DNA by droplet digital PCR (Intact Proviral DNA Assay)., Results: Obatoclax reduced intact HIV DNA [median = 27-30% of dimethyl sulfoxide control (DMSO)] but not defective or total HIV DNA. Other drugs showed no statistically significant effects., Conclusion: Obatoclax and other Bcl-2 inhibitors deserve further study in combination therapies aimed at reducing the intact HIV reservoir in order to achieve a functional cure and/or reduce HIV-associated immune activation.

In most people living with HIV (PLWH) on effective antiretroviral therapy (ART), cell-associated viral transcripts are readily detectable in CD4+ T cells despite the absence of viremia. Quantification of HIV RNA species provides insights into the transcriptional activity of proviruses that persist in cells and tissues throughout the body during ART ('HIV reservoir'). One such technique for HIV RNA quantitation, 'HIV transcription profiling', developed in the Yukl laboratory, measures a series of HIV RNA species using droplet digital PCR. To take advantage of advances in digital (d)PCR, we adapted the 'HIV transcription profiling' technique to Qiagen's dPCR platform (QIAcuity) and compared its performance to droplet digital (dd)PCR (Bio-Rad QX200 system). Using RNA standards, the two technologies were tested in parallel and assessed for multiple parameters including sensitivity, specificity, linearity, and intra- and inter-assay variability. The newly validated dPCR assays were then applied to samples from PLWH to determine HIV transcriptional activity relative to HIV reservoir size. We report that HIV transcriptional profiling was readily adapted to dPCR and assays performed similarly to ddPCR, with no differences in assay characteristics. We applied these assays in a cohort of 23 PLWH and found that HIV reservoir size, based on genetically intact proviral DNA, does not predict HIV transcriptional activity. In contrast, levels of total DNA correlated with levels of most HIV transcripts (initiated, proximally and distally elongated, unspliced, and completed, but not multiply spliced), suggesting that a considerable proportion of HIV transcripts likely originate from defective proviruses. These findings may have implications for measuring and assessing curative strategies and clinical trial outcomes.

Quantification of intact proviruses is a critical measurement in HIV cure studies both in vitro and in vivo . The widely adopted 'intact proviral DNA assay' (IPDA), designed to discriminate and quantify genetically intact HIV proviruses based on detection of two HIV sequence-specific targets, was originally validated using Bio-Rad's droplet digital PCR technology (ddPCR). Despite its advantages, ddPCR is limited in multiplexing capability (two-channel) and is both labor- and time intensive. To overcome some of these limitations, we utilized a nanowell-based digital PCR platform (dPCR, QIAcuity from Qiagen) which is a fully automated system that partitions samples into nanowells rather than droplets. In this study we adapted the IPDA assay to the QIAcuity platform and assessed its performance relative to ddPCR. The dPCR could differentiate between intact, 5' defective and 3' defective proviruses and was sensitive to single HIV copy input. We found the intra-assay and inter-assay variability was within acceptable ranges (with coefficient of variation at or below 10%). When comparing the performance of the IPDA in ex vivo CD4 + T cells from people with HIV on antiretroviral therapy, there was a strong correlation in the quantification of intact (rs = 0.93; p < 0.001) and 3' defective proviruses (rs = 0.96; p < 0.001) with a significant but less strong correlation for 5' defective proviruses (rs = 0.7; p = 0.04). We demonstrate that the dPCR platform enables sensitive and accurate quantification of genetically intact and defective proviruses similar to the ddPCR system but with greater speed and efficiency. This flexible system can be further optimized in the future, to detect up to 5 targets, enabling a more precise detection of intact and potentially replication-competent proviruses., Competing Interests: The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: SRL has received investigator-initiated grant funding from Gilead, Merck and ViiV Healthcare. She has been a paid member of advisory boards to Immunocore, Abivax, Abbvie and Gilead., (© 2023 The Authors.)

Posttreatment controllers (PTCs) are rare HIV-infected individuals who can limit viral rebound after antiretroviral therapy interruption (ATI), but the mechanisms of this remain unclear. To investigate these mechanisms, we quantified various HIV RNA transcripts (via reverse transcription droplet digital PCR [RT-ddPCR]) and cellular transcriptomes (via RNA-seq) in blood cells from PTCs and noncontrollers (NCs) before and two time points after ATI. HIV transcription initiation did not significantly increase after ATI in PTCs or in NCs, whereas completed HIV transcripts increased at early ATI in both groups and multiply-spliced HIV transcripts increased only in NCs. Compared to NCs, PTCs showed lower levels of HIV DNA, more cell-associated HIV transcripts per total RNA at all times, no increase in multiply-spliced HIV RNA at early or late ATI, and a reduction in the ratio of completed/elongated HIV RNA after early ATI. NCs expressed higher levels of the IL-7 pathway before ATI and expressed higher levels of multiple cytokine, inflammation, HIV transcription, and cell death pathways after ATI. Compared to the baseline, the NCs upregulated interferon and cytokine (especially TNF) pathways during early and late ATI, whereas PTCs upregulated interferon and p53 pathways only at early ATI and downregulated gene translation during early and late ATI. In NCs, viral rebound after ATI is associated with increases in HIV transcriptional completion and splicing, rather than initiation. Differences in HIV and cellular transcription may contribute to posttreatment control, including an early limitation of spliced HIV RNA, a delayed reduction in completed HIV transcripts, and the differential expression of the IL-7, p53, and TNF pathways. IMPORTANCE The findings presented here provide new insights into how HIV and cellular gene expression change after stopping ART in both noncontrollers and posttreatment controllers. Posttreatment control is associated with an early ability to limit increases in multiply-spliced HIV RNA, a delayed (and presumably immune-mediated) ability to reverse an initial rise in processive/completed HIV transcripts, and multiple differences in cellular gene expression pathways. These differences may represent correlates or mechanisms of posttreatment control and may provide insight into the development and/or monitoring of therapeutic strategies that are aimed at a functional HIV cure.

Most of the HIV DNA in infected individuals is noninfectious because of deleterious mutations. However, it is unclear how much of the transcribed HIV RNA is potentially infectious or defective. To address this question, we developed and validated a novel intact viral RNA assay (IVRA) that uses droplet digital reverse transcriptase PCR (dd-RT-PCR) for the commonly mutated packaging signal (Psi) and Rev response element (RRE) regions (from the intact proviral DNA assay [IPDA]) to quantify likely intact (Psi + RRE + ), 3' defective (Psi + RRE - ), and 5' defective (Psi - RRE + ) HIV RNA. We then applied the IPDA and IVRA to quantify intact and defective HIV DNA and RNA from peripheral CD4 + T cells from 9 antiretroviral therapy (ART)-suppressed individuals. Levels of 3' defective HIV DNA were not significantly different from those of 5' defective HIV DNA, and both were higher than intact HIV DNA. In contrast, 3' defective HIV RNA (median 86 copies/10 6 cells; 94% of HIV RNA) was much more abundant than 5' defective (2.1 copies/10 6 cells; 5.6%) or intact (0.6 copies/10 6 cells; <1%) HIV RNA. Likewise, the frequency of CD4 + T cells with 3' defective HIV RNA was greater than the frequency with 5' defective or intact HIV RNA. Intact HIV RNA was transcribed by a median of 0.018% of all proviruses and 2.2% of intact proviruses. The vast excess of 3' defective RNA over 5' defective or intact HIV RNA, which was not observed for HIV DNA, suggests that HIV transcription is completely blocked prior to the RRE in most cells with intact proviruses and/or that cells transcribing intact HIV RNA are cleared at very high rates. IMPORTANCE We developed a new assay that can distinguish and quantify intact (potentially infectious) as well as defective HIV RNA. In ART-treated individuals, we found that the vast majority of all HIV RNA is defective at the 3' end, possibly due to incomplete transcriptional processivity. Only a very small percentage of all HIV RNA is intact, and very few total or intact proviruses transcribe intact HIV RNA. Though rare, this intact HIV RNA is tremendously important because it is necessary to serve as the genome of infectious virions that allow transmission and spread, including rebound after stopping ART. Moreover, intact viral RNA may contribute disproportionately to the immune activation, inflammation, and organ damage observed with untreated and treated HIV infection. The intact viral RNA assay can be applied to many future studies aimed at better understanding HIV pathogenesis and barriers to HIV cure.

The replication of SARS-CoV-2 and other coronaviruses depends on transcription of negative-sense RNA intermediates that serve as the templates for the synthesis of positive-sense genomic RNA (gRNA) and multiple different subgenomic mRNAs (sgRNAs) encompassing fragments arising from discontinuous transcription. Recent studies have aimed to characterize the expression of subgenomic SARS-CoV-2 transcripts in order to investigate their clinical significance. Here, we describe a novel panel of reverse transcription droplet digital PCR (RT-ddPCR) assays designed to specifically quantify multiple different subgenomic SARS-CoV-2 transcripts and distinguish them from transcripts that do not arise from discontinuous transcription at each locus. These assays can be applied to samples from SARS-CoV-2 infected patients to better understand the regulation of SARS-CoV-2 transcription and how different sgRNAs may contribute to viral pathogenesis and clinical disease severity., (Published by Elsevier Inc.)

Although there have been great advancements in the field of HIV treatment and prevention, there is no cure. There are two types of HIV: HIV-1 and HIV-2. In addition to genetic differences between the two types of HIV, HIV-2 infection causes a slower disease progression, and the rate of new HIV-2 infections has dramatically decreased since 2003. Like HIV-1, HIV-2 is capable of establishing latent infection in CD4+ T cells, thereby allowing the virus to evade viral cytopathic effects and detection by the immune system. The mechanisms underlying HIV latency are not fully understood, rendering this a significant barrier to development of a cure. Using RT-ddPCR, we previously demonstrated that latent infection with HIV-1 may be due to blocks to HIV transcriptional elongation, distal transcription/polyadenylation, and multiple splicing. In this study, we describe the development of seven highly-specific RT-ddPCR assays for HIV-2 that can be applied to the study of HIV-2 infections and latency. We designed and validated seven assays targeting different HIV-2 RNA regions along the genome that can be used to measure the degree of progression through different blocks to HIV-2 transcription and splicing. Given that HIV-2 is vastly understudied relative to HIV-1 and that it can be considered a model of a less virulent infection, application of these assays to studies of HIV-2 latency may inform new therapies for HIV-2, HIV-1, and other retroviruses., Competing Interests: The authors have declared that no competing interests exist.

A hallmark of coronavirus transcription is the generation of negative-sense RNA intermediates that serve as the templates for the synthesis of positive-sense genomic RNA (gRNA) and an array of subgenomic mRNAs (sgRNAs) encompassing sequences arising from discontinuous transcription. Existing PCR-based diagnostic assays for SAR-CoV-2 are qualitative or semi-quantitative and do not provide the resolution needed to assess the complex transcription dynamics of SARS-CoV-2 over the course of infection. We developed and validated a novel panel of sensitive, quantitative RT-ddPCR assays designed to target regions spanning the genome of SARS-CoV-2. Our assays target untranslated regions (5', 3') as well as different coding regions, including non-structural genes that are only found in full length (genomic) RNA and structural genes that are found in genomic as well as different subgenomic RNAs. Application of these assays to clinically relevant samples will enhance our understanding of SARS-CoV-2 gene expression and may also inform the development of improved diagnostic tools and therapeutics., (Published by Elsevier B.V.)

Latent HIV infection is the main barrier to cure, and most HIV-infected cells reside in the gut, where distinct but unknown mechanisms may promote viral latency. Transforming growth factor β (TGF-β), which induces the expression of CD103 on tissue-resident memory T cells, has been implicated in HIV latency. Using CD103 as a surrogate marker to identify cells that have undergone TGF-β signaling, we compared the HIV RNA/DNA contents and cellular transcriptomes of CD103 + and CD103 - CD4 T cells from the blood and rectum of HIV-negative (HIV - ) and antiretroviral therapy (ART)-suppressed HIV-positive (HIV + ) individuals. Like gut CD4 + T cells, circulating CD103 + cells harbored more HIV DNA than did CD103 - cells but transcribed less HIV RNA per provirus. Circulating CD103 + cells also shared a gene expression profile that is closer to that of gut CD4 T cells than to that of circulating CD103 - cells, with significantly lower expression levels of ribosomal proteins and transcriptional and translational pathways associated with HIV expression but higher expression levels of a subset of genes implicated in suppressing HIV transcription. These findings suggest that blood CD103 + CD4 T cells can serve as a model to study the molecular mechanisms of HIV latency in the gut and reveal new cellular factors that may contribute to HIV latency. IMPORTANCE The ability of HIV to establish a reversibly silent, "latent" infection is widely regarded as the main barrier to curing HIV. Most HIV-infected cells reside in tissues such as the gut, but it is unclear what mechanisms maintain HIV latency in the blood or gut. We found that circulating CD103 + CD4 + T cells are enriched for HIV-infected cells in a latent-like state. Using RNA sequencing (RNA-seq), we found that CD103 + T cells share a cellular transcriptome that more closely resembles that of CD4 + T cells from the gut, suggesting that they are homing to or from the gut. We also identified the cellular genes whose expression distinguishes gut CD4 + or circulating CD103 + T cells from circulating CD103 - T cells, including some genes that have been implicated in HIV expression. These genes may contribute to latent HIV infection in the gut and may serve as new targets for therapies aimed at curing HIV., (Copyright © 2020 American Society for Microbiology.)

It is unclear what mechanisms govern latent HIV infection in vivo or in primary cell models. To investigate these questions, we compared the HIV and cellular transcription profile in three primary cell models and peripheral CD4+ T cells from HIV-infected ART-suppressed individuals using RT-ddPCR and RNA-seq. All primary cell models recapitulated the block to HIV multiple splicing seen in cells from ART-suppressed individuals, suggesting that this may be a key feature of HIV latency in primary CD4+ T cells. Blocks to HIV transcriptional initiation and elongation were observed more variably among models. A common set of 234 cellular genes, including members of the minor spliceosome pathway, was differentially expressed between unstimulated and activated cells from primary cell models and ART-suppressed individuals, suggesting these genes may play a role in the blocks to HIV transcription and splicing underlying latent infection. These genes may represent new targets for therapies designed to reactivate or silence latently-infected cells., Competing Interests: The authors have declared that no competing interests exist.

Objective: While latently HIV-infected cells have been described in the blood, it is unclear whether a similar inducible reservoir exists in the gut, where most HIV-infected cells reside. Tissue-specific environments may contribute to differences in the mechanisms that govern latent HIV infection and amenability to reactivation. We sought to determine whether HIV-infected cells from the blood and gut differ in their responses to T-cell activation and mechanistically distinct latency reversing agents (LRAs)., Design: Cross sectional study using samples from HIV-infected individuals (n = 11)., Methods: Matched peripheral blood mononuclear cells (PBMC) and dissociated total cells from rectum ± ileum were treated ex vivo for 24 h with anti-CD3/CD28 or LRAs in the presence of antiretrovirals. HIV DNA and 'read-through', initiated, 5' elongated, completed, and multiply-spliced HIV transcripts were quantified using droplet digital PCR., Results: T-cell activation increased levels of all HIV transcripts in PBMC and gut cells, and was the only treatment that increased multiply-spliced HIV RNA. Disulfiram increased initiated HIV transcripts in PBMC but not gut cells, while ingenol mebutate increased HIV transcription more in gut cells. Romidepsin increased HIV transcription in PBMC and gut cells, but the increase in transcription initiation was greater in PBMC., Conclusion: The gut harbors HIV-infected cells in a latent-like state that can be reversed by T-cell activation involving CD3/CD28 signaling. Histone deacetylation and protein kinase B may contribute less to HIV transcriptional initiation in the gut, whereas protein kinase C may contribute more. New LRAs or combinations are needed to induce multiply-spliced HIV and should be tested on both blood and gut.

The latent reservoir is a major barrier to HIV cure. As latently infected cells cannot be phenotyped directly, the features of the in vivo reservoir have remained elusive. Here, we describe a method that leverages high-dimensional phenotyping using CyTOF to trace latently infected cells reactivated ex vivo to their original pre-activation states. Our results suggest that, contrary to common assumptions, the reservoir is not randomly distributed among cell subsets, and is remarkably conserved between individuals. However, reservoir composition differs between tissues and blood, as do cells successfully reactivated by different latency reversing agents. By selecting 8-10 of our 39 original CyTOF markers, we were able to isolate highly purified populations of unstimulated in vivo latent cells. These purified populations were highly enriched for replication-competent and intact provirus, transcribed HIV, and displayed clonal expansion. The ability to isolate unstimulated latent cells from infected individuals enables previously impossible studies on HIV persistence., Competing Interests: JN, XL, JF, GX, FH, TM, VM, AL, ST, RT, WT, BW, RH, MS, PV, JM, KJ, NA, PH, SD, SY, SP, WG, NR No competing interests declared, (© 2020, Neidleman et al.)

The primary reservoir for HIV is within memory CD4+ T cells residing within tissues, yet the features that make some of these cells more susceptible than others to infection by HIV is not well understood. Recent studies demonstrated that CCR5-tropic HIV-1 efficiently enters tissue-derived memory CD4+ T cells expressing CD127, the alpha chain of the IL7 receptor, but rarely completes the replication cycle. We now demonstrate that the inability of HIV to replicate in these CD127-expressing cells is not due to post-entry restriction by SAMHD1. Rather, relative to other memory T cell subsets, these cells are highly prone to undergoing latent infection with HIV, as revealed by the high levels of integrated HIV DNA in these cells. Host gene expression profiling revealed that CD127-expressing memory CD4+ T cells are phenotypically distinct from other tissue memory CD4+ T cells, and are defined by a quiescent state with diminished NFκB, NFAT, and Ox40 signaling. However, latently-infected CD127+ cells harbored unspliced HIV transcripts and stimulation of these cells with anti-CD3/CD28 reversed latency. These findings identify a novel subset of memory CD4+ T cells found in tissue and not in blood that are preferentially targeted for latent infection by HIV, and may serve as an important reservoir to target for HIV eradication efforts., Competing Interests: The authors have declared that no competing interests exist.

Background: Most HIV-infected cells during antiretroviral therapy (ART) persist in lymphoid tissues. Studies disagree on whether suboptimal tissue ART concentrations contribute to ongoing HIV replication during viral suppression., Methods: We performed a cross-sectional study in virally-suppressed HIV+ participants measuring lymphoid tissue ART [darunavir (DRV), atazanavir (ATV), and raltegravir (RAL)] concentrations by LC-MS/MS assay. Tissue and plasma ART concentrations were used to estimate TPRs and drug-specific tissue:inhibitory concentration ratios (TICs). HIV DNA and sequentially produced HIV RNA transcripts were quantified from rectal biopsies using droplet digital PCR (ddPCR) assays., Results: Tissue samples were collected in duplicate from 19 participants: 38 rectal, 8 ileal (4 RAL, 2 DRV, 2 ATV), and 6 lymph node (4 RAL, 2 DRV) samples. Overall, median TICs were higher for RAL than DRV or ATV (both P = 0.006). Median TICs were lower in lymph nodes vs. ileum (0.49 vs. 143, P = 0.028) or rectum (33, P = 0.019), and all ART levels were below target concentrations. Higher rectal TICs were associated with lower HIV RNA transcripts (read-through, long LTR, and Nef, P all < 0.026) and a lower long LTR RNA/long LTR DNA ratio (P = 0.021)., Conclusions: We observed higher tissue ART concentrations in ileum and rectum compared with lymph nodes. We observed higher HIV transcription in participants with lower rectal ART concentrations. These findings add to the limited data supporting the idea that viral transcription may be influenced by ART concentrations in lymphoid tissues. Further exploration of tissue pharmacokinetics is needed in future HIV eradication strategies.

Background: HIV-infected cell lines are widely used to study latent HIV infection, which is considered the main barrier to HIV cure. We hypothesized that these cell lines differ from each other and from cells from HIV-infected individuals in the mechanisms underlying latency., Results: To quantify the degree to which HIV expression is inhibited by blocks at different stages of HIV transcription, we employed a recently-described panel of RT-ddPCR assays to measure levels of 7 HIV transcripts ("read-through," initiated, 5' elongated, mid-transcribed/unspliced [Pol], distal-transcribed [Nef], polyadenylated, and multiply-sliced [Tat-Rev]) in bulk populations of latently-infected (U1, ACH-2, J-Lat) and productively-infected (8E5, activated J-Lat) cell lines. To assess single-cell variation and investigate cellular genes associated with HIV transcriptional blocks, we developed a novel multiplex qPCR panel and quantified single cell levels of 7 HIV targets and 89 cellular transcripts in latently- and productively-infected cell lines. The bulk cell HIV transcription profile differed dramatically between cell lines and cells from ART-suppressed individuals. Compared to cells from ART-suppressed individuals, latent cell lines showed lower levels of HIV transcriptional initiation and higher levels of polyadenylation and splicing. ACH-2 and J-Lat cells showed different forms of transcriptional interference, while U1 cells showed a block to elongation. Single-cell studies revealed marked variation between/within cell lines in expression of HIV transcripts, T cell phenotypic markers, antiviral factors, and genes implicated in latency. Expression of multiply-spliced HIV Tat-Rev was associated with expression of cellular genes involved in activation, tissue retention, T cell transcription, and apoptosis/survival., Conclusions: HIV-infected cell lines differ from each other and from cells from ART-treated individuals in the mechanisms governing latent HIV infection. These differences in viral and cellular gene expression must be considered when gauging the suitability of a given cell line for future research on HIV. At the same time, some features were shared across cell lines, such as low expression of antiviral defense genes and a relationship between productive infection and genes involved in survival. These features may contribute to HIV latency or persistence in vivo, and deserve further study using novel single cell assays such as those described in this manuscript.

Human immunodeficiency virus (HIV) continues to be a major contributor to morbidity and mortality worldwide, particularly in developing nations where high cost and logistical issues severely limit the use of current HIV therapeutics. This, combined HIV's high propensity to develop resistance, means that new antiviral agents against novel targets are still urgently required. We previously identified novel anti-HIV agents directed against the nuclear import of the HIV integrase (IN) protein, which plays critical roles in the HIV lifecycle inside the cell nucleus, as well as in transporting the HIV preintegration complex (PIC) into the nucleus. Here we investigate the structure activity relationship of a series of these compounds for the first time, including a newly identified anti-IN compound, budesonide, showing that the extent of binding to the IN core domain correlates directly with the ability of the compound to inhibit IN nuclear transport in a permeabilised cell system. Importantly, compounds that inhibited the nuclear transport of IN were found to significantly decrease HIV viral replication, even in a dividing cell system. Significantly, budesonide or its analogue flunisolide, were able to effect a significant reduction in the presence of specific nuclear forms of the HIV DNA (2-LTR circles), suggesting that the inhibitors work though blocking IN, and potentially PIC, nuclear import. The work presented here represents a platform for further development of these specific inhibitors of HIV replication with therapeutic and prophylactic potential., (© 2018 The Authors Cellular Microbiology Published by John Wiley & Sons Ltd.)

Latently-infected CD4+ T cells are widely considered to be the major barrier to a cure for HIV. Much of our understanding of HIV latency comes from latency models and blood cells, but most HIV-infected cells reside in lymphoid tissues such as the gut. We hypothesized that tissue-specific environments may impact the mechanisms that govern HIV expression. To assess the degree to which different mechanisms inhibit HIV transcription in the gut and blood, we quantified HIV transcripts suggestive of transcriptional interference (U3-U5; "Read-through"), initiation (TAR), 5' elongation (R-U5-pre-Gag; "Long LTR"), distal transcription (Nef), completion (U3-polyA; "PolyA"), and multiple splicing (Tat-Rev) in matched peripheral blood mononuclear cells (PBMCs) and rectal biopsies, and matched FACS-sorted CD4+ T cells from blood and rectum, from two cohorts of ART-suppressed individuals. Like the PBMCs, rectal biopsies showed low levels of read-through transcripts (median = 23 copies/106 cells) and a gradient of total (679)>elongated(75)>Nef(16)>polyadenylated (11)>multiply-spliced HIV RNAs(<1) [p<0.05 for all], demonstrating blocks to HIV transcriptional elongation, completion, and splicing. Rectal CD4+ T cells showed a similar gradient of total>polyadenylated>multiply-spliced transcripts, but the ratio of total to elongated transcripts was 6-fold lower than in blood CD4+ T cells (P = 0.016), suggesting less of a block to HIV transcriptional elongation in rectal CD4+ T cells. Levels of total transcripts per provirus were significantly lower in rectal biopsies compared to PBMCs (median 3.5 vs. 15.4; P = 0.008) and in sorted CD4+ T cells from rectum compared to blood (median 2.7 vs. 31.8; P = 0.016). The lower levels of HIV transcriptional initiation and of most HIV transcripts per provirus in the rectum suggest that this site may be enriched for latently-infected cells, cells in which latency is maintained by different mechanisms, or cells in a "deeper" state of latency. These are important considerations for designing therapies that aim to disrupt HIV latency in all tissue compartments., Competing Interests: The authors have declared that no competing interests exist.

Latently infected CD4 + T cells are the main barrier to complete clearance of HIV infection, but it is unclear what mechanisms govern latent HIV infection in vivo. To address this question, we developed a new panel of reverse transcription droplet digital polymerase chain reaction (RT-ddPCR) assays specific for different HIV transcripts that define distinct blocks to transcription. We applied this panel of assays to CD4 + T cells freshly isolated from HIV-infected patients on suppressive antiretroviral therapy (ART) to quantify the degree to which different mechanisms inhibit HIV transcription. In addition, we measured the degree to which these transcriptional blocks could be reversed ex vivo by T cell activation (using anti-CD3/CD28 antibodies) or latency-reversing agents. We found that the main reversible block to HIV RNA transcription was not inhibition of transcriptional initiation but rather a series of blocks to proximal elongation, distal transcription/polyadenylation (completion), and multiple splicing. Cell dilution experiments suggested that these mechanisms operated in most of the HIV-infected CD4 + T cells examined. Latency-reversing agents exerted differential effects on the three blocks to HIV transcription, suggesting that these blocks may be governed by different mechanisms., (Copyright © 2018 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

The gastrointestinal (GI) tract harbors most of the body's immune cells and is also a major HIV reservoir in ART-treated patients. To achieve a cure, most HIV-infected cells must be identified and eliminated. While obtaining gut biopsies is a relatively noninvasive method of sampling relevant tissue for monitoring HIV activity, immune cell isolation from these limited tissue samples has proven to be challenging. Enzymatic tissue digestion is required for maximal immune cell isolation from gut biopsies. However, these enzymatic digestions can also be detrimental for preservation of cellular surface markers that are required for accurate identification of various subsets of leukocytes. In this study, we describe an optimized protocol for isolation of lymphocytes from human gut biopsies. We also discuss our validation results, which show that compared with several other collagenase preparations, the use of CSLPA maintains high lymphocyte recovery while preserving the integrity of most cellular surface antigens that we tested. Importantly, chemokine receptors that are used to characterize various subsets of T cells, which are notorious for being digested during a typical enzymatic tissue digestion, are highly preserved using this protocol.

The single greatest challenge to an HIV cure is the persistence of latently infected cells containing inducible, replication-competent proviral genomes, which constitute only a small fraction of total or infected cells in the body. Although resting CD4 + T cells in the blood are a well-known source of viral rebound, more than 90% of the body's lymphocytes reside elsewhere. Many are in gut tissue, where HIV DNA levels per million CD4 + T cells are considerably higher than in the blood. Despite the significant contribution of gut tissue to viral replication and persistence, little is known about the cell types that support persistence of HIV in the gut; importantly, T cells in the gut have phenotypic, functional, and survival properties that are distinct from T cells in other tissues. The mechanisms by which latency is established and maintained will likely depend on the location and cytokine milieu surrounding the latently infected cells in each compartment. Therefore, successful HIV cure strategies require identification and characterization of the exact cell types that support viral persistence, particularly in the gut. In this review, we describe the seeding of the latent HIV reservoir in the gut mucosa; highlight the evidence for compartmentalization and depletion of T cells; summarize the immunologic consequences of HIV infection within the gut milieu; propose how the damaged gut environment may promote the latent HIV reservoir; and explore several immune cell targets in the gut and their place on the path toward HIV cure.

Objective: Synonymous substitutions K65K/K66K in HIV-1 reverse transcriptase alleviate fitness and fidelity defects in HIV-1 molecular clones harboring thymidine analogue mutations (TAMs); however, their potential for transmission and persistence is unknown. Here, we investigated the temporal appearance of K65K/K66K relative to TAMs in a HIV-1 cohort, their prevalence over time, and their impact on viral fitness in the context of patient-derived reverse transcriptase sequences., Methods: Retrospective analyses of the temporal appearance and longitudinal prevalence of synonymous substitutions and drug resistance mutations were performed using the British Columbia Centre for Excellence in HIV/AIDS Drug Treatment Program (DTP) database. Plasma-derived HIV-1 from the DTP was used to generate infectious molecular clones. Growth competition assays were performed to determine viral fitness., Results: The prevalence of K65K/K66K in drug-naïve individuals tripled from 11% in 1997 to 37% in 2014 (P < 0.0001, n = 5221), with K66K mainly accounting for the increase. These mutations emerged in drug-treated individuals without TAMs in 14% of the cohort and conferred a fitness advantage in the context of patient-derived multidrug-resistant (MDR) virus in the absence of drug., Conclusion: The appearance of K65K/K66K in drug-treated individuals was largely independent of TAMs, suggesting alternative factors are likely associated with their emergence. The increasing K65K/K66K prevalence to over a third of treatment-naïve individuals in the mostly subtype B DTP cohort and their ability to confer a fitness advantage to multidrug-resistant virus might explain the transmission and persistence of virus harbouring K65K/K66K in untreated individuals, and highlights their role in adaptive HIV-1 evolution.

Resistance to combined antiretroviral therapy (cART) in HIV-1-infected individuals is typically due to nonsynonymous mutations that change the protein sequence; however, the selection of synonymous or 'silent' mutations in the HIV-1 genome with cART has been reported. These silent K65K and K66K mutations in the HIV-1 reverse transcriptase (RT) occur in over 35% of drug-experienced individuals and are highly associated with the thymidine analog mutations D67N and K70R, which confer decreased susceptibility to most nucleoside and nucleotide RT inhibitors. However, the basis for selection of these silent mutations under selective drug pressure is unknown. Using Illumina next-generation sequencing, we demonstrate that the D67N/K70R substitutions in HIV-1 RT increase indel frequency by 100-fold at RT codons 65-67, consequently impairing viral fitness. Introduction of either K65K or K66K into HIV-1 containing D67N/K70R reversed the error-prone DNA synthesis at codons 65-67 in RT and improved viral replication fitness, but did not impact RT inhibitor drug susceptibility. These data provide new mechanistic insights into the role of silent mutations selected during antiretroviral therapy and have broader implications for the relevance of silent mutations in the evolution and fitness of RNA viruses., (© The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Topical microbicides for use by women to prevent the transmission of human immunodeficiency virus (HIV) and other sexually transmitted infections are urgently required. Dendrimers are highly branched nanoparticles being developed as microbicides. SPL7013 is a dendrimer with broad-spectrum activity against HIV type I (HIV-1) and -2 (HIV-2), herpes simplex viruses type-1 (HSV-1) and -2 (HSV-2) and human papillomavirus. SPL7013 [3% (w/w)] has been formulated in a mucoadhesive carbopol gel (VivaGel®) for use as a topical microbicide. Previous studies showed that SPL7013 has similar potency against CXCR4-(X4) and CCR5-using (R5) strains of HIV-1 and that it blocks viral entry. However, the ability of SPL7013 to directly inactivate HIV-1 is unknown. We examined whether SPL7013 demonstrates virucidal activity against X4 (NL4.3, MBC200, CMU02 clade EA and 92UG046 clade D), R5 (Ba-L, NB25 and 92RW016 clade A) and dual-tropic (R5X4; MACS1-spln) HIV-1 using a modified HLA-DR viral capture method and by polyethylene glycol precipitation. Evaluation of virion integrity was determined by ultracentrifugation through a sucrose cushion and detection of viral proteins by Western blot analysis. SPL7013 demonstrated potent virucidal activity against X4 and R5X4 strains, although virucidal activity was less potent for the 92UG046 X4 clade D isolate. Where potent virucidal activity was observed, the 50% virucidal concentrations were similar to the 50% effective concentrations previously reported in drug susceptibility assays, indicating that the main mode of action of SPL7013 is by direct viral inactivation for these strains. In contrast, SPL7013 lacked potent virucidal activity against R5 HIV-1 strains. Evaluation of the virucidal mechanism showed that SPL7013-treated NL4.3, 92UG046 and MACS1-spln virions were intact with no significant decrease in gp120 surface protein with respect to p24 capsid content compared to the corresponding untreated virus. These studies demonstrate that SPL7013 is virucidal against HIV-1 strains that utilize the CXCR4 coreceptor but not viruses tested in this study that solely use CCR5 by a mechanism that is distinct from virion disruption or loss of gp120. In addition, the mode of action by which SPL7013 prevents infection of cells with X4 and R5X4 strains is likely to differ from R5 strains of HIV-1., (Copyright © 2011 Elsevier B.V. All rights reserved.)