Chronic HIV Transcription, Translation, and Persistent Inflammation.

People with HIV exhibit persistent inflammation that correlates with HIV-associated comorbidities including accelerated aging, increased risk of cardiovascular disease, and neuroinflammation. Mechanisms that perpetuate chronic inflammation in people with HIV undergoing antiretroviral treatments are poorly understood. One hypothesis is that the persistent low-level expression of HIV proviruses, including RNAs generated from defective proviral genomes, drives the immune dysfunction that is responsible for chronic HIV pathogenesis. We explore factors during HIV infection that contribute to the generation of a pool of defective proviruses as well as how HIV-1 mRNA and proteins alter immune function in people living with HIV.

Virally Suppressed People Living with HIV Who Use Opioids Have Diminished Latency Reversal.

Of the 12 million people who inject drugs worldwide, 13% live with HIV. Whether opioid use impacts HIV pathogenesis and latency is an outstanding question. To gain insight into whether opioid use influences the proviral landscape and latent HIV reservoir, we performed intact proviral DNA assays (IPDA) on peripheral blood mononuclear cells (PBMCs) from antiretroviral therapy (ART)-suppressed people living with HIV (PWH) with or without current opioid use. No differences were observed between PWH with and without opioid use in the frequency of HIV intact and defective proviral genomes. To evaluate the latent reservoir, we activated PBMCs from ART-suppressed PWH with or without opioid use and assessed the induction of HIV RNA. PWH using opioids had diminished responses to ex vivo HIV reactivation, suggesting a smaller reversible reservoir of HIV-1 latently infected cells. However, in vitro studies using primary CD4+ T cells treated with morphine showed no effect of opioids on HIV-1 infection, replication or latency establishment. The discrepancy in our results from in vitro and clinical samples suggests that while opioids may not directly impact HIV replication, latency and reactivation in CD4+ T cells, opioid use may indirectly shape the HIV reservoir in vivo by modulating general immune functions.

Defective HIV-1 genomes and their potential impact on HIV pathogenesis.

Defective HIV-1 proviruses represent a population of viral genomes that are selected for by immune pressures, and clonally expanded to dominate the persistent HIV-1 proviral genome landscape. There are examples of RNA and protein expression from these compromised genomes which are generated by a variety of mechanisms. Despite the evidence that these proviruses are transcribed and translated, their role in HIV pathogenesis has not been fully explored. The potential for these genomes to participate in immune stimulation is particularly relevant considering the accumulation of cells harboring these defective proviruses over the course of antiretroviral therapy in people living with HIV. The expression of defective proviruses in different cells and tissues could drive innate sensing mechanisms and inflammation. They may also alter antiviral T cell responses and myeloid cell functions that directly contribute to HIV-1 associated chronic comorbidities. Understanding the impact of these defective proviruses needs to be considered as we advance cure strategies that focus on targeting the diverse population of HIV-1 proviral genomes.

Host T Cell Dedifferentiation Effects Drive HIV-1 Latency Stability.

The development of therapies to eliminate the latent HIV-1 reservoir is hampered by our incomplete understanding of the biomolecular mechanism governing HIV-1 latency. To further complicate matters, recent single-cell RNA sequencing (scRNA-seq) studies reported extensive heterogeneity between latently HIV-1-infected primary T cells, implying that latent HIV-1 infection can persist in greatly differing host cell environments. We show here that transcriptomic heterogeneity is also found between latently infected T cell lines, which allowed us to study the underlying mechanisms of intercell heterogeneity at high signal resolution. Latently infected T cells exhibited a dedifferentiated phenotype, characterized by the loss of T cell-specific markers and gene regulation profiles reminiscent of hematopoietic stem cells (HSC). These changes had functional consequences. As reported for stem cells, latently HIV-1-infected T cells efficiently forced lentiviral superinfections into a latent state and favored glycolysis. As a result, metabolic reprogramming or cell redifferentiation destabilized latent infection. Guided by these findings, data mining of single-cell RNA-seq data of latently HIV-1-infected primary T cells from patients revealed the presence of similar dedifferentiation motifs. More than 20% of the highly detectable genes that were differentially regulated in latently infected cells were associated with hematopoietic lineage development (e.g., HUWE1, IRF4, PRDM1, BATF3, TOX, ID2, IKZF3, and CDK6) or were hematopoietic markers (SRGN; hematopoietic proteoglycan core protein). The data add to evidence that the biomolecular phenotype of latently HIV-1-infected cells differs from that of normal T cells and strategies to address their differential phenotype need to be considered in the design of therapeutic cure interventions. IMPORTANCE HIV-1 persists in a latent reservoir in memory CD4 T cells for the lifetime of a patient. Understanding the biomolecular mechanisms used by the host cells to suppress viral expression will provide essential insights required to develop curative therapeutic interventions. Unfortunately, our current understanding of these control mechanisms is still limited. By studying gene expression profiles, we demonstrated that latently HIV-1-infected T cells have a dedifferentiated T cell phenotype. Software-based data integration allowed the identification of drug targets that would redifferentiate viral host cells and, by extension, destabilize latent HIV-1 infection events. The importance of the presented data lies within the clear demonstration that HIV-1 latency is a host cell phenomenon. As such, therapeutic strategies must first restore proper host cell functionality to accomplish efficient HIV-1 reactivation.

Intragenic proviral elements support transcription of defective HIV-1 proviruses.

HIV-1 establishes a persistent proviral reservoir by integrating into the genome of infected host cells. Current antiretroviral treatments do not target this persistent population of proviruses which include latently infected cells that upon treatment interruption can be reactivated to contribute to HIV-1 rebound. Deep sequencing of persistent HIV proviruses has revealed that greater than 90% of integrated HIV genomes are defective and unable to produce infectious virions. We hypothesized that intragenic elements in the HIV genome support transcription of aberrant HIV-1 RNAs from defective proviruses that lack long terminal repeats (LTRs). Using an intact provirus detection assay, we observed that resting CD4+ T cells and monocyte-derived macrophages (MDMs) are biased towards generating defective HIV-1 proviruses. Multiplex reverse transcription droplet digital PCR identified env and nef transcripts which lacked 5' untranslated regions (UTR) in acutely infected CD4+ T cells and MDMs indicating transcripts are generated that do not utilize the promoter within the LTR. 5'UTR-deficient env transcripts were also identified in a cohort of people living with HIV (PLWH) on ART, suggesting that these aberrant RNAs are produced in vivo. Using 5' rapid amplification of cDNA ends (RACE), we mapped the start site of these transcripts within the Env gene. This region bound several cellular transcription factors and functioned as a transcriptional regulatory element that could support transcription and translation of downstream HIV-1 RNAs. These studies provide mechanistic insights into how defective HIV-1 proviruses are persistently expressed to potentially drive inflammation in PLWH.

Pandemic Response Requires Research Samples: A U.S. Safety-Net Hospital's Experience and Call for National Action.

Biorepositories provide a critical resource for gaining knowledge of emerging infectious diseases and offer a mechanism to rapidly respond to outbreaks; the emergence of the novel coronavirus, SARS-CoV-2, has proved their importance. During the COVID-19 pandemic, the absence of centralized, national biorepository efforts meant that the onus fell on individual institutions to establish sample repositories. As a safety-net hospital, Boston Medical Center (BMC) recognized the importance of creating a COVID-19 biorepository to both support critical science at BMC and ensure representation in research for its urban patient population, most of whom are from underserved communities. This article offers a realistic overview of the authors' experience in establishing this biorepository at the onset of the COVID-19 pandemic during the height of the first surge of cases in Boston, Massachusetts, with the hope that the challenges and solutions described are useful to other institutions. Going forward, funders, policymakers, and infectious disease and public health communities must support biorepository implementation as an essential element of future pandemic preparedness.

Activity of CcpA-Regulated GH18 Family Glycosyl Hydrolases That Contributes to Nutrient Acquisition and Fitness in Enterococcus faecalis.

The ability of Enterococcus faecalis to colonize host anatomical sites is dependent on its adaptive response to host conditions. Three glycosyl hydrolase gene clusters, each belonging to glycosyl hydrolase family 18 (GH18) (ef0114, ef0361, and ef2863), in E. faecalis were previously found to be upregulated under glucose-limiting conditions. The GH18 catalytic domain is present in proteins that are classified as either chitinases or ß-1,4 endo-ß-N-acetylglucosaminidases (ENGases) based on their ß-1,4 endo-N-acetyl-ß-d-glucosaminidase activity, and ENGase activity is commonly associated with cleaving N-linked glycoprotein, an abundant glycan structure on host epithelial surfaces. Here, we show that all three hydrolases are negatively regulated by the transcriptional regulator carbon catabolite protein A (CcpA). Additionally, we demonstrate that a constitutively active CcpA variant represses the expression of CcpA-regulated genes irrespective of glucose availability. Previous studies showed that the GH18 catalytic domains of EndoE (EF0114) and EfEndo18A (EF2863) were capable of deglycosylating RNase B, a model high-mannose-type glycoprotein. However, it remained uncertain which glycosidase is primarily responsible for the deglycosylation of high-mannose-type glycoproteins. In this study, we show by mutation analysis as well as a dose-dependent analysis of recombinant protein expression that EfEndo18A is primarily responsible for deglycosylating high-mannose glycoproteins and that the glycans removed by EfEndo18A support growth under nutrient-limiting conditions in vitro. In contrast, IgG is representative of a complex-type glycoprotein, and we demonstrate that the GH18 domain of EndoE is primarily responsible for the removal of this glycan decoration. Finally, our data highlight the combined contribution of glycosidases to the virulence of E. faecalis in vivo.

A functional screen identifies transcriptional networks that regulate HIV-1 and HIV-2.

The molecular networks involved in the regulation of HIV replication, transcription, and latency remain incompletely defined. To expand our understanding of these networks, we performed an unbiased high-throughput yeast one-hybrid screen, which identified 42 human transcription factors and 85 total protein-DNA interactions with HIV-1 and HIV-2 long terminal repeats. We investigated a subset of these transcription factors for transcriptional activity in cell-based models of infection. KLF2 and KLF3 repressed HIV-1 and HIV-2 transcription in CD4+ T cells, whereas PLAGL1 activated transcription of HIV-2 through direct protein-DNA interactions. Using computational modeling with interacting proteins, we leveraged the results from our screen to identify putative pathways that define intrinsic transcriptional networks. Overall, we used a high-throughput functional screen, computational modeling, and biochemical assays to identify and confirm several candidate transcription factors and biochemical processes that influence HIV-1 and HIV-2 transcription and latency.

Building on Antimicrobial Stewardship Programs Through Integration with Electronic Medical Records: The Australian Experience.

Antimicrobial stewardship (AMS) is well established in Australian hospitals. Electronic medical record (EMR) implementation has lagged in Australia, with two Healthcare Information and Management Systems Society (HIMSS) Stage 6 hospitals and one Stage 7 hospital as of September 2020. Specific barriers faced by AMS teams with paper-based prescribing and medical records include real-time identification of antimicrobials orders; the ability to prospectively monitor antimicrobial use; and the integration of fundamental point of prescribing AMS principles into routine clinical practice. There are few local guidelines to assist Australian hospitals and AMS teams beyond "out of the box" EMR functionality. EMR implementation has enormous potential to positively impact AMS teams through more efficient workflows and the ability to expand the reach and coverage of AMS activities. There are inevitable limitations associated with EMR implementation that must be considered. In this paper, four Australian hospitals share their experience with EMR roll out, AMS customisation and how they have overcome specific barriers in local AMS practice.

Targeted Chromatinization and Repression of HIV-1 Provirus Transcription with Repurposed CRISPR/Cas9.

The major barrier to HIV-1 cure is the persistence of latent provirus, which is not eradicated by antiretroviral therapy. The "shock and kill" approach entails stimulating viral production with latency-reversing agents followed by the killing of cells actively producing the virus by immune clearance. However, this approach does not induce all intact proviruses, leaving a residual reservoir. CRISPR/Cas9 has been utilized to excise integrated Human Immunodeficiency Virus (HIV) DNA from infected cells in an RNA-guided, sequence-specific manner. Here, we seek to epigenetically silence the proviral DNA by introducing nuclease-deficient disabled Cas9 (dCas9) coupled with a transcriptional repressor domain derived from Kruppel-associated box (KRAB). We show that specific guide RNAs (gRNAs) and dCas9-KRAB repress HIV-1 transcription and reactivation of latent HIV-1 provirus. This repression is correlated with chromatin changes, including decreased H3 histone acetylation and increased histone H3 lysine 9 trimethylation, histone marks that are associated with transcriptional repression. dCas9-KRAB-mediated inhibition of HIV-1 transcription suggests that CRISPR can be engineered as a tool for block-and-lock strategies.

Enhanced Human Immunodeficiency Virus-1 Replication in CD4+ T Cells Derived From Individuals With Latent Mycobacterium tuberculosis Infection.

BACKGROUND: Mycobacterium tuberculosis (Mtb) and human immunodeficiency virus (HIV) coinfection increases mortality, accelerates progression to acquired immune deficiency syndrome, and exacerbates tuberculosis disease. However, the impact of pre-existing Mtb infection on subsequent HIV infection has not been fully explored. We hypothesized that Mtb infection creates an immunological environment that influences the course of HIV infection, and we investigated whether pre-existing Mtb infection impacts the susceptibility of CD4+ T cells to HIV-1 infection. METHODS: Plasma and blood CD4+ T cells isolated from HIV-negative individuals across the Mtb infection spectrum and non-Mtb-infected control individuals were analyzed for inflammation markers and T-cell phenotypes. CD4+ T cells were infected with HIV-1 in vitro and were monitored for viral replication. RESULTS: We observed differences in proinflammatory cytokines and the relative proportion of memory T-cell subsets depending on Mtb infection status. CD4+ T cells derived from individuals with latent Mtb infection supported more efficient HIV-1 transcription, release, and replication. Enhanced HIV-1 replication correlated with higher percentages of CD4+ TEM and TTD cells. CONCLUSIONS: Pre-existing Mtb infection creates an immunological environment that reflects Mtb infection status and influences the susceptibility of CD4+ T cells to HIV-1 replication. These findings provide cellular and molecular insights into how pre-existing Mtb infection influences HIV-1 pathogenesis.

Single cell transcriptomics reveals opioid usage evokes widespread suppression of antiviral gene program.

Chronic opioid usage not only causes addiction behavior through the central nervous system, but also modulates the peripheral immune system. However, how opioid impacts the immune system is still barely characterized systematically. In order to understand the immune modulatory effect of opioids in an unbiased way, here we perform single-cell RNA sequencing (scRNA-seq) of peripheral blood mononuclear cells from opioid-dependent individuals and controls to show that chronic opioid usage evokes widespread suppression of antiviral gene program in naive monocytes, as well as in multiple immune cell types upon stimulation with the pathogen component lipopolysaccharide. Furthermore, scRNA-seq reveals the same phenomenon after a short in vitro morphine treatment. These findings indicate that both acute and chronic opioid exposure may be harmful to our immune system by suppressing the antiviral gene program. Our results suggest that further characterization of the immune modulatory effects of opioid is critical to ensure the safety of clinical opioids.

Targeting HIV-1 proviral transcription.

Despite the success of antiretroviral therapies, there is no cure for HIV-1 infection due to the establishment of a long-lived latent reservoir that fuels viral rebound upon treatment interruption. 'Shock-and-kill' strategies to diminish the latent reservoir have had modest impact on the reservoir leading to considerations of alternative approaches to target HIV-1 proviruses. This review explores approaches to target HIV-1 transcription as a way to block the provirus expression.

Modified measles with an atypical presentation

We report symptomatic confirmed modified measles infection in a person with one documented MMR (measles, mumps, rubella) vaccination and travel to Indonesia. No secondary cases were identified, consistent with other case reports of modified measles infection. The infectivity of modified measles for contact tracing requirements requires further elucidation.

Strength of T cell signaling regulates HIV-1 replication and establishment of latency.

A major barrier to curing HIV-1 is the long-lived latent reservoir that supports re-emergence of HIV-1 upon treatment interruption. Targeting this reservoir will require mechanistic insights into the establishment and maintenance of HIV-1 latency. Whether T cell signaling at the time of HIV-1 infection influences productive replication or latency is not fully understood. We used a panel of chimeric antigen receptors (CARs) with different ligand binding affinities to induce a range of signaling strengths to model differential T cell receptor signaling at the time of HIV-1 infection. Stimulation of T cell lines or primary CD4+ T cells expressing chimeric antigen receptors supported HIV-1 infection regardless of affinity for ligand; however, only signaling by the highest affinity receptor facilitated HIV-1 expression. Activation of chimeric antigen receptors that had intermediate and low binding affinities did not support provirus transcription, suggesting that a minimal signal is required for optimal HIV-1 expression. In addition, strong signaling at the time of infection produced a latent population that was readily inducible, whereas latent cells generated in response to weaker signals were not easily reversed. Chromatin immunoprecipitation showed HIV-1 transcription was limited by transcriptional elongation and that robust signaling decreased the presence of negative elongation factor, a pausing factor, by more than 80%. These studies demonstrate that T cell signaling influences HIV-1 infection and the establishment of different subsets of latently infected cells, which may have implications for targeting the HIV-1 reservoir.

Mechanisms of HIV-1 cell-to-cell transmission and the establishment of the latent reservoir.

HIV-1 spreads through both the release of cell-free particles and by cell-to-cell transmission. Mounting evidence indicates that cell-to-cell transmission is more efficient than cell-free transmission of particles and likely influences the pathogenesis of HIV-1 infection. This mode of viral transmission also influences the generation and maintenance of the latent reservoir, which represents the main obstacle for curing the infection. In this review we will discuss general cell contact-dependent mechanisms that HIV-1 utilizes for its spread and the evidence pointing to cell-to-cell transmission as a mechanism for the establishment and maintenance of latent infection.

HIV-1-Infected CD4+ T Cells Facilitate Latent Infection of Resting CD4+ T Cells through Cell-Cell Contact.

HIV-1 is transmitted between T cells through the release of cell-free particles and through cell-cell contact. Cell-to-cell transmission is more efficient than cell-free virus transmission, mediates resistance to immune responses, and facilitates the spread of virus among T cells. However, whether HIV cell-to-cell transmission influences the establishment of HIV-1 latency has not been carefully explored. We developed an HIV-1 latency model based on the transmission of HIV-1 directly to resting CD4+ T cells by cell-cell contact. This model recapitulates the spread of HIV-1 in T-cell-dense anatomical compartments. We demonstrate that productively infected activated CD4+ T cells transmit HIV-1 to resting CD4+ T cells in a cell-contact-dependent manner. However, proviruses generated in this fashion are more difficult to induce compared to proviruses generated by cell-free infection, suggesting that cell-to-cell transmission influences the establishment and maintenance of latent infection in resting CD4+ T cells.

CD4+ T Cell Subsets and Pathways to HIV Latency.

Latent infection of CD4+ T cells is the main barrier to eradicating HIV-1 infection from infected patients. The cellular and molecular mechanisms involved in the establishment and maintenance of latent infection are directly linked to the transcriptional program of the different CD4+ T cell subsets targeted by the virus. In this review, we provide an overview of how T cell activation, T cell differentiation into functional subsets, and the mode of initial viral infection influence HIV proviral transcription and entry into latency.

Identification of benzazole compounds that induce HIV-1 transcription.

Despite advances in antiretroviral therapy, HIV-1 infection remains incurable in patients and continues to present a significant public health burden worldwide. While a number of factors contribute to persistent HIV-1 infection in patients, the presence of a stable, long-lived reservoir of latent provirus represents a significant hurdle in realizing an effective cure. One potential strategy to eliminate HIV-1 reservoirs in patients is reactivation of latent provirus with latency reversing agents in combination with antiretroviral therapy, a strategy termed "shock and kill". This strategy has shown limited clinical effectiveness thus far, potentially due to limitations of the few therapeutics currently available. We have identified a novel class of benzazole compounds effective at inducing HIV-1 expression in several cellular models. These compounds do not act via histone deacetylase inhibition or T cell activation, and show specificity in activating HIV-1 in vitro. Initial exploration of structure-activity relationships and pharmaceutical properties indicates that these compounds represent a potential scaffold for development of more potent HIV-1 latency reversing agents.

Virion-Associated Vpr Alleviates a Postintegration Block to HIV-1 Infection of Dendritic Cells.

Viral protein R (Vpr) is an HIV-1 accessory protein whose function remains poorly understood. In this report, we sought to determine the requirement of Vpr for facilitating HIV-1 infection of monocyte-derived dendritic cells (MDDCs), one of the first cell types to encounter virus in the peripheral mucosal tissues. In this report, we characterize a significant restriction of Vpr-deficient virus replication and spread in MDDCs alone and in cell-to-cell spread in MDDC-CD4+ T cell cocultures. This restriction of HIV-1 replication in MDDCs was observed in a single round of virus replication and was rescued by the expression of Vpr in trans in the incoming virion. Interestingly, infections of MDDCs with viruses that encode Vpr mutants unable to interact with either the DCAF1/DDB1 E3 ubiquitin ligase complex or a host factor hypothesized to be targeted for degradation by Vpr also displayed a significant replication defect. While the extent of proviral integration in HIV-1-infected MDDCs was unaffected by the absence of Vpr, the transcriptional activity of the viral long terminal repeat (LTR) from Vpr-deficient proviruses was significantly reduced. Together, these results characterize a novel postintegration restriction of HIV-1 replication in MDDCs and show that the interaction of Vpr with the DCAF1/DDB1 E3 ubiquitin ligase complex and the yet-to-be-identified host factor might alleviate this restriction by inducing transcription from the viral LTR. Taken together, these findings identify a robust in vitro cell culture system that is amenable to addressing mechanisms underlying Vpr-mediated enhancement of HIV-1 replication.IMPORTANCE Despite decades of work, the function of the HIV-1 protein Vpr remains poorly understood, primarily due to the lack of an in vitro cell culture system that demonstrates a deficit in replication upon infection with viruses in the absence of Vpr. In this report, we describe a novel cell infection system that utilizes primary human dendritic cells, which display a robust decrease in viral replication upon infection with Vpr-deficient HIV-1. We show that this replication difference occurs in a single round of infection and is due to decreased transcriptional output from the integrated viral genome. Viral transcription could be rescued by virion-associated Vpr. Using mutational analysis, we show that domains of Vpr involved in binding to the DCAF1/DDB1/E3 ubiquitin ligase complex and prevention of cell cycle progression into mitosis are required for LTR-mediated viral expression, suggesting that the evolutionarily conserved G2 cell cycle arrest function of Vpr is essential for HIV-1 replication.