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Interactions between human immunodeficiency virus type 1 (HIV-1) and the host factors or restriction factors of its target cells determine the cell’s susceptibility to, and outcome of, infection. Factors intrinsic to the cell are involved at every step of the HIV-1 replication cycle, contributing to productive infection and replication, or severely attenuating the chances of success. Furthermore, factors unique to certain cell types contribute to the differences in infection between these cell types. Understanding the involvement of these factors in HIV-1 infection is a key requirement for the development of anti-HIV-1 therapies. As the list of factors grows, and the dynamic interactions between these factors and the virus are elucidated, comprehensive and up-to-date summaries that recount the knowledge gathered after decades of research are beneficial to the field, displaying what is known so that researchers can build off the groundwork of others to investigate what is unknown. Herein, we aim to provide a review focusing on protein host factors, both well-known and relatively new, that impact HIV-1 replication in a positive or negative manner at each stage of the replication cycle, highlighting factors unique to the various HIV-1 target cell types where appropriate.

Antiretroviral therapy can reduce HIV-1 to undetectable levels in peripheral blood, but the effectiveness of treatment in suppressing replication in lymphoid tissue reservoirs has not been determined. Here we show in lymph node samples obtained before and during 6 mo of treatment that the tissue concentrations of five of the most frequently used antiretroviral drugs are much lower than in peripheral blood. These lower concentrations correlated with continued virus replication measured by the slower decay or increases in the follicular dendritic cell network pool of virions and with detection of viral RNA in productively infected cells. The evidence of persistent replication associated with apparently suboptimal drug concentrations argues for development and evaluation of novel therapeutic strategies that will fully suppress viral replication in lymphatic tissues. These strategies could avert the long-term clinical consequences of chronic immune activation driven directly or indirectly by low-level viral replication to thereby improve immune reconstitution.

Significance Antiretroviral therapy (ART) effectively suppresses HIV replication; however, treatment cannot be stopped, because latently infected CD4+ T cells will rekindle infection. As one estimate of the size of the pool of latently infected cells that must be purged for cure, we asked whether recrudescent infection is the result of reactivation from one or a larger number latently infected cells. We briefly stopped ART in fully suppressed patients to see how widespread new infections were in the lymphoid tissues (LTs) and how diverse rebounding/founder viruses were in peripheral blood. Recrudescent infection was detectable in multiple different LTs, and the population was genetically diverse, consistent with reactivation from a larger number of cells. These findings underscore the challenges facing strategies to eradicate HIV infection. Antiretroviral therapy (ART) suppresses HIV replication in most individuals but cannot eradicate latently infected cells established before ART was initiated. Thus, infection rebounds when treatment is interrupted by reactivation of virus production from this reservoir. Currently, one or a few latently infected resting memory CD4 T cells are thought be the principal source of recrudescent infection, but this estimate is based on peripheral blood rather than lymphoid tissues (LTs), the principal sites of virus production and persistence before initiating ART. We, therefore, examined lymph node (LN) and gut-associated lymphoid tissue (GALT) biopsies from fully suppressed subjects, interrupted therapy, monitored plasma viral load (pVL), and repeated biopsies on 12 individuals as soon as pVL became detectable. Isolated HIV RNA-positive (vRNA+) cells were detected by in situ hybridization in LTs obtained before interruption in several patients. After interruption, multiple foci of vRNA+ cells were detected in 6 of 12 individuals as soon as pVL was measureable and in some subjects, in more than one anatomic site. Minimal estimates of the number of rebounding/founder (R/F) variants were determined by single-gene amplification and sequencing of viral RNA or DNA from peripheral blood mononuclear cells and plasma obtained at or just before viral recrudescence. Sequence analysis revealed a large number of R/F viruses representing recrudescent viremia from multiple sources. Together, these findings are consistent with the origins of recrudescent infection by reactivation from many latently infected cells at multiple sites. The inferred large pool of cells and sites to rekindle recrudescent infection highlights the challenges in eradicating HIV.

RNA interference (RNAi) is the process by which double-stranded RNA (dsRNA) directs sequence-specific degradation of messenger RNA in animal and plant cells. In mammalian cells, RNAi can be triggered by 21-nucleotide duplexes of small interfering RNA (siRNA). Here we describe inhibition of early and late steps of HIV-1 replication in human cell lines and primary lymphocytes by siRNAs targeted to various regions of the HIV-1 genome. We demonstrate that synthetic siRNA duplexes or plasmid-derived siRNAs inhibit HIV-1 infection by specifically degrading genomic HIV-1 RNA, thereby preventing formation of viral complementary-DNA intermediates. These results demonstrate the utility of RNAi for modulating the HIV replication cycle and provide evidence that genomic HIV-1 RNA, as it exists within a nucleoprotein reverse-transcription complex, is amenable to siRNA-mediated degradation.

HIV-1 persists in cellular reservoirs that can reignite viremia if antiretroviral therapy (ART) is interrupted. Therefore, insight into the nature of those reservoirs may be revealed from the composition of recrudescing viremia following treatment cessation. A minor population of macrophage-tropic (M-tropic) viruses was identified in a library of recombinant viruses constructed with individual envelope genes that were obtained from plasma of six individuals undergoing analytic treatment interruption (ATI). M-tropic viruses could also be enriched from post-ATI plasma using macrophagespecific (CD14) but not CD4+ T cell-specific (CD3) antibodies, suggesting that M-tropic viruses had a macrophage origin. Molecular clock analysis indicated that the establishment of M-tropic HIV-1 variants predated ATI. Collectively, these data suggest that macrophages are a viral reservoir in HIV-1–infected individuals on effective ART and that M-tropic variants can appear in rebounding viremia when treatment is interrupted. These findings have implications for the design of curative strategies for HIV-1.

Despite highly active antiretroviral therapy, active replication persists and drives immune activation in some individuals with HIV. This activation is higher if therapy is intensified by additional antiretroviral drugs ( pages 373–374 ). Highly active antiretroviral therapy (HAART) results in potent and durable suppression of HIV-1 viremia. However, HIV-1 replication resumes if therapy is interrupted 1 , 2 . Although it is generally believed that active replication has been halted in individuals on HAART, immune activation and inflammation continue at abnormal levels 3 , suggesting continued, low-level viral replication. To assess whether active replication might be driving immune activation in HAART, we examined the impact of treatment intensification with the integrase inhibitor raltegravir on viral complementary DNA and immune activation parameters. In the presence of raltegravir, linear HIV-1 cDNA is prevented from integrating into chromatin and is subsequently converted to episomal cDNAs 4 , 5 . Raltegravir intensification of a three-drug suppressive HAART regimen resulted in a specific and transient increase in episomal DNAs in a large percentage of HAART-suppressed subjects. Furthermore, in subjects with these episomal DNAs, immune activation was higher at baseline and was subsequently normalized after raltegravir intensification. These results suggest that, despite suppressive HAART, active replication persists in some infected individuals and drives immune activation. The ability of raltegravir intensification to perturb the reservoir that supports active replication has implications for therapeutic strategies aimed at achieving viral eradication.

Reverse transcription of HIV-1 results in the generation of a linear cDNA that serves as the precursor to the integrated provirus. Other classes of extrachromosomal viral cDNA molecules can be found in acutely infected cells including the 1-LTR and 2-LTR circles of viral DNA, also referred as episomal HIV-1 DNA. Circulating CD4 + T-cells of treatment-naïve individuals contain significant levels of unintegrated forms of HIV-1 DNA. However, the importance of episomal HIV-1 DNA in the study of viral persistence during antiviral therapy (ART) is debatable. 2-LTR circles are preferentially observed in the effector memory CD4 + T cell subset of long-term treated subjects. Treatment intensification of standard regimens has been used to determine if more potent ART can impact viral reservoir activity. Adding a potent antiretroviral drug to a stable triple-drug regimen has no measurable impact on plasma HIV-1 RNA levels, suggesting that ongoing cycles of HIV-1 replication are not a major mechanism driving persistent plasma viremia during triple-drug ART. However, in randomized clinical trials of HIV-1-infected adults on apparently effective ART, the addition of an integrase inhibitor (raltegravir) to stable regimens resulted in a transient increase in 2-LTR circles in some patients, suggesting a pre-intensification steady-state in which the processes of virion generation and de novo infection were occurring. Mathematical modeling of 2-LTR production during integrase inhibitor intensification suggests the coexistence, at different levels, of ongoing de novo infection and de novo replication mechanisms, specifically in inflamed lymphoid drug sanctuaries. Most reports looking into potential changes in 2-LTR circles in interventional clinical studies have simultaneously assessed other potential surrogate markers of viral persistence. Transient increases in 2-LTR circles have been correlated to decreases in CD8 + T-cell activation, transient CD45RA − CD4 + T-cell redistribution, and decreases in the hypercoagulation biomarker D-dimer in ART-intensified individuals. It is difficult, however, to establish a systematic association because the level of correlation with different types of markers differs significantly among studies. In conclusion, despite suppressive ART, a steady-state of de novo infection may persist in some infected individuals and that this may drive immune activation and inflammation changes reflecting residual viral reservoir activity during otherwise apparently suppressive ART.

Wastewater is a geospatially- and temporally-linked microbial fingerprint of a given population, making it a potentially valuable tool for tracking public health across locales and time. Here, we integrate targeted and bulk RNA sequencing (N = 2238 samples) to track the viral, bacterial, and functional content over geospatially distinct areas within Miami Dade County, USA, from 2020-2022. We used targeted amplicon sequencing to track diverse SARS-CoV-2 variants across space and time, and we found a tight correspondence with positive PCR tests from University students and Miami-Dade hospital patients. Additionally, in bulk metatranscriptomic data, we demonstrate that the bacterial content of different wastewater sampling locations serving small population sizes can be used to detect putative, host-derived microorganisms that themselves have known associations with human health and diet. We also detect multiple enteric pathogens (e.g., Norovirus ) and characterize viral diversity across sites. Moreover, we observed an enrichment of antimicrobial resistance genes (ARGs) in hospital wastewater; antibiotic-specific ARGs correlated to total prescriptions of those same antibiotics (e.g Ampicillin, Gentamicin). Overall, this effort lays the groundwork for systematic characterization of wastewater that can potentially influence public health decision-making. Wastewater is a promising source of data for continuous monitoring of pathogens in communities, but analysis protocols and methods are still being established. Here, the authors develop sequencing and analysis protocols and use them to evaluate the microbial content of longitudinal wastewater samples from Miami-Dade County, USA.

Chronic infection with human immunodeficiency virus (HIV) and hepatitis C virus (HCV) affects an estimated 35 million and 75 million individuals worldwide, respectively. These viruses induce persistent inflammation which often drives the development or progression of organ-specific diseases and even cancer including Hepatocellular Carcinoma (HCC). In this study, we sought to examine inflammatory responses following HIV or HCV stimulation of macrophages or Kupffer cells (KCs), that may contribute to virus mediated inflammation and subsequent liver disease. KCs are liver-resident macrophages and reports have provided evidence that HIV can stimulate and infect them. In order to characterize HIV-intrinsic innate immune responses that may occur in the liver, we performed microarray analyses on KCs following HIV stimulation. Our data demonstrate that KCs upregulate several innate immune signaling pathways involved in inflammation, myeloid cell maturation, stellate cell activation, and Triggering Receptor Expressed on Myeloid cells 1 (TREM1) signaling. TREM1 is a member of the immunoglobulin superfamily of receptors and it is reported to be involved in systemic inflammatory responses due to its ability to amplify activation of host defense signaling pathways. Our data demonstrate that stimulation of KCs with HIV or HCV induces the upregulation of TREM1. Additionally, HIV viral proteins can upregulate expression of TREM1 mRNA through NF-кB signaling. Furthermore, activation of the TREM1 signaling pathway, with a targeted agonist, increased HIV or HCV-mediated inflammatory responses in macrophages due to enhanced activation of the ERK1/2 signaling cascade. Silencing TREM1 dampened inflammatory immune responses elicited by HIV or HCV stimulation. Finally, HIV and HCV infected patients exhibit higher expression and frequency of TREM1 and CD68 positive cells. Taken together, TREM1 induction by HIV contributes to chronic inflammation in the liver and targeting TREM1 signaling may be a therapeutic option to minimize HIV induced chronic inflammation.

Introduction Barriers to pre‐exposure prophylaxis (PrEP) access have limited its reach to priority populations. Community‐based mobile clinics have potential to broaden PrEP engagement. We evaluated reach and persistence for fixed and mobile clinic cohorts in Miami‐Dade County, Florida. Methods This observational cohort study analysed data from 1896 clients engaged through our fixed or mobile clinic from August 2018 to March 2023. Services were offered at no cost to clients. The same staff and package of barrier‐lowering strategies was deployed across fixed and mobile clinic sites. Chi‐square and Fisher's exact test or the Kruskal–Wallis test were used to test for differences in characteristics across sites as well as across services sought. Kaplan–Meier curves were generated to evaluate persistence on PrEP and in care, defined as completion of at least one clinic visit (including PrEP prescribing, for PrEP persistence, or for any reason, for persistence in care) within 24 weeks of the prior visit. Cox proportional hazards models were used to evaluate risk factors for discontinuation of PrEP or clinic care by 48 weeks by gender, race, ethnicity, insurance status and site. Results The fixed and mobile clinics reached 781 and 1109 clients, respectively, during the study period. The median client age was 35 years; the majority (70.4%) of clients were cisgender men, identified as Hispanic/Latino (62.5%) and were men who have sex with men (54.5%). The mobile clinic extended reach to a higher proportion of cisgender women (32.1% mobile vs. 12.9% for fixed clinic), Black clients (34.5% vs. 13.1%) and older clients (median 37 vs. 33 years) compared with the fixed setting. Uninsured individuals, men and those who initiated services in the mobile clinic were more likely to continue PrEP to 48 weeks (HR: 1.20, p = 0.01; HR: 2.02, p<0.01; HR: 1.68, p<0.01, respectively). Persistence did not differ by race or ethnicity. Conclusions A mobile clinic strategy for PrEP engagement can increase reach to key populations underrepresented in HIV prevention care including cisgender women and Black clients. Persistence in PrEP was increased for the mobile clinic cohort, suggesting an additional benefit to this modality beyond other barrier‐lowering strategies employed in our fixed and mobile clinics.