Magnitude of Type I Interferon Responses by Plasmacytoid Dendritic Cells After TLR7 Stimulation Is Associated With Human Immunodeficiency Virus Type 1 (HIV-1) Reservoir Sizes in Cisgender Women With HIV-1 on Antiretroviral Therapy.

Human immunodeficiency virus type 1 (HIV-1) disease manifestations differ between cisgender women and men, including better control of viral replication during primary infection and less frequent residual HIV-1 replication on antiretroviral therapy (ART) in cisgender women with HIV-1 (WWH). Investigating plasmacytoid dendritic cell (pDC) functions and HIV-1 reservoir sizes in 20 WWH on stable ART, we observed inverse correlations between interferon-α and tumor necrosis factor responses of pDCs to Toll-like receptor 7/8 stimulation and intact/total proviral HIV-1 DNA levels. Additionally, ISG15 mRNA levels in peripheral blood mononuclear cells correlated with cytokine responses of pDCs. These findings demonstrate an association between higher type I interferon responses and lower HIV-1 reservoir sizes in WWH on ART, warranting studies to identify the underlying mechanisms.

Long non-coding RNAs in biomarking COVID-19: a machine learning-based approach.

BACKGROUND: The coronavirus pandemic that started in 2019 has caused the highest mortality and morbidity rates worldwide. Data on the role of long non-coding RNAs (lncRNAs) in coronavirus disease 2019 (COVID-19) is scarce. We aimed to elucidate the relationship of three important lncRNAs in the inflammatory states, H19, taurine upregulated gene 1 (TUG1), and colorectal neoplasia differentially expressed (CRNDE) with key factors in inflammation and fibrosis induction including signal transducer and activator of transcription3 (STAT3), alpha smooth muscle actin (α-SMA), tumor necrosis factor-alpha (TNF-α), and interleukin-6 (IL-6) in COVID-19 patients with moderate to severe symptoms. METHODS: Peripheral blood mononuclear cells from 28 COVID-19 patients and 17 healthy controls were collected. The real-time quantitative polymerase chain reaction (RT-qPCR) was performed to evaluate the expression of RNAs and lncRNAs. Western blotting analysis was also performed to determine the expression levels of STAT3 and α-SMA proteins. Machine learning and receiver operating characteristic (ROC) curve analysis were carried out to evaluate the distinguishing ability of lncRNAs. RESULTS: The expression levels of H19, TUG1, and CRNDE were significantly overexpressed in COVID-19 patients compared to healthy controls. Moreover, STAT3 and α-SMA expression levels were remarkedly increased at both transcript and protein levels in patients with COVID-19 compared to healthy subjects and were correlated with Three lncRNAs. Likewise, IL-6 and TNF-α were considerably upregulated in COVID-19 patients. Machine learning and ROC curve analysis showed that CRNDE-H19 panel has the proper ability to distinguish COVID-19 patients from healthy individuals (area under the curve (AUC) = 0.86). CONCLUSION: The overexpression of three lncRNAs in COVID-19 patients observed in this study may align with significant manifestations of COVID-19. Furthermore, their co-expression with STAT3 and α-SMA, two critical factors implicated in inflammation and fibrosis induction, underscores their potential involvement in exacerbating cardiovascular, pulmonary and common symptoms and complications associated with COVID-19. The combination of CRNDE and H19 lncRNAs seems to be an impressive host-based biomarker panel for screening and diagnosis of COVID-19 patients from healthy controls. Research into lncRNAs can provide a robust platform to find new viral infection-related mediators and propose novel therapeutic strategies for viral infections and immune disorders.

Reduction in ACE2 expression in peripheral blood mononuclear cells during COVID-19 - implications for post COVID-19 conditions.

BACKGROUND: Severe COVID-19 is uncommon, restricted to 19% of the total population. In response to the first virus wave (alpha variant of SARS-CoV-2), we investigated whether a biomarker indicated severity of disease and, in particular, if variable expression of angiotensin converting enzyme 2 (ACE2) in blood might clarify this difference in risk and of post COVID -19 conditions (PCC). METHODS: The IRB-approved study compared patients hospitalized with severe COVID-19 to healthy controls. Severe infection was defined requiring oxygen or increased oxygen need from baseline at admission with positive COVID-19 PCR. A single blood sample was obtained from patients within a day of admission. ACE2 RNA expression in blood cells was measured by an RT-PCR assay. Plasma ACE1 and ACE2 enzyme activities were quantified by fluorescent peptides. Plasma TIMP-1, PIIINP and MMP-9 antigens were quantified by ELISA. Data were entered into REDCap and analyzed using STATA v 14 and GraphPad Prism v 10. RESULTS: Forty-eight patients and 72 healthy controls were recruited during the pandemic. ACE2 RNA expression in peripheral blood mononuclear cells (PBMC) was rarely detected acutely during severe COVID-19 but common in controls (OR for undetected ACE2: 12.4 [95% CI: 2.62-76.1]). ACE2 RNA expression in PBMC did not determine plasma ACE1 and ACE2 activity, suggesting alternative cell-signaling pathways. Markers of fibrosis (TIMP-1 and PIIINP) and vasculopathy (MMP-9) were additionally elevated. ACE2 RNA expression during severe COVID-19 often responded within hours to convalescent plasma. Analogous to oncogenesis, we speculate that potent, persistent, cryptic processes following COVID-19 (the renin-angiotensin system (RAS), fibrosis and vasculopathy) initiate or promote post-COVID-19 conditions (PCC) in susceptible individuals. CONCLUSIONS: This work elucidates biological and temporal plausibility for ACE2, TIMP1, PIIINP and MMP-9 in the pathogenesis of PCC. Intersection of these independent systems is uncommon and may in part explain the rarity of PCC.

Longitudinal dynamic single-cell mass cytometry analysis of peripheral blood mononuclear cells in COVID-19 patients within 6 months after viral RNA clearance.

This study investigates the longitudinal dynamic changes in immune cells in COVID-19 patients over an extended period after recovery, as well as the interplay between immune cells and antibodies. Leveraging single-cell mass spectrometry, we selected six COVID-19 patients and four healthy controls, dissecting the evolving landscape within six months post-viral RNA clearance, alongside the levels of anti-spike protein antibodies. The T cell immunophenotype ascertained via single-cell mass spectrometry underwent validation through flow cytometry in 37 samples. Our findings illuminate that CD8 + T cells, gamma-delta (gd) T cells, and NK cells witnessed an increase, in contrast to the reduction observed in monocytes, B cells, and double-negative T (DNT) cells over time. The proportion of monocytes remained significantly elevated in COVID-19 patients compared to controls even after six-month. Subpopulation-wise, an upsurge manifested within various T effector memory subsets, CD45RA + T effector memory, gdT, and NK cells, whereas declines marked the populations of DNT, naive and memory B cells, and classical as well as non-classical monocytes. Noteworthy associations surfaced between DNT, gdT, CD4 + T, NK cells, and the anti-S antibody titer. This study reveals the changes in peripheral blood mononuclear cells of COVID-19 patients within 6 months after viral RNA clearance and sheds light on the interactions between immune cells and antibodies. The findings from this research contribute to a better understanding of immune transformations during the recovery from COVID-19 and offer guidance for protective measures against reinfection in the context of viral variants.

Deep Sequencing Analysis of Individual HIV-1 Proviruses Reveals Frequent Asymmetric Long Terminal Repeats.

Effective strategies to eliminate human immunodeficiency virus type 1 (HIV-1) reservoirs are likely to require more thorough characterizations of proviruses that persist on antiretroviral therapy (ART). The rarity of infected CD4+ T-cells and related technical challenges have limited the characterization of integrated proviruses. Current approaches using next-generation sequencing can be inefficient and limited sequencing depth can make it difficult to link proviral sequences to their respective integration sites. Here, we report on an efficient method by which HIV-1 proviruses and their sites of integration are amplified and sequenced. Across five HIV-1-positive individuals on clinically effective ART, a median of 41.2% (n = 88 of 209) of amplifications yielded near-full-length proviruses and their 5'-host-virus junctions containing a median of 430 bp (range, 18 to 1,363 bp) of flanking host sequence. Unexpectedly, 29.5% (n = 26 of 88) of the sequenced proviruses had structural asymmetries between the 5' and 3' long terminal repeats (LTRs), commonly in the form of major 3' deletions. Sequence-intact proviruses were detected in 3 of 5 donors, and infected CD4+ T-cell clones were detected in 4 of 5 donors. The accuracy of the method was validated by amplifying and sequencing full-length proviruses and flanking host sequences directly from peripheral blood mononuclear cell DNA. The individual proviral sequencing assay (IPSA) described here can provide an accurate, in-depth, and longitudinal characterization of HIV-1 proviruses that persist on ART, which is important for targeting proviruses for elimination and assessing the impact of interventions designed to eradicate HIV-1. IMPORTANCE The integration of human immunodeficiency virus type 1 (HIV-1) into chromosomal DNA establishes the long-term persistence of HIV-1 as proviruses despite effective antiretroviral therapy (ART). Characterizing proviruses is difficult because of their rarity in individuals on long-term suppressive ART, their highly polymorphic sequences and genetic structures, and the need for efficient amplification and sequencing of the provirus and its integration site. Here, we describe a novel, integrated, two-step method (individual proviral sequencing assay [IPSA]) that amplifies the host-virus junction and the full-length provirus except for the last 69 bp of the 3' long terminal repeat (LTR). Using this method, we identified the integration sites of proviruses, including those that are sequence intact and replication competent or defective. Importantly, this new method identified previously unreported asymmetries between LTRs that have implications for how proviruses are detected and quantified. The IPSA method reported is unaffected by LTR asymmetries, permitting a more accurate and comprehensive characterization of the proviral landscape.

Porcine Epidemic Diarrhea Virus Infection Disrupts the Nasal Endothelial Barrier To Favor Viral Dissemination.

Crossing the endothelium from the entry site and spreading in the bloodstream are crucial but obscure steps in the pathogenesis of many emerging viruses. Previous studies confirmed that porcine epidemic diarrhea virus (PEDV) caused intestinal infection by intranasal inoculation. However, the role of the nasal endothelial barrier in PEDV translocation remains unclear. Here, we demonstrated that PEDV infection causes nasal endothelial dysfunction to favor viral dissemination. Intranasal inoculation with PEDV compromised the integrity of endothelial cells (ECs) in nasal microvessels. The matrix metalloproteinase 7 (MMP-7) released from the PEDV-infected nasal epithelial cells (NECs) contributed to the destruction of endothelial integrity by degrading the tight junctions, rather than direct PEDV infection. Moreover, the proinflammatory cytokines released from PEDV-infected NECs activated ECs to upregulate ICAM-1 expression, which favored peripheral blood mononuclear cells (PBMCs) migration. PEDV could further exploit migrated cells to favor viral dissemination. Together, our results reveal the mechanism by which PEDV manipulates the endothelial dysfunction to favor viral dissemination and provide novel insights into how coronavirus interacts with the endothelium. IMPORTANCE The endothelial barrier is the last but vital defense against systemic viral transmission. Porcine epidemic diarrhea virus (PEDV) can cause severe atrophic enteritis and acute viremia. However, the mechanisms by which the virus crosses the endothelial barrier and causes viremia are poorly understood. In this study, we revealed the mechanisms of endothelial dysfunction in PEDV infection. The viral infection activates NECs and causes the upregulation of MMP-7 and proinflammatory cytokines. Using NECs, ECs, and PBMCs as in vitro models, we determined that the released MMP-7 contributed to the destruction of endothelial barrier, and the released proinflammatory cytokines activated ECs to facilitate PBMCs migration. Moreover, the virus further exploited the migrated cells to promote viral dissemination. Thus, our results provide new insights into the mechanisms underlying endothelial dysfunction induced by coronavirus infection.

Changes to the Simian Immunodeficiency Virus (SIV) Reservoir and Enhanced SIV-Specific Responses in a Rhesus Macaque Model of Functional Cure after Serial Rounds of Romidepsin Administrations.

HIV persistence requires lifelong antiretroviral therapy (ART), calling for a cure. The histone deacetylase inhibitor, romidepsin, is used in the "shock and kill" approach with the goal of reactivating virus and subsequently clearing infected cells through cell-mediated immune responses. We tested serial and double infusions of romidepsin in a rhesus macaque (RM) model of SIV functional cure, which controls virus without ART. Off ART, romidepsin reactivated SIV in all RMs. Subsequent infusions resulted in diminished reactivation, and two RMs did not reactivate the virus after the second or third infusions. Therefore, those two RMs received CD8-depleting antibody to assess the replication competence of the residual reservoir. The remaining RMs received double infusions, i.e., two doses separated by 48-h. Double infusions were well tolerated, induced immune activation, and effectively reactivated SIV. Although reactivation was gradually diminished, cell-associated viral DNA was minimally changed, and viral outgrowth occurred in 4/5 RMs. In the RM which did not reactivate after CD8 depletion, viral outgrowth was not detected in peripheral blood mononuclear cells (PBMC)-derived CD4+ cells. The frequency of SIV-specific CD8+ T cells increased after romidepsin administration, and the increased SIV-specific immune responses were associated, although not statistically, with the diminished reactivation. Thus, our data showing sequential decreases in viral reactivation with repeated romidepsin administrations with all RMs and absence of viral reactivation after CD8+ T-cell depletion in one animal suggest that, in the context of healthy immune responses, romidepsin affected the inducible viral reservoir and gradually increased immune-mediated viral control. Given the disparities between the results of romidepsin administration to ART-suppressed SIVmac239-infected RMs and HIV-infected normal progressors compared to our immune-healthy model, our data suggest that improving immune function for greater SIV-specific responses should be the starting point of HIV cure strategies. IMPORTANCE HIV cure is sought after due to the prevalence of comorbidities that occur in persons with HIV. One of the most investigated HIV cure strategies is the "shock and kill" approach. Our study investigated the use of romidepsin, a histone deacetylase (HDAC) inhibitor, in our rhesus macaque model of functional cure, which allows for better resolution of viral reactivation due to the lack of antiretroviral therapy. We found that repeated rounds of romidepsin resulted in gradually diminished viral reactivation. One animal inevitably lacked replication-competent virus in the blood. With the accompanying enhancement of the SIV-specific immune response, our data suggest that there is a reduction of the viral reservoir in one animal by the cell-mediated immune response. With the differences observed between our model and persons living with HIV (PWH) treated with romidepsin, specifically in the context of a healthy immune system in our model, our data thereby indicate the importance of restoring the immune system for cure strategies.

Discovering common pathogenetic processes between COVID-19 and diabetes mellitus by differential gene expression pattern analysis.

Coronavirus disease 2019 (COVID-19) is an infectious disease caused by the newly discovered coronavirus, SARS-CoV-2. Increased severity of COVID-19 has been observed in patients with diabetes mellitus (DM). This study aimed to identify common transcriptional signatures, regulators and pathways between COVID-19 and DM. We have integrated human whole-genome transcriptomic datasets from COVID-19 and DM, followed by functional assessment with gene ontology (GO) and pathway analyses. In peripheral blood mononuclear cells (PBMCs), among the upregulated differentially expressed genes (DEGs), 32 were found to be commonly modulated in COVID-19 and type 2 diabetes (T2D), while 10 DEGs were commonly downregulated. As regards type 1 diabetes (T1D), 21 DEGs were commonly upregulated, and 29 DEGs were commonly downregulated in COVID-19 and T1D. Moreover, 35 DEGs were commonly upregulated in SARS-CoV-2 infected pancreas organoids and T2D islets, while 14 were commonly downregulated. Several GO terms were found in common between COVID-19 and DM. Prediction of the putative transcription factors involved in the upregulation of genes in COVID-19 and DM identified RELA to be implicated in both PBMCs and pancreas. Here, for the first time, we have characterized the biological processes and pathways commonly dysregulated in COVID-19 and DM, which could be in the next future used for the design of personalized treatment of COVID-19 patients suffering from DM as comorbidity.

Integration in oncogenes plays only a minor role in determining the in vivo distribution of HIV integration sites before or during suppressive antiretroviral therapy.

HIV persists during antiretroviral therapy (ART) as integrated proviruses in cells descended from a small fraction of the CD4+ T cells infected prior to the initiation of ART. To better understand what controls HIV persistence and the distribution of integration sites (IS), we compared about 15,000 and 54,000 IS from individuals pre-ART and on ART, respectively, with approximately 395,000 IS from PBMC infected in vitro. The distribution of IS in vivo is quite similar to the distribution in PBMC, but modified by selection against proviruses in expressed genes, by selection for proviruses integrated into one of 7 specific genes, and by clonal expansion. Clones in which a provirus integrated in an oncogene contributed to cell survival comprised only a small fraction of the clones persisting in on ART. Mechanisms that do not involve the provirus, or its location in the host genome, are more important in determining which clones expand and persist.

Temporally integrated single cell RNA sequencing analysis of PBMC from experimental and natural primary human DENV-1 infections.

Dengue human infection studies present an opportunity to address many longstanding questions in the field of flavivirus biology. However, limited data are available on how the immunological and transcriptional response elicited by an attenuated challenge virus compares to that associated with a wild-type DENV infection. To determine the kinetic transcriptional signature associated with experimental primary DENV-1 infection and to assess how closely this profile correlates with the transcriptional signature accompanying natural primary DENV-1 infection, we utilized scRNAseq to analyze PBMC from individuals enrolled in a DENV-1 human challenge study and from individuals experiencing a natural primary DENV-1 infection. While both experimental and natural primary DENV-1 infection resulted in overlapping patterns of inflammatory gene upregulation, natural primary DENV-1 infection was accompanied with a more pronounced suppression in gene products associated with protein translation and mitochondrial function, principally in monocytes. This suggests that the immune response elicited by experimental and natural primary DENV infection are similar, but that natural primary DENV-1 infection has a more pronounced impact on basic cellular processes to induce a multi-layered anti-viral state.

Infection of humanized mice with a novel phlebovirus presented pathogenic features of severe fever with thrombocytopenia syndrome.

Severe fever with thrombocytopenia syndrome virus (SFTSV) is a tick-borne emerging phlebovirus with high mortality rates of 6.0 to 30%. SFTSV infection is characterized by high fever, thrombocytopenia, leukopenia, hemorrhage and multiple organ failures. Currently, specific therapies and vaccines remain elusive. Suitable small animal models are urgently needed to elucidate the pathogenesis and evaluate the potential drug and vaccine for SFTSV infection. Previous models presented only mild or no pathogenesis of SFTS, limiting their applications in SFTSV infection. Therefore, it is an urgent need to develop a small animal model for the investigation of SFTSV pathogenesis and evaluation of therapeutics. In the current report, we developed a SFTSV infection model based on the HuPBL-NCG mice that recapitulates many pathological characteristics of SFTSV infection in humans. Virus-induced histopathological changes were identified in spleen, lung, kidney, and liver. SFTSV was colocalized with macrophages in the spleen and liver, suggesting that the macrophages in the spleen and liver could be the principle target cells of SFTSV. In addition, histological analysis showed that the vascular endothelium integrity was severely disrupted upon viral infection along with depletion of platelets. In vitro cellular assays further revealed that SFTSV infection increased the vascular permeability of endothelial cells by promoting tyrosine phosphorylation and internalization of the adhesion molecule vascular endothelial (VE)-cadherin, a critical component of endothelial integrity. In addition, we found that both virus infection and pathogen-induced exuberant cytokine release dramatically contributed to the vascular endothelial injury. We elucidated the pathogenic mechanisms of hemorrhage syndrome and developed a humanized mouse model for SFTSV infection, which should be helpful for anti-SFTSV therapy and pathogenesis study.

Disrupted Resolution Mechanisms Favor Altered Phagocyte Responses in COVID-19.

[Figure: see text].

Longitudinal Peripheral Blood Transcriptional Analysis Reveals Molecular Signatures of Disease Progression in COVID-19 Patients.

Coronavirus disease 2019 (COVID-19) is caused by a novel coronavirus named severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), with some patients developing severe illness or even death. Disease severity has been associated with increased levels of proinflammatory cytokines and lymphopenia. To elucidate the atlas of peripheral immune response and pathways that might lead to immunopathology during COVID-19 disease course, we performed a peripheral blood RNA sequencing analysis of the same patient's samples collected from symptom onset to full recovery. We found that PBMCs at different disease stages exhibited unique transcriptome characteristics. We observed that SARS-CoV-2 infection caused excessive release of inflammatory cytokines and lipid mediators as well as an aberrant increase of low-density neutrophils. Further analysis revealed an increased expression of RNA sensors and robust IFN-stimulated genes expression but a repressed type I IFN production. SARS-CoV-2 infection activated T and B cell responses during the early onset but resulted in transient adaptive immunosuppression during severe disease state. Activation of apoptotic pathways and functional exhaustion may contribute to the reduction of lymphocytes and dysfunction of adaptive immunity, whereas increase in IL2, IL7, and IL15 may facilitate the recovery of the number and function of lymphocytes. Our study provides comprehensive transcriptional signatures of peripheral blood response in patients with moderate COVID-19.

Silencing HIV-1 In Vivo.

RNA interference holds great promise for antiviral therapy, but delivering effective quantities of small interfering RNAs (siRNAs) into the right target cells in vivo represents a major challenge. Kumar et al. (2008) now report a technique to target siRNAs specifically to T cells to suppress viral infection in a humanized mouse model of HIV/AIDS.

T cell-specific siRNA delivery suppresses HIV-1 infection in humanized mice.

Evaluation of the therapeutic potential of RNAi for HIV infection has been hampered by the challenges of siRNA delivery and lack of suitable animal models. Using a delivery method for T cells, we show that siRNA treatment can dramatically suppress HIV infection. A CD7-specific single-chain antibody was conjugated to oligo-9-arginine peptide (scFvCD7-9R) for T cell-specific siRNA delivery in NOD/SCIDIL2rgamma-/- mice reconstituted with human lymphocytes (Hu-PBL) or CD34+ hematopoietic stem cells (Hu-HSC). In HIV-infected Hu-PBL mice, treatment with anti-CCR5 (viral coreceptor) and antiviral siRNAs complexed to scFvCD7-9R controlled viral replication and prevented the disease-associated CD4 T cell loss. This treatment also suppressed endogenous virus and restored CD4 T cell counts in mice reconstituted with HIV+ peripheral blood mononuclear cells. Moreover, scFvCD7-9R could deliver antiviral siRNAs to naive T cells in Hu-HSC mice and effectively suppress viremia in infected mice. Thus, siRNA therapy for HIV infection appears to be feasible in a preclinical animal model.

Fc-mediated effector function contributes to the in vivo antiviral effect of an HIV neutralizing antibody.

Treatment of HIV infection with either antiretroviral (ARV) therapy or neutralizing monoclonal antibodies (NAbs) leads to a reduction in HIV plasma virus. Both ARVs and NAbs prevent new rounds of viral infection, but NAbs may have the additional capacity to accelerate the loss of virus-infected cells through Fc gamma receptor (FcγR)-mediated effector functions, which should affect the kinetics of plasma-virus decline. Here, we formally test the role of effector function in vivo by comparing the rate and timing of plasma-virus clearance in response to a single-dose treatment with either unmodified NAb or those with either reduced or augmented Fc function. When infused into viremic simian HIV (SHIV)-infected rhesus macaques, there was a 21% difference in slope of plasma-virus decline between NAb and NAb with reduced Fc function. NAb engineered to increase FcγRIII binding and improve antibody-dependent cellular cytotoxicity (ADCC) in vitro resulted in arming of effector cells in vivo, yet led to viral-decay kinetics similar to NAbs with reduced Fc function. These studies show that the predominant mechanism of antiviral activity of HIV NAbs is through inhibition of viral entry, but that Fc function can contribute to the overall antiviral activity, making them distinct from standard ARVs.

Defective HIV-1 proviruses produce viral proteins.

HIV-1 proviruses persist in the CD4+ T cells of HIV-infected individuals despite years of combination antiretroviral therapy (cART) with suppression of HIV-1 RNA levels <40 copies/mL. Greater than 95% of these proviruses detected in circulating peripheral blood mononuclear cells (PBMCs) are referred to as "defective" by virtue of having large internal deletions and lethal genetic mutations. As these defective proviruses are unable to encode intact and replication-competent viruses, they have long been thought of as biologically irrelevant "graveyard" of viruses with little significance to HIV-1 pathogenesis. Contrary to this notion, we have recently demonstrated that these defective proviruses are not silent, are capable of transcribing novel unspliced forms of HIV-RNA transcripts with competent open reading frames (ORFs), and can be found in the peripheral blood CD4+ T cells of patients at all stages of HIV-1 infection. In the present study, by an approach of combining serial dilutions of CD4+ T cells and T cell-cloning technologies, we are able to demonstrate that defective proviruses that persist in HIV-infected individuals during suppressive cART are translationally competent and produce the HIV-1 Gag and Nef proteins. The HIV-RNA transcripts expressed from these defective proviruses may trigger an element of innate immunity. Likewise, the viral proteins coded in the defective proviruses may form extracellular virus-like particles and may trigger immune responses. The persistent production of HIV-1 proteins in the absence of viral replication helps explain persistent immune activation despite HIV-1 levels below detection, and also presents new challenges to HIV-1 eradication.

The HIV-1 accessory protein Nef increases surface expression of the checkpoint receptor Tim-3 in infected CD4+ T cells.

Prolonged immune activation drives the upregulation of multiple checkpoint receptors on the surface of virus-specific T cells, inducing their exhaustion. Reversing HIV-1-induced T cell exhaustion is imperative for efficient virus clearance; however, viral mediators of checkpoint receptor upregulation remain largely unknown. The enrichment of checkpoint receptors on T cells upon HIV-1 infection severely constrains the generation of an efficient immune response. Herein, we examined the role of HIV-1 Nef in mediating the upregulation of checkpoint receptors on peripheral blood mononuclear cells. We demonstrate that the HIV-1 accessory protein Nef upregulates cell surface levels of the checkpoint receptor T-cell immunoglobulin mucin domain-3 (Tim-3) and that this is dependent on Nef's dileucine motif LL164/165. Furthermore, we used a bimolecular fluorescence complementation assay to demonstrate that Nef and Tim-3 form a complex within cells that is abrogated upon mutation of the Nef dileucine motif. We also provide evidence that Nef moderately promotes Tim-3 shedding from the cell surface in a dileucine motif-dependent manner. Treating HIV-1-infected CD4+ T cells with a matrix metalloprotease inhibitor enhanced cell surface Tim-3 levels and reduced Tim-3 shedding. Finally, Tim-3-expressing CD4+ T cells displayed a higher propensity to release the proinflammatory cytokine interferon-gamma. Collectively, our findings uncover a novel mechanism by which HIV-1 directly increases the levels of a checkpoint receptor on the surface of infected CD4+ T cells.

Deep Gene Sequence Cluster Analyses of Multi-Virus-Infected Mucosal Tissue Reveal Enhanced Transmission of Acute HIV-1.

Exposure of the genital mucosa to a genetically diverse viral swarm from the donor HIV-1 can result in breakthrough and systemic infection by a single transmitted/founder (TF) virus in the recipient. The highly diverse HIV-1 envelope (Env) in this inoculating viral swarm may have a critical role in transmission and subsequent immune response. Thus, chronic (Envchronic) and acute (Envacute) Env chimeric HIV-1 were tested using multivirus competition assays in human mucosal penile and cervical tissues. Viral competition analysis revealed that Envchronic viruses resided and replicated mainly in the tissue, while Envacute viruses penetrated the human tissue and established infection of CD4+ T cells more efficiently. Analysis of the replication fitness, as tested in peripheral blood mononuclear cells (PBMCs), showed similar replication fitness of Envacute and Envchronic viruses, which did not correlate with transmission fitness in penile tissue. Further, we observed that chimeric Env viruses with higher replication in genital mucosal tissue (chronic Env viruses) had higher binding affinity to C-type lectins. Data presented herein suggest that the inoculating HIV-1 may be sequestered in the genital mucosal tissue (represented by chronic Env HIV-1) but that a single HIV-1 clone (e.g., acute Env HIV-1) can escape this trapped replication for systemic infection.IMPORTANCE During heterosexual HIV-1 transmission, a genetic bottleneck occurs in the newly infected individual as the virus passes from the mucosa, leading to systemic infection with a single transmitted HIV-1 clone in the recipient. This bottleneck in the recipient has just been described (K. Klein et al., PLoS Pathog 14:e1006754, https://doi.org/10.1371/journal.ppat.1006754), and the mechanisms involved in this selection process have not been elucidated. However, understanding mucosal restriction is of the utmost importance for understanding dynamics of infections and for designing focused vaccines. Using our human penile and cervical mucosal tissue models for mixed HIV infections, we provide evidence that HIV-1 from acute/early infection, compared to that from chronic infection, can more efficiently traverse the mucosal epithelium and be transmitted to T cells, suggesting higher transmission fitness. This study focused on the role of the HIV-1 envelope in transmission and provides strong evidence that HIV transmission may involve breaking the mucosal lectin trap.

The Balance between p53 Isoforms Modulates the Efficiency of HIV-1 Infection in Macrophages.

Several host factors influence HIV-1 infection and replication. The p53-mediated antiviral role in monocyte-derived macrophages (MDMs) was previously highlighted. Indeed, an increase in p53 level results in a stronger restriction against HIV-1 early replication steps through SAMHD1 activity. In this study, we investigated the potential role of some p53 isoforms in HIV-1 infection. Transfection of isoform-specific small interfering RNA (siRNA) induced distinctive effects on the virus life cycle. For example, in contrast to an siRNA targeting all isoforms, a knockdown of Δ133p53 transcripts reduced virus replication in MDMs that was correlated with a decrease in phosphorylated inactive SAMHD1. Combination of Δ133p53 knockdown and nutlin-3, a pharmacological inhibitor of MDM2 that stabilizes p53, further reduced susceptibility of MDMs to HIV-1 infection, thus suggesting an inhibitory role of Δ133p53 toward p53 antiviral activity. In contrast, p53ß knockdown in MDMs increased the viral production independently of SAMHD1. Moreover, experiments with a Nef-deficient virus showed that this viral protein plays a protective role against the antiviral environment mediated by p53. Finally, HIV-1 infection affected the expression pattern of p53 isoforms by increasing p53ß and p53γ mRNA levels while stabilizing the protein level of p53α and some isoforms from the p53ß subclass. The balance between the various p53 isoforms is therefore an important factor in the overall susceptibility of macrophages to HIV-1 infection, fine-tuning the p53 response against HIV-1. This study brings a new understanding of the complex role of p53 in virus replication processes in myeloid cells. IMPORTANCE As of today, HIV-1 infection is still considered a global pandemic without a functional cure, partly because of the presence of stable viral reservoirs. Macrophages constitute one of these cell reservoirs, contributing to the viral persistence. Studies investigating the host factors involved in cell susceptibility to HIV-1 infection might lead to a better understanding of reservoir formation and will eventually allow the development of an efficient cure. Our team previously showed the antiviral role of p53 in macrophages, which acts by compromising the early steps of HIV-1 replication. In this study, we demonstrate the involvement of p53 isoforms, which regulate p53 activity and define the cellular environment influencing viral replication. In addition, the results concerning the potential role of p53 in antiviral innate immunity could be transposed to other fields of virology and suggest that knowledge in oncology can be applied to HIV-1 research.