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SARS-CoV-2 is an enveloped virus responsible for the Coronavirus Disease 2019 (COVID-19) pandemic. Here, single viruses were analyzed by atomic force microscopy (AFM) operating directly in a level 3 biosafety (BSL3) facility, which appeared as a fast and powerful method to assess at the nanoscale level and in 3D infectious virus morphology in its native conformation, or upon inactivation treatments. AFM imaging reveals structurally intact infectious and inactivated SARS-CoV-2 upon low concentration of formaldehyde treatment. This protocol combining AFM and plaque assays allows the preparation of intact inactivated SARS-CoV-2 particles for safe use of samples out of level 3 laboratory to accelerate researches against the COVID-19 pandemic. Overall, we illustrate how adapted BSL3-AFM is a remarkable toolbox for rapid and direct virus analysis based on nanoscale morphology.

How macrophages avoid HIV-1 infection HIV-1 is unable to replicate efficiently in dendritic cells, the antigen-presenting tissue cells that function in both innate and adaptive immunity. Other primate lentiviruses, including HIV-2 and some simian immunodeficiency viruses, express a protein called Vpx that is able to overcome the block to replication. Two groups now report the identification of the restriction factor in dendritic cells and macrophages that is overcome by Vpx. Vpx is found to induce degradation of the protein SAMHD1. Mutations in SAMHD1 cause Aicardi–Goutières syndrome, a disorder characterized by inappropriate activation of the immune system. Knockdown of SAMHD1 increases HIV-1 replication in dendritic cells, which could be important for generating appropriate immune responses to the virus. The primate lentivirus auxiliary protein Vpx counteracts an unknown restriction factor that renders human dendritic and myeloid cells largely refractory to HIV-1 infection 1 , 2 , 3 , 4 , 5 , 6 . Here we identify SAMHD1 as this restriction factor. SAMHD1 is a protein involved in Aicardi–Goutières syndrome, a genetic encephalopathy with symptoms mimicking congenital viral infection, that has been proposed to act as a negative regulator of the interferon response 7 . We show that Vpx induces proteasomal degradation of SAMHD1. Silencing of SAMHD1 in non-permissive cell lines alleviates HIV-1 restriction and is associated with a significant accumulation of viral DNA in infected cells. Concurrently, overexpression of SAMHD1 in sensitive cells inhibits HIV-1 infection. The putative phosphohydrolase activity of SAMHD1 is probably required for HIV-1 restriction. Vpx-mediated relief of restriction is abolished in SAMHD1-negative cells. Finally, silencing of SAMHD1 markedly increases the susceptibility of monocytic-derived dendritic cells to infection. Our results demonstrate that SAMHD1 is an antiretroviral protein expressed in cells of the myeloid lineage that inhibits an early step of the viral life cycle.

The rate of HIV-1 gene expression is a key step that determines the kinetics of virus spread and AIDS progression. Viral entry and gene expression were described to be the key determinants for cell permissiveness to HIV. Recent reports highlighted the involvement of miRNA in regulating HIV-1 replication post-transcriptionally. In this study we explored the role of cellular factors required for miRNA-mediated mRNA translational inhibition in regulating HIV-1 gene expression. Here we show that HIV-1 mRNAs associate and co-localize with components of the RNA Induced Silencing Complex (RISC), and we characterize some of the proteins required for miRNA-mediated silencing (miRNA effectors). RCK/p54, GW182, LSm-1 and XRN1 negatively regulate HIV-1 gene expression by preventing viral mRNA association with polysomes. Interestingly, knockdown of RCK/p54 or DGCR8 resulted in virus reactivation in PBMCs isolated from HIV infected patients treated with suppressive HAART.

Background Quiescent CD4 + T lymphocytes are highly refractory to HIV-1 infection due to a block at reverse transcription. Results Examination of SAMHD1 expression in peripheral blood lymphocytes shows that SAMHD1 is expressed in both CD4+ and CD8+ T cells at levels comparable to those found in myeloid cells. Treatment of CD4+ T cells with Virus-Like Particles (VLP) containing Vpx results in the loss of SAMHD1 expression that correlates with an increased permissiveness to HIV-1 infection and accumulation of reverse transcribed viral DNA without promoting transcription from the viral LTR. Importantly, CD4 + T-cells from patients with Aicardi-Goutières Syndrome harboring mutation in the SAMHD1 gene display an increased susceptibility to HIV-1 infection that is not further enhanced by VLP-Vpx-treatment. Conclusion Here, we identified SAMHD1 as the restriction factor preventing efficient viral DNA synthesis in non-cycling resting CD4 + T-cells. These results highlight the crucial role of SAMHD1 in mediating restriction of HIV-1 infection in quiescent CD4 + T-cells and could impact our understanding of HIV-1 mediated CD4 + T-cell depletion and establishment of the viral reservoir, two of the HIV/AIDS hallmarks.

Various neurological symptoms have been associated to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection including headache, fever, anosmia, ageusia, but also, encephalitis, Guillain-Barre syndrome and ischemic stroke. Responsible for the current coronavirus disease (COVID-19) pandemic, SARS-CoV-2 may access and affect the central nervous system (CNS) by several pathways such as axonal retrograde transport or through interaction with the blood-brain barrier (BBB) or blood-cerebrospinal fluid (CSF) barrier. Here, we explored the molecular and cellular effects of direct SARS-CoV-2 infection of human BBB cells. We observed low replication of SARS-CoV-2 that was accompanied by very moderate inflammatory response. Using a human in vitro BBB model, we also described low replication levels without strong inflammatory response or modulation of endothelium integrity. Finally, using serum samples from COVID-19 patients, we highlighted strong concentrations of pro-inflammatory factors that did not perturb BBB integrity after short term exposure. Altogether, our results show that the main mechanism of brain access following SARS-CoV-2 infection does not seem to be directed by brain infection through endothelial cells.

MicroRNAs (miRNAs) are single-stranded noncoding RNAs of 19 to 25 nucleotides that function as gene regulators and as a host cell defense against both RNA and DNA viruses. We provide evidence for a physiological role of the miRNA-silencing machinery in controlling HIV-1 replication. Type III RNAses Dicer and Drosha, responsible for miRNA processing, inhibited virus replication both in peripheral blood mononuclear cells from HIV-1-infected donors and in latently infected cells. In turn, HIV-1 actively suppressed the expression of the polycistronic miRNA cluster miR-17/92. This suppression was found to be required for efficient viral replication and was dependent on the histone acetyltransferase Tat cofactor PCAF. Our results highlight the involvement of the miRNA-silencing pathway in HIV-1 replication and latency.

HIV‐1 gene expression is the major determinant regulating the rate of virus replication and, consequently, AIDS progression. Following primary infection, most infected cells produce virus. However, a small population becomes latently infected and constitutes the viral reservoir. This stable viral reservoir seriously challenges the hope of complete viral eradication. Viewed in this context, it is critical to define the molecular mechanisms involved in the establishment of transcriptional latency and the reactivation of viral expression. We show that Suv39H1, HP1γ and histone H3Lys9 trimethylation play a major role in chromatin‐mediated repression of integrated HIV‐1 gene expression. Suv39H1, HP1γ and histone H3Lys9 trimethylation are reversibly associated with HIV‐1 in a transcription‐dependent manner. Finally, we show in different cellular models, including PBMCs from HIV‐1‐infected donors, that HIV‐1 reactivation could be achieved after HP1γ RNA interference.

Exportin-5 is involved in microRNA precursor nuclear export. Dicer mRNA is now found to compete with pre-miRNAs for exportin-5 binding, suggesting that cytoplasmic levels of Dicer and its pre-miRNA substrate engage in a feedback loop. Analysis of adenoviral RNA indicates that it can also compete for exportin-5, suggesting that this might be a mechanism by which adenovirus can interfere with host miRNA processing. MicroRNAs (miRNAs) are a class of small, noncoding RNAs that function by regulating gene expression post-transcriptionally. Alterations in miRNA expression can strongly influence cellular physiology. Here we demonstrated cross-regulation between two components of the RNA interference (RNAi) machinery in human cells. Inhibition of exportin-5, the karyopherin responsible for pre-miRNA export, downregulated expression of Dicer, the RNase III required for pre-miRNA maturation. This effect was post-transcriptional and resulted from an increased nuclear localization of Dicer mRNA. In vitro assays and cellular RNA immunoprecipitation experiments showed that exportin-5 interacted directly with Dicer mRNA. Titration of exportin-5 by overexpression of either pre-miRNA or the adenoviral VA1 RNA resulted in loss of Dicer mRNA–exportin-5 interaction and reduction of Dicer level. This saturation also occurred during adenoviral infection and enhanced viral replication. Our study reveals an important cross-regulatory mechanism between pre-miRNA or viral small RNAs and Dicer through exportin-5.

Unlocking the potential of broadly reactive coronavirus monoclonal antibodies (mAbs) and their derivatives offers a transformative therapeutic avenue against severe COVID-19, especially crucial for safeguarding high-risk populations. Novel mAb-based immunotherapies may help address the reduced efficacy of current vaccines and neutralizing mAbs caused by the emergence of variants of concern (VOCs). Using phage display technology, we discovered a pan-SARS-CoV-2 mAb (C10) that targets a conserved region within the receptor-binding domain (RBD) of the virus. Noteworthy, C10 demonstrates exceptional efficacy in recognizing all assessed VOCs, including recent Omicron variants. While C10 lacks direct neutralization capacity, it efficiently binds to infected lung epithelial cells and induces their lysis via natural killer (NK) cell-mediated antibody-dependent cellular cytotoxicity (ADCC). Building upon this pan-SARS-CoV-2 mAb, we engineered C10-based, Chimeric Antigen Receptor (CAR)-T cells endowed with efficient killing capacity against SARS-CoV-2-infected lung epithelial cells. Notably, NK and CAR-T-cell mediated killing of lung infected cells effectively reduces viral titers. These findings highlight the potential of non-neutralizing mAbs in providing immune protection against emerging infectious diseases. Our work reveals a pan-SARS-CoV-2 mAb effective in targeting infected cells and demonstrates the proof-of-concept for the potential application of CAR-T cell therapy in combating SARS-CoV-2 infections. Furthermore, it holds promise for the development of innovative antibody-based and cell-based therapeutic strategies against severe COVID-19 by expanding the array of therapeutic options available for high-risk populations. ClinicalTrials.gov identifier: NCT04093596.

The p300–CBP-associated factor (PCAF) is a histone acetyltransferase (HAT) involved in the reversible acetylation of various transcriptional regulators 1 , including the tumour suppressor p53. It is implicated in many cellular processes, such as transcription, differentiation, proliferation and apoptosis. We observed that knockdown of PCAF expression in HeLa or U2OS cell lines induces stabilization of the oncoprotein Hdm2, a RING finger E3 ligase primarily known for its role in controlling p53 stability 2 , 3 . To investigate the molecular basis of this effect, we examined whether PCAF is involved in Hdm2 ubiquitination. Here, we show that PCAF, in addition to its acetyltransferase activity, possesses an intrinsic ubiquitination activity that is critical for controlling Hdm2 expression levels, and thus p53 functions. Our data highlight a regulatory crosstalk between PCAF and Hdm2 activities, which is likely to have a central role in the subtle control of p53 activity after DNA damage.