Although refrigerated storage slows the metabolism of volunteer donor RBCs, which is essential in transfusion medicine, cellular aging still occurs throughout this in vitro process. Storage-induced microerythrocytes (SMEs) are morphologically altered senescent RBCs that accumulate during storage and are cleared from circulation following transfusion. However, the molecular and cellular alterations that trigger clearance of this RBC subset remain to be identified. Using a staining protocol that sorts long-stored SMEs (i.e., CFSEhi) and morphologically normal RBCs (CFSElo), these in vitro aged cells were characterized. Metabolomics analysis identified depletion of energy, lipid-repair, and antioxidant metabolites in CFSEhi RBCs. By redox proteomics, irreversible protein oxidation primarily affected CFSEhi RBCs. By proteomics, 96 proteins, mostly in the proteostasis family, had relocated to CFSEhi RBC membranes. CFSEhi RBCs exhibited decreased proteasome activity and deformability; increased phosphatidylserine exposure, osmotic fragility, and endothelial cell adherence; and were cleared from the circulation during human spleen perfusion ex vivo. Conversely, molecular, cellular, and circulatory properties of long-stored CFSElo RBCs resembled those of short-stored RBCs. CFSEhi RBCs are morphologically and metabolically altered, have irreversibly oxidized and membrane-relocated proteins, and exhibit decreased proteasome activity. In vitro aging during storage selectively alters metabolism and proteostasis in these storage-induced senescent RBCs targeted for clearance.

We recently developed an original HCV-permissive cell culture model based on both partial differentiation and physiological oxygen pressure (Hypo-Diff cells) that produces highly infectious lipid-rich and native-like HCV-lipoviroparticles (LVPs). Here, we explored the precise role of physiological hypoxia and related specific transcription factors, hypoxia-inducible factors 1α and 2α (HIF-1α and HIF-2α) to better understand the mechanism governing viral morphogenesis. Knocking-down HIF-2α specifically reduced both the number and size of neutral lipid-rich droplets in Huh7.5 Hypo-Diff cells, suggesting a central role for HIF-2 in controlling lipid metabolism under physiological hypoxia. In HCV-infected siHIF-2α Hypo-Diff cells, both HCV replication and the specific infectious viral efficacy of progeny viruses were significantly impaired. Interestingly, the ectopic expression of a mutated form of the HIF-2α protein, stabilized in normoxia, both increased the number and size of neutral lipid-rich droplets and restored the production of highly infectious HCV viruses in the absence of cell differentiation. Finally, by iodixanol fractionation of supernatants to determine the mean buoyant density of infectious HCV LVPs, we established that HIF-2α is exclusively responsible for producing highly lipidated and broadly infectious HCV-LVPs by Hypo-Diff cells. These findings thus clearly establish the central role of physiological hypoxia, and notably HIF-2, in the production of highly infectious lipid-rich native-like HCV particles. Since physiological hypoxia is a shared characteristic in mammalian tissues, we propose to reconsider the role of natural oxygen tension and especially the role of HIF-2 in the life cycle of other lipid-associated viruses, whether hepatotropic or not.

Like many viruses, HIV relies on the microtubule (MT) cytoskeleton for successful infection. MT-associated proteins (MAPs) regulate MT functions and thus bear the potential to modulate viral infection. However, while several MAPs are known to exert proviral effects on HIV, little is known about antiviral ones. We previously described the Tripartite motif protein 69 (Trim69) as an innate immune factor that remodels MTs, leading to inhibition of a broad spectrum of viruses, including HIV. Through in silico modeling, TIRF microscopy, and cell-based assays, we determine that Trim69 binding to MTs is determined by a basic surface in its SPRY domain that interacts with the C-terminal tails of tubulins. This surface is conserved among mammalian Trim69s and is critical for its functions. We demonstrate that by binding and remodeling MTs, Trim69 inhibits the docking and the migration of virion cores on MTs by promoting the stalling of the dynein/dynactin motor complexes. Altogether, these findings shed light on a mechanism of viral defense that involves an innate immune regulation of the MT cytoskeleton.

A crucial step of HBV (Hepatitis B Virus) virion morphogenesis is the envelopment of the nucleocapsid by the viral envelope proteins, which is triggered by an interaction between the HBV core protein and the large HBV envelope protein. To document this protein-protein interaction, we co-expressed core and large HBV envelope (LHBs) in Huh-7 cells and subjected the cells to microscopy examination by Fluorescence Resonance Energy Transfer (FRET) and Transmission Electron Microscopy (TEM). Our results show that the sole expression of the core protein leads to assembly of capsids that remain individually isolated within the whole cell, but particularly within the nucleus. In the presence of LHBs, capsids were observed as large clusters in a membrane rich region peripheral to the nucleus. In this context, core-LHBs complex co-localize with markers of the late endosome/multivesicular bodies, this co-localization being driven by LHBs. These results thus show that LHBs binds to the core proteins when preassembled into capsid, at membranes of the late endosome, where the inner capsid and the outer envelope meet to assemble a virion.

The Crimean-Congo hemorrhagic fever virus (CCHFV) is an emerging pathogen of the Orthonairovirus genus that can cause severe and often lethal hemorrhagic diseases in humans. CCHFV has a broad tropism and can infect a variety of species and tissues. Here, by using gene silencing, blocking antibodies or soluble receptor fragments, we identify the low-density lipoprotein receptor (LDL-R) as a CCHFV entry factor. The LDL-R facilitates binding of CCHFV particles but does not allow entry of Hazara virus (HAZV), another member of the genus. In addition, we show that apolipoprotein E (apoE), an exchangeable protein that mediates LDL/LDL-R interaction, is incorporated on CCHFV particles, though not on HAZV particles, and enhances their specific infectivity by promoting an LDL-R dependent entry. Finally, we show that molecules that decrease LDL-R from the surface of target cells could inhibit CCHFV infection. Our study highlights that CCHFV takes advantage of a lipoprotein receptor and recruits its natural ligand to promote entry into cells. This study shows that Crimean-Congo hemorrhagic fever virus (CCHFV) recruits apoE, an exchangeable apolipoprotein that mediates LDL/LDL-R interaction, to promote virion entry via the LDLR. Molecules that down-regulate LDL-R inhibit CCHFV infection.

Although refrigerated storage slows the metabolism of volunteer donor RBCs, cellular aging still occurs throughout this in vitro process, which is essential in transfusion medicine. Storage-induced microerythrocytes (SMEs) are morphologically-altered senescent RBCs that accumulate during storage and which are cleared from circulation following transfusion. However, the molecular and cellular alterations that trigger clearance of this RBC subset remain to be identified. Using a staining protocol that sorts long-stored SMEs (i.e., CFSE high ) and morphologically-normal RBCs (CFSE low ), these in vitro aged cells were characterized. Metabolomics analysis identified depletion of energy, lipid-repair, and antioxidant metabolites in CFSE high RBCs. By redox proteomics, irreversible protein oxidation primarily affected CFSE high RBCs. By proteomics, 96 proteins, mostly in the proteostasis family, had relocated to CFSE high RBC membranes. CFSE high RBCs exhibited decreased proteasome activity and deformability; increased phosphatidylserine exposure, osmotic fragility, and endothelial cell adherence; and were cleared from the circulation during human spleen ex vivo perfusion. Conversely, molecular, cellular, and circulatory properties of long-stored CFSE low RBCs resembled those of short-stored RBCs. CFSE high RBCs are morphologically and metabolically altered, have irreversibly oxidized and membrane-relocated proteins, and exhibit decreased proteasome activity. In vitro aging during storage selectively alters metabolism and proteostasis in SMEs, targeting these senescent cells for clearance.

Abstract Entry of viral capsids into the nucleus induces the formation of biomolecular condensates called HIV-1 membraneless organelles (HIV-1-MLOs). Several questions remain about their persistence, in vivo formation, composition, and function. Our study reveals that HIV-1-MLOs persisted for several weeks in infected cells, and their abundance correlated with viral infectivity. Using an appropriate animal model, we show that HIV-1-MLOs were formed in vivo during acute infection. To explore the viral structures present within these biomolecular condensates, we used a combination of double immunogold labeling, electron microscopy and tomography, and unveiled a diverse array of viral core structures. Our functional analyses showed that HIV-1-MLOs remained stable during treatment with a reverse transcriptase inhibitor, maintaining the virus in a dormant state. Drug withdrawal restored reverse transcription, promoting efficient virus replication akin to that observed in latently infected patients on antiretroviral therapy. However, when HIV-1 MLOs were deliberately disassembled by pharmacological treatment, we observed a complete loss of viral infectivity. Our findings show that HIV-1 MLOs shield the final reverse transcription product from host immune detection.

Positive single -strand RNA (+RNA) viruses can remodel host cell membranes to induce replication organelles isolating the replication of their genome from cellular innate immunity mechanisms. Some of these viruses, including the severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2), induce double-membrane vesicles (DMV) to behave as replication organelles. As for the hepatitis C virus (HCV), pores crossing both membranes of SARS-CoV-2-induced DMV have been identified. These pores likely allow the supply of metabolites essential for viral replication within the DMV, and allow the export of the newly synthesized viral RNA to form the genome of the neo-synthesized virions. However, it remains unknown whether DMV with open pores can coexist with fully sealed DMV allowing the storage of large amounts of viral RNA, as seen for the HCV model. Interestingly, recent studies have revealed many similarities in the mechanisms of DMV biogenesis between these two phylogenetically distant viruses. Understanding the mechanisms of DMV formation and function will be essential for the development of new broad-spectrum antiviral approaches, able to inhibit DMV-inducing viruses from different families. (c) 2023 l'Academie nationale de medecine. Published by Elsevier Masson SAS. All rights reserved.

Human papillomaviruses (HPVs) are commensal viruses with pathogenic potential. Their life cycle requires the proliferation and differentiation of keratinocytes (KCs) to form pluristratified epithelia. Based on the original organotypic epithelial raft cultures protocol, we provide an updated workflow to optimally generate pluristratified human epithelia supporting the complete HPV replicative life cycle, here called 3D full-thickness epithelial cultures (3Deps). We describe steps for HPV genome preparation, KC transfection, and dermal equivalent preparation. We then detail procedures for 3Deps culture, harvesting, and analysis.

Alphaviruses, such as chikungunya virus (CHIKV), are mosquito-borne viruses that represent a significant threat to human health due to the current context of global warming. Efficient alphavirus infection relies on the activity of the non-structural protein 3 (nsP3), a puzzling multifunctional molecule whose role in infection remains largely unknown. NsP3 is a component of the plasma membrane-bound viral RNA replication complex (vRC) essential for RNA amplification and is also found in large cytoplasmic aggregates of unknown function. Here, we report the cryo-electron microscopy (cryo-EM) structure of the CHIKV nsP3 at 2.35 & Aring; resolution. We show that nsP3 assembles into tubular structures made by a helical arrangement of its alphavirus unique domain (AUD). The nsP3 helical scaffolds are consistent with crown structures found on tomographic reconstructions of the mature viral RCs. In addition, nsP3 helices assemble into cytoplasmic granules organized in a network of tubular structures that contain viral genomic RNA and capsid as well as host factors required for productive infection. Structure-guided mutagenesis identified residues that prevent or disturb nsP3 assemblies, resulting in impaired viral replication or transcription. Altogether, our results reveal an unexpected nsP3-dependent molecular organization essential for different phases of alphavirus infection. The role of alphavirus nsP3 protein is not entirely clear. Here, through structural analysis the authors show that CHIKV nsP3 polymerizes to form tubular scaffolds in both replication complexes and alpha-granules and that these scaffolds are important for virus RNA synthesis and infectivity.

Background & Aims: Elimination of chronic HBV/HDV infection remains a major global health challenge. Targeting excessive hepatitis B surface antigen (HBsAg) release may provide an interesting window of opportunity to break immune tolerance and to achieve a functional cure using additional antivirals.Methods: We evaluated a HBsAg-specific human monoclonal antibody, as part of either a prophylactic or therapeutic strategy, against HBV/HDV infection in cell culture models and in human-liver chimeric mice. To assess prophylactic efficacy, mice were passively immunized prior to infection with HBV or HBV/HDV (coinfection and superinfection setting). Therapeutic efficacy was assessed in HBV and HBV/HDV-coinfected mice receiving 4 weeks of treatment. Viral parameters (HBV DNA, HDV RNA and HBsAg) were assessed in mouse plasma.Results: The antibody could effectively prevent HBV/HDV infection in a dose-dependent manner with IC50 values of-3.5 ng/ ml. Passive immunization showed complete protection of mice from both HBV and HBV/HDV coinfection. Moreover, HDV superinfection was either completely prevented or at least attenuated in HBV-infected mice. Finally, antibody treatment in mice with established HBV/HDV infection resulted in a significant decline in viremia and a concomitant drop in on-treatment HBsAg, with a moderate viral rebound following treatment cessation.Conclusion: We present data on a valuable antibody candidate that could complement other antivirals in strategies aimed at achieving functional cure of chronic HBV and HDV infection.Impact and implications: Patients chronically infected with HBV may eventually develop liver cancer and are at great risk of being superinfected with HDV, which worsens and accelerates disease progression. Unfortunately, current treatments can rarely eliminate both viruses from chronically infected patients. In this study, we present data on a novel antibody that is able to prevent chronic HBV/HDV infection in a mouse model with a humanized liver. Moreover, antibody treatment of HBV/HDV-infected mice strongly diminishes viral loads during therapy. This antibody is a valuable candidate for further clinical development.(c) 2022 The Authors. Published by Elsevier B.V. on behalf of European Association for the Study of the Liver (EASL). This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

ABSTRACT The human T-lymphotropic virus type 1 (HTLV-1) is an oncogenic retrovirus whose transmission relies primarily on cell-to-cell contacts as cell-free viruses are poorly infectious. Among the intercellular transmission routes described, HTLV-1 biofilms are adhesive structures polarized at the cell surface that confine virions in a protective environment, which is believed to promote their simultaneous delivery during infection. Here, we show that several tetraspanins are enriched in HTLV-1 biofilms and incorporated into the viral envelope. However, we report that only the tetraspanin CD82 interacts with HTLV-1 Gag proteins which initiates their polarization into viral biofilms. Also, we demonstrate that CD82 maintains HTLV-1 biofilm polarization and favors viral transmission, as its silencing induces a complete reorganization of viral clusters at the cell surface and reduces the ability of infected T-cells to transmit the virus. Our results highlight the crucial role of CD82 and its glycosylation state in the architectural organization of HTLV-1 biofilms and their subsequent transfer through intercellular contacts. IMPORTANCE In the early stages of infection, human T-lymphotropic virus type 1 (HTLV-1) dissemination within its host is believed to rely mostly on cell-to-cell contacts. Past studies unveiled a novel mechanism of HTLV-1 intercellular transmission based on the remodeling of the host-cell extracellular matrix and the generation of cell-surface viral assemblies whose structure, composition, and function resemble bacterial biofilms. These polarized aggregates of infectious virions, identified as viral biofilms, allow the bulk delivery of viruses to target cells and may help to protect virions from immune attacks. However, viral biofilms’ molecular and functional description is still in its infancy, although it is crucial to fully decipher retrovirus pathogenesis. Here, we explore the function of cellular tetraspanins (CD9, CD81, CD82) that we detect inside HTLV-1 particles within biofilms. Our results demonstrate specific roles for CD82 in the cell-surface distribution and intercellular transmission of HTLV-1 biofilms, which we document as two essential parameters for efficient viral transmission. At last, our findings indicate that N-glycosylation of cell-surface molecules, including CD82, is required for the polarization of HTLV-1 biofilms and for the efficient transmission of HTLV-1 between T-lymphocytes.

The introduction of direct-acting antivirals (DAAs) has revolutionized hepatitis C treatment. Short courses of treatment with these drugs are highly beneficial to patients, eliminating hepatitis C virus (HCV) without adverse effects. However, this outstanding success is tempered by the continuing difficulty of eradicating the virus worldwide. Thus, access to an effective vaccine against HCV is strongly needed to reduce the burden of the disease and contribute to the elimination of viral hepatitis. The recent failure of a T-cell vaccine based on the use of viral vectors expressing the HCV non-structural protein sequences to prevent chronic hepatitis C in drug users has pointed out that the induction of neutralizing antibodies (NAbs) will be essential in future vaccine candidates. To induce NAbs, vaccines must contain the main target of this type of antibody, the HCV envelope glycoproteins (E1 and E2). In this review, we summarize the structural regions in E1 and E2 proteins that are targeted by NAbs and how these proteins are presented in the vaccine candidates currently under development.

Human papillomaviruses (HPVs) are highly prevalent commensal viruses that require epithelial stratification to complete their replicative cycle. While HPV infections are most often asymptomatic, certain HPV types can cause lesions, that are usually benign. In rare cases, these infections may progress to non-replicative viral cycles associated with high HPV oncogene expression promoting cell transformation, and eventually cancer when not cleared by host responses. While the consequences of HPV-induced transformation on keratinocytes have been extensively explored, the impact of viral replication on epithelial homeostasis remains largely unexplored. Gap junction intercellular communication (GJIC) is critical for stratified epithelium integrity and function. This process is ensured by a family of proteins named connexins (Cxs), including 8 isoforms that are expressed in stratified squamous epithelia. GJIC was reported to be impaired in HPV-transformed cells, which was attributed to the decreased expression of the Cx43 isoform. However, it remains unknown whether and how HPV replication might impact on the expression of Cx isoforms and GJIC in stratified squamous epithelia. To address this question, we have used 3D-epithelial cell cultures (3D-EpCs), the only model supporting the productive HPV life cycle. We report a transcriptional downregulation of most epithelial Cx isoforms except Cx45 in HPV-replicating epithelia. At the protein level, HPV replication results in a reduction of Cx43 expression while that of Cx45 increases and displays a topological shift toward the cell membrane. To quantify GJIC, we pioneered quantitative gap-fluorescence loss in photobleaching (FLIP) assay in 3D-EpCs, which allowed us to show that the reprogramming of Cx landscape in response to HPV replication translates into accelerated GJIC in living epithelia. Supporting the pathophysiological relevance of our observations, the HPV-associated Cx43 and Cx45 expression pattern was confirmed in human cervical biopsies harboring HPV. In conclusion, the reprogramming of Cx expression and distribution in HPV-replicating epithelia fosters accelerated GJIC, which may participate in epithelial homeostasis and host immunosurveillance.

Les virus à ARN simple brin positif (ARN+) peuvent remodeler les membranes des cellules hôtes qu’ils infectent pour induire des organelles de réplication. Ces organelles viro-induites servent à isoler la réplication du génome viral des capteurs déclenchant les mécanismes de l’immunité cellulaire innée. Certains de ces virus, dont le coronavirus du syndrome respiratoire aigu sévère de type 2 (SARS-CoV-2), induisent des vésicules à double membrane (DMV) qui jouent ce rôle d’organelle de réplication virale. Comme dans le cas du virus de l’hépatite C (VHC), des pores traversant les deux membranes des DMV induites par le SRAS-CoV-2 ont été identifiés. Ces pores permettent vraisemblablement l’apport de métabolites essentiels à la réplication virale au sein d’une DMV, ainsi que l’exportation de l’ARN viral nouvellement synthétisé pour former le génome des futurs virions. On ne sait pas encore si, comme pour le VHC, des DMV à pores ouverts peuvent coexister avec des DMV entièrement scellées qui sont sans doute nécessaires au stockage de grandes quantités d’ARN viral. Cependant, de façon remarquable, des études récentes ont révélé de nombreuses similitudes dans les mécanismes de biogenèse des DMV entre ces deux virus pourtant phylogénétiquement très éloignés. La compréhension des mécanismes de formation et de fonctionnement des DMV pourrait être essentielle pour le développement de nouvelles approches antivirales de large spectre, pouvant agir sur des virus de différentes familles inducteurs de ces DMV.

IFITMs are a family of highly related interferon-induced transmembrane proteins that interfere with the processes of fusion between viral and cellular membranes and are thus endowed with broad antiviral properties. A number of studies have shown how the antiviral potency of IFITMs is highly dependent on their steady-state levels, their intracellular distribution and a complex pattern of post-translational modifications, parameters that are overall tributary of a number of cellular partners. In an effort to identify additional protein partners involved in the biology of IFITMs, we devised a proteomics-based approach based on the piggyback incorporation of IFITM3 partners into extracellular vesicles. MS analysis of the proteome of vesicles bearing or not bearing IFITM3 identified the NDFIP2 protein adaptor protein as an important regulator of IFITM3 levels. NDFIP2 is a membrane-anchored adaptor protein of the E3 ubiquitin ligases of the NEDD4 family that have already been found to be involved in IFITM3 regulation. We show here that NDFIP2 acts as a recruitment factor for both IFITM3 and NEDD4 and mediates their distribution in lysosomal vesicles. The genetic inactivation and overexpression of NDFIP2 drive, respectively, lower and higher levels of IFITM3 accumulation in the cell, overall suggesting that NDFIP2 locally competes with IFITM3 for NEDD4 binding. Given that NDFIP2 is itself tightly regulated and highly responsive to external cues, our study sheds light on a novel and likely dynamic layer of regulation of IFITM3.

Purpose The objective of the present study was to provide a detailed histopathological description of fatal coronavirus disease 2019 (COVID 19), and compare the lesions in Intensive Care Unit (ICU) and non-ICU patients. Methods In this prospective study we included adult patients who died in hospital after presenting with confirmed COVID-19. Multiorgan biopsies were performed. Data generated with light microscopy, transmission electron microscopy (TEM) and RT-PCR assays were reviewed. Results 20 patients were enrolled in the study and the main pulmonary finding was alveolar damage, which was focal in 11 patients and diffuse in 8 patients. Chronic fibrotic and inflammatory lesions were observed in 18 cases, with acute inflammatory lesions in 12 cases. Diffuse lesions, collapsed alveoli and dystrophic pneumocytes were more frequent in the ICU group (62.5%, vs. 25%; 63%, vs. 55%; 87.5%, vs. 54%). Acute lesions (82%, vs. 37.5%; p = 0.07) with neutrophilic alveolitis (63.6% vs. 0%, respectively; p = 0.01) were observed more frequently in the non-ICU group. Viral RNA was detected in 12 lung biopsies (60%) up to 56 days after disease upset. TEM detected viral particles in the lung and kidney biopsy samples up to 27 days after disease upset. Furthermore, abundant networks of double-membrane vesicles (DMVs, a hallmark of viral replication) were observed in proximal tubular epithelial cells. Conclusion Lung injury was different in ICU and non-ICU patients. Extrapulmonary damage consisting in kidney and myocardial injury were more frequent in ICU patients. Our TEM experiments provided the first description of SARS-CoV-2-induced DMVs in kidney biopsy samples—a sign of intense viral replication in this organ.

Bats are host ancestors of several viruses that cause serious disease in humans, as illustrated by the ongoing SARS-CoV-2 pandemic. Progress in investigating bat-virus interactions has been hampered by a limited number of available bat cellular models.

Sickle cell disease (SCD) is an inherited blood disorder characterized by sickled red blood cells (RBCs), which are more sensitive to haemolysis and can contribute to disease pathophysiology. Although treatment of SCD can include RBC transfusion, patients with SCD have high rates of alloimmunization. We hypothesized that RBCs from patients with SCD have functionally active mitochondria and can elicit a type 1 interferon response. We evaluated blood samples from more than 100 patients with SCD and found elevated frequencies of mitochondria in reticulocytes and mature RBCs, as compared to healthy blood donors. The presence of mitochondria in mature RBCs was confirmed by flow cytometry, electron microscopy, and proteomic analysis. The mitochondria in mature RBCs were metabolically competent, as determined by enzymatic activities and elevated levels of mitochondria-derived metabolites. Metabolically-active mitochondria in RBCs may increase oxidative stress, which could facilitate and/or exacerbate SCD complications. Coculture of mitochondria-positive RBCs with neutrophils induced production of type 1 interferons, which are known to increase RBC alloimmunization rates. These data demonstrate that mitochondria retained in mature RBCs are functional and can elicit immune responses, suggesting that inappropriate retention of mitochondria in RBCs may play an underappreciated role in SCD complications and be an RBC alloimmunization risk factor.