TMPRSS2 is a functional receptor for human coronavirus HKU1.

Four endemic seasonal human coronaviruses causing common colds circulate worldwide: HKU1, 229E, NL63 and OC43 (ref. 1). After binding to cellular receptors, coronavirus spike proteins are primed for fusion by transmembrane serine protease 2 (TMPRSS2) or endosomal cathepsins2-9. NL63 uses angiotensin-converting enzyme 2 as a receptor10, whereas 229E uses human aminopeptidase-N11. HKU1 and OC43 spikes bind cells through 9-O-acetylated sialic acid, but their protein receptors remain unknown12. Here we show that TMPRSS2 is a functional receptor for HKU1. TMPRSS2 triggers HKU1 spike-mediated cell-cell fusion and pseudovirus infection. Catalytically inactive TMPRSS2 mutants do not cleave HKU1 spike but allow pseudovirus infection. Furthermore, TMPRSS2 binds with high affinity to the HKU1 receptor binding domain (Kd 334 and 137 nM for HKU1A and HKU1B genotypes) but not to SARS-CoV-2. Conserved amino acids in the HKU1 receptor binding domain are essential for binding to TMPRSS2 and pseudovirus infection. Newly designed anti-TMPRSS2 nanobodies potently inhibit HKU1 spike attachment to TMPRSS2, fusion and pseudovirus infection. The nanobodies also reduce infection of primary human bronchial cells by an authentic HKU1 virus. Our findings illustrate the various evolution strategies of coronaviruses, which use TMPRSS2 to either directly bind to target cells or prime their spike for membrane fusion and entry.

Considerable escape of SARS-CoV-2 Omicron to antibody neutralization.

The SARS-CoV-2 Omicron variant was first identified in November 2021 in Botswana and South Africa1-3. It has since spread to many countries and is expected to rapidly become dominant worldwide. The lineage is characterized by the presence of around 32 mutations in spike-located mostly in the N-terminal domain and the receptor-binding domain-that may enhance viral fitness and enable antibody evasion. Here we isolated an infectious Omicron virus in Belgium from a traveller returning from Egypt. We examined its sensitivity to nine monoclonal antibodies that have been clinically approved or are in development4, and to antibodies present in 115 serum samples from COVID-19 vaccine recipients or individuals who have recovered from COVID-19. Omicron was completely or partially resistant to neutralization by all monoclonal antibodies tested. Sera from recipients of the Pfizer or AstraZeneca vaccine, sampled five months after complete vaccination, barely inhibited Omicron. Sera from COVID-19-convalescent patients collected 6 or 12 months after symptoms displayed low or no neutralizing activity against Omicron. Administration of a booster Pfizer dose as well as vaccination of previously infected individuals generated an anti-Omicron neutralizing response, with titres 6-fold to 23-fold lower against Omicron compared with those against Delta. Thus, Omicron escapes most therapeutic monoclonal antibodies and, to a large extent, vaccine-elicited antibodies. However, Omicron is neutralized by antibodies generated by a booster vaccine dose.

Reduced sensitivity of SARS-CoV-2 variant Delta to antibody neutralization.

The SARS-CoV-2 B.1.617 lineage was identified in October 2020 in India1-5. Since then, it has become dominant in some regions of India and in the UK, and has spread to many other countries6. The lineage includes three main subtypes (B1.617.1, B.1.617.2 and B.1.617.3), which contain diverse mutations in the N-terminal domain (NTD) and the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein that may increase the immune evasion potential of these variants. B.1.617.2-also termed the Delta variant-is believed to spread faster than other variants. Here we isolated an infectious strain of the Delta variant from an individual with COVID-19 who had returned to France from India. We examined the sensitivity of this strain to monoclonal antibodies and to antibodies present in sera from individuals who had recovered from COVID-19 (hereafter referred to as convalescent individuals) or who had received a COVID-19 vaccine, and then compared this strain with other strains of SARS-CoV-2. The Delta variant was resistant to neutralization by some anti-NTD and anti-RBD monoclonal antibodies, including bamlanivimab, and these antibodies showed impaired binding to the spike protein. Sera collected from convalescent individuals up to 12 months after the onset of symptoms were fourfold less potent against the Delta variant relative to the Alpha variant (B.1.1.7). Sera from individuals who had received one dose of the Pfizer or the AstraZeneca vaccine had a barely discernible inhibitory effect on the Delta variant. Administration of two doses of the vaccine generated a neutralizing response in 95% of individuals, with titres three- to fivefold lower against the Delta variant than against the Alpha variant. Thus, the spread of the Delta variant is associated with an escape from antibodies that target non-RBD and RBD epitopes of the spike protein.

SARS-CoV-2 Alpha, Beta, and Delta variants display enhanced Spike-mediated syncytia formation.

Severe COVID-19 is characterized by lung abnormalities, including the presence of syncytial pneumocytes. Syncytia form when SARS-CoV-2 spike protein expressed on the surface of infected cells interacts with the ACE2 receptor on neighboring cells. The syncytia forming potential of spike variant proteins remain poorly characterized. Here, we first assessed Alpha (B.1.1.7) and Beta (B.1.351) spread and fusion in cell cultures, compared with the ancestral D614G strain. Alpha and Beta replicated similarly to D614G strain in Vero, Caco-2, Calu-3, and primary airway cells. However, Alpha and Beta formed larger and more numerous syncytia. Variant spike proteins displayed higher ACE2 affinity compared with D614G. Alpha, Beta, and D614G fusion was similarly inhibited by interferon-induced transmembrane proteins (IFITMs). Individual mutations present in Alpha and Beta spikes modified fusogenicity, binding to ACE2 or recognition by monoclonal antibodies. We further show that Delta spike also triggers faster fusion relative to D614G. Thus, SARS-CoV-2 emerging variants display enhanced syncytia formation.

High fusion and cytopathy of SARS-CoV-2 variant B.1.640.1.

SARS-CoV-2 variants with undetermined properties have emerged intermittently throughout the COVID-19 pandemic. Some variants possess unique phenotypes and mutations which allow further characterization of viral evolution and Spike functions. Around 1,100 cases of the B.1.640.1 variant were reported in Africa and Europe between 2021 and 2022, before the expansion of Omicron. Here, we analyzed the biological properties of a B.1.640.1 isolate and its Spike. Compared to the ancestral Spike, B.1.640.1 carried 14 amino acid substitutions and deletions. B.1.640.1 escaped binding by some anti-N-terminal domain and anti-receptor-binding domain monoclonal antibodies, and neutralization by sera from convalescent and vaccinated individuals. In cell lines, infection generated large syncytia and a high cytopathic effect. In primary airway cells, B.1.640.1 replicated less than Omicron BA.1 and triggered more syncytia and cell death than other variants. The B.1.640.1 Spike was highly fusogenic when expressed alone. This was mediated by two poorly characterized and infrequent mutations located in the Spike S2 domain, T859N and D936H. Altogether, our results highlight the cytopathy of a hyper-fusogenic SARS-CoV-2 variant, supplanted upon the emergence of Omicron BA.1. (This study has been registered at ClinicalTrials.gov under registration no. NCT04750720.)IMPORTANCEOur results highlight the plasticity of SARS-CoV-2 Spike to generate highly fusogenic and cytopathic strains with the causative mutations being uncharacterized in previous variants. We describe mechanisms regulating the formation of syncytia and the subsequent consequences in a primary culture model, which are poorly understood.

Syncytia formation by SARS-CoV-2-infected cells.

Severe cases of COVID-19 are associated with extensive lung damage and the presence of infected multinucleated syncytial pneumocytes. The viral and cellular mechanisms regulating the formation of these syncytia are not well understood. Here, we show that SARS-CoV-2-infected cells express the Spike protein (S) at their surface and fuse with ACE2-positive neighboring cells. Expression of S without any other viral proteins triggers syncytia formation. Interferon-induced transmembrane proteins (IFITMs), a family of restriction factors that block the entry of many viruses, inhibit S-mediated fusion, with IFITM1 being more active than IFITM2 and IFITM3. On the contrary, the TMPRSS2 serine protease, which is known to enhance infectivity of cell-free virions, processes both S and ACE2 and increases syncytia formation by accelerating the fusion process. TMPRSS2 thwarts the antiviral effect of IFITMs. Our results show that SARS-CoV-2 pathological effects are modulated by cellular proteins that either inhibit or facilitate syncytia formation.

IRF8 regulates efficacy of therapeutic anti-CD20 monoclonal antibodies.

Anti-CD20 monoclonal antibodies such as Rituximab, Ofatumumab, and Obinutuzumab are widely used to treat lymphomas and autoimmune diseases. They act by depleting B cells, mainly through Fc-dependent effectors functions. Some patients develop resistance to treatment but the underlying mechanisms are poorly understood. Here, we performed a genome-wide CRISPR/Cas9 screen to identify genes regulating the efficacy of anti-CD20 antibodies. We used as a model the killing of RAJI B cells by Rituximab through complement-dependent-cytotoxicity (CDC). As expected, the screen identified MS4A1, encoding CD20, the target of Rituximab. Among other identified genes, the role of Interferon Regulatory Factor 8 (IRF8) was validated in two B-cell lines. IRF8 KO also decreased the efficacy of antibody-dependent cellular cytotoxicity and phagocytosis (ADCC and ADCP) induced by anti-CD20 antibodies. We further show that IRF8 is necessary for efficient CD20 transcription. Levels of IRF8 and CD20 RNA or proteins correlated in normal B cells and in hundreds of malignant B cells. Therefore, IRF8 regulates CD20 expression and controls the depleting capacity of anti-CD20 antibodies. Our results bring novel insights into the pathways underlying resistance to CD20-targeting immunotherapies.

Zika virus induces massive cytoplasmic vacuolization and paraptosis-like death in infected cells.

The cytopathic effects of Zika virus (ZIKV) are poorly characterized. Innate immunity controls ZIKV infection and disease in most infected patients through mechanisms that remain to be understood. Here, we studied the morphological cellular changes induced by ZIKV and addressed the role of interferon-induced transmembrane proteins (IFITM), a family of broad-spectrum antiviral factors, during viral replication. We report that ZIKV induces massive vacuolization followed by "implosive" cell death in human epithelial cells, primary skin fibroblasts and astrocytes, a phenomenon which is exacerbated when IFITM3 levels are low. It is reminiscent of paraptosis, a caspase-independent, non-apoptotic form of cell death associated with the formation of large cytoplasmic vacuoles. We further show that ZIKV-induced vacuoles are derived from the endoplasmic reticulum (ER) and dependent on the PI3K/Akt signaling axis. Inhibiting the Sec61 ER translocon in ZIKV-infected cells blocked vacuole formation and viral production. Our results provide mechanistic insight behind the ZIKV-induced cytopathic effect and indicate that IFITM3, by acting as a gatekeeper for incoming virus, restricts virus takeover of the ER and subsequent cell death.

Atlastin Endoplasmic Reticulum-Shaping Proteins Facilitate Zika Virus Replication.

The endoplasmic reticulum (ER) is the site for Zika virus (ZIKV) replication and is central to the cytopathic effects observed in infected cells. ZIKV induces the formation of ER-derived large cytoplasmic vacuoles followed by "implosive" cell death. Little is known about the nature of the ER factors that regulate flavivirus replication. Atlastins (ATL1, -2, and -3) are dynamin-related GTPases that control the structure and the dynamics of the ER membrane. We show here that ZIKV replication is significantly decreased in the absence of ATL proteins. The appearance of infected cells is delayed, the levels of intracellular viral proteins and released virus are reduced, and the cytopathic effects are strongly impaired. We further show that ATL3 is recruited to viral replication sites and interacts with the nonstructural viral proteins NS2A and NS2B3. Thus, proteins that shape and maintain the ER tubular network ensure efficient ZIKV replication.IMPORTANCE Zika virus (ZIKV) is an emerging virus associated with Guillain-Barré syndrome, and fetal microcephaly as well as other neurological complications. There is no vaccine or specific antiviral treatment against ZIKV. We found that endoplasmic reticulum (ER)-shaping atlastin proteins (ATL1, -2, and -3), which induce ER membrane fusion, facilitate ZIKV replication. We show that ATL3 is recruited to the viral replication site and colocalize with the viral proteins NS2A and NS2B3. The results provide insights into host factors used by ZIKV to enhance its replication.

SUN2 Silencing Impairs CD4 T Cell Proliferation and Alters Sensitivity to HIV-1 Infection Independently of Cyclophilin A.

Linker of nucleoskeleton and cytoskeleton (LINC) complexes connect the nucleus to the cytoskeleton in eukaryotic cells. We previously reported that the overexpression of SUN2, an inner nuclear membrane protein and LINC complex component, inhibits HIV infection between the steps of reverse transcription and nuclear import in a capsid-specific manner. We also reported that SUN2 silencing does not modulate HIV infection in several cell lines. Silencing of SUN2 was recently reported to decrease HIV infection of CD4 T cells, an effect which was suggested to result from modulation of cyclophilin A (CypA)-dependent steps of HIV infection. We confirm here that HIV infection of primary CD4 T cells is compromised in the absence of endogenous SUN2, and we extend these findings to additional viral strains. However, we find that CypA is not required for the decreased infection observed in SUN2-silenced cells and, conversely, that endogenous SUN2 is not required for the well-documented positive modulation of HIV infection by CypA. In contrast, CD4 T cells lacking SUN2 exhibit a considerable defect in proliferative capacity and display reduced levels of activation markers and decreased viability. Additionally, SUN2-silenced CD4 T cells that become infected support reduced levels of viral protein expression. Our results demonstrate that SUN2 is required for the optimal activation and proliferation of primary CD4 T cells and suggest that the disruption of these processes explains the contribution of endogenous SUN2 to HIV infection in primary lymphocytes.IMPORTANCE Linker of nucleoskeleton and cytoskeleton (LINC) complexes connect the nucleus to the cytoskeleton. We previously reported that the overexpression of the LINC complex protein SUN2 inhibits HIV infection by targeting the viral capsid and blocking infection before the virus enters the nucleus. A recent report showed that the depletion of endogenous SUN2 in primary CD4 T cells results in decreased HIV infection and that this involves cyclophilin A (CypA), a host protein that interacts with the capsid of HIV to promote infection. We confirm that HIV infection is reduced in CD4 T cells lacking SUN2, but we find no role for CypA. Instead, SUN2 silencing results in CD4 T cells with decreased viability and much lower proliferation rates. Our results show that SUN2 is required for optimal CD4 T cell activation and proliferation and explain the reduced level of HIV infection in the absence of SUN2.

HIV Fusion in Dendritic Cells Occurs Mainly at the Surface and Is Limited by Low CD4 Levels.

HIV-1 poorly infects monocyte-derived dendritic cells (MDDCs). This is in large part due to SAMHD1, which restricts viral reverse transcription. Pseudotyping HIV-1 with vesicular stomatitis virus G protein (VSV-G) strongly enhances infection, suggesting that earlier steps of viral replication, including fusion, are also inefficient in MDDCs. The site of HIV-1 fusion remains controversial and may depend on the cell type, with reports indicating that it occurs at the plasma membrane or, conversely, in an endocytic compartment. Here, we examined the pathways of HIV-1 entry in MDDCs. Using a combination of temperature shift and fusion inhibitors, we show that HIV-1 fusion mainly occurs at the cell surface. We then asked whether surface levels or intracellular localization of CD4 modulates HIV-1 entry. Increasing CD4 levels strongly enhanced fusion and infection with various HIV-1 isolates, including reference and transmitted/founder strains, but not with BaL, which uses low CD4 levels for entry. Overexpressing coreceptors did not facilitate viral infection. To further study the localization of fusion events, we generated CD4 mutants carrying heterologous cytoplasmic tails (LAMP1 or Toll-like receptor 7 [TLR7]) to redirect the molecule to intracellular compartments. The intracellular CD4 mutants did not facilitate HIV-1 fusion and replication in MDDCs. Fusion of an HIV-2 isolate with MDDCs was also enhanced by increasing surface CD4 levels. Our results demonstrate that MDDCs are inefficiently infected by various HIV-1 and HIV-2 strains, in part because of low CD4 levels. In these cells, viral fusion occurs mainly at the surface, and probably not after internalization.IMPORTANCE Dendritic cells (DCs) are professional antigen-presenting cells inducing innate and adaptive immune responses. DCs express the HIV receptor CD4 and are potential target cells for HIV. There is debate about the sensitivity of DCs to productive HIV-1 and HIV-2 infection. The fusion step of the viral replication cycle is inefficient in DCs, and the underlying mechanisms are poorly characterized. We show that increasing the levels of CD4 at the plasma membrane allows more HIV fusion and productive infection in DCs. We further demonstrate that HIV fusion occurs mainly at the cell surface and not in an intracellular compartment. Our results help us understand why DCs are poorly sensitive to HIV infection.

Natural mutations in IFITM3 modulate post-translational regulation and toggle antiviral specificity.

The interferon-induced transmembrane (IFITM) proteins protect host cells from diverse virus infections. IFITM proteins also incorporate into HIV-1 virions and inhibit virus fusion and cell-to-cell spread, with IFITM3 showing the greatest potency. Here, we report that amino-terminal mutants of IFITM3 preventing ubiquitination and endocytosis are more abundantly incorporated into virions and exhibit enhanced inhibition of HIV-1 fusion. An analysis of primate genomes revealed that IFITM3 is the most ancient antiviral family member of the IFITM locus and has undergone a repeated duplication in independent host lineages. Some IFITM3 genes in nonhuman primates, including those that arose following gene duplication, carry amino-terminal mutations that modify protein localization and function. This suggests that "runaway" IFITM3 variants could be selected for altered antiviral activity. Furthermore, we show that adaptations in IFITM3 result in a trade-off in antiviral specificity, as variants exhibiting enhanced activity against HIV-1 poorly restrict influenza A virus. Overall, we provide the first experimental evidence that diversification of IFITM3 genes may boost the antiviral coverage of host cells and provide selective functional advantages.

SUN2 Overexpression Deforms Nuclear Shape and Inhibits HIV.

UNLABELLED: In a previous screen of putative interferon-stimulated genes, SUN2 was shown to inhibit HIV-1 infection in an uncharacterized manner. SUN2 is an inner nuclear membrane protein belonging to the linker of nucleoskeleton and cytoskeleton complex. We have analyzed here the role of SUN2 in HIV infection. We report that in contrast to what was initially thought, SUN2 is not induced by type I interferon, and that SUN2 silencing does not modulate HIV infection. However, SUN2 overexpression in cell lines and in primary monocyte-derived dendritic cells inhibits the replication of HIV but not murine leukemia virus or chikungunya virus. We identified HIV-1 and HIV-2 strains that are unaffected by SUN2, suggesting that the effect is specific to particular viral components or cofactors. Intriguingly, SUN2 overexpression induces a multilobular flower-like nuclear shape that does not impact cell viability and is similar to that of cells isolated from patients with HTLV-I-associated adult T-cell leukemia or with progeria. Nuclear shape changes and HIV inhibition both mapped to the nucleoplasmic domain of SUN2 that interacts with the nuclear lamina. This block to HIV replication occurs between reverse transcription and nuclear entry, and passaging experiments selected for a single-amino-acid change in capsid (CA) that leads to resistance to overexpressed SUN2. Furthermore, using chemical inhibition or silencing of cyclophilin A (CypA), as well as CA mutant viruses, we implicated CypA in the SUN2-imposed block to HIV infection. Our results demonstrate that SUN2 overexpression perturbs both nuclear shape and early events of HIV infection. IMPORTANCE: Cells encode proteins that interfere with viral replication, a number of which have been identified in overexpression screens. SUN2 is a nuclear membrane protein that was shown to inhibit HIV infection in such a screen, but how it blocked HIV infection was not known. We show that SUN2 overexpression blocks the infection of certain strains of HIV before nuclear entry. Mutation of the viral capsid protein yielded SUN2-resistant HIV. Additionally, the inhibition of HIV infection by SUN2 involves cyclophilin A, a protein that binds the HIV capsid and directs subsequent steps of infection. We also found that SUN2 overexpression substantially changes the shape of the cell's nucleus, resulting in many flower-like nuclei. Both HIV inhibition and deformation of nuclear shape required the domain of SUN2 that interacts with the nuclear lamina. Our results demonstrate that SUN2 interferes with HIV infection and highlight novel links between nuclear shape and viral infection.

Vpr Enhances Tumor Necrosis Factor Production by HIV-1-Infected T Cells.

UNLABELLED: The HIV-1 accessory protein Vpr displays different activities potentially impacting viral replication, including the arrest of the cell cycle in the G2 phase and the stimulation of apoptosis and DNA damage response pathways. Vpr also modulates cytokine production by infected cells, but this property remains partly characterized. Here, we investigated the effect of Vpr on the production of the proinflammatory cytokine tumor necrosis factor (TNF). We report that Vpr significantly increases TNF secretion by infected lymphocytes. De novo production of Vpr is required for this effect. Vpr mutants known to be defective for G2 cell cycle arrest induce lower levels of TNF secretion, suggesting a link between these two functions. Silencing experiments and the use of chemical inhibitors further implicated the cellular proteins DDB1 and TAK1 in this activity of Vpr. TNF secreted by HIV-1-infected cells triggers NF-κB activity in bystander cells and allows viral reactivation in a model of latently infected cells. Thus, the stimulation of the proinflammatory pathway by Vpr may impact HIV-1 replication in vivo. IMPORTANCE: The role of the HIV-1 accessory protein Vpr remains only partially characterized. This protein is important for viral pathogenesis in infected individuals but is dispensable for viral replication in most cell culture systems. Some of the functions described for Vpr remain controversial. In particular, it remains unclear whether Vpr promotes or instead prevents proinflammatory and antiviral immune responses. In this report, we show that Vpr promotes the release of TNF, a proinflammatory cytokine associated with rapid disease progression. Using Vpr mutants or inhibiting selected cellular genes, we show that the cellular proteins DDB1 and TAK1 are involved in the release of TNF by HIV-infected cells. This report provides novel insights into how Vpr manipulates TNF production and helps clarify the role of Vpr in innate immune responses and inflammation.

SAMHD1 Limits HIV-1 Antigen Presentation by Monocyte-Derived Dendritic Cells.

UNLABELLED: Monocyte-derived dendritic cells (MDDC) stimulate CD8 cytotoxic T lymphocytes (CTL) by presenting endogenous and exogenous viral peptides via major histocompatibility complex class I (MHC-I) molecules. MDDC are poorly susceptible to HIV-1, in part due to the presence of SAMHD1, a cellular enzyme that depletes intracellular deoxynucleoside triphosphates (dNTPs) and degrades viral RNA. Vpx, an HIV-2/SIVsm protein absent from HIV-1, antagonizes SAMHD1 by inducing its degradation. The impact of SAMHD1 on the adaptive cellular immune response remains poorly characterized. Here, we asked whether SAMHD1 modulates MHC-I-restricted HIV-1 antigen presentation. Untreated MDDC or MDDC pretreated with Vpx were exposed to HIV-1, and antigen presentation was examined by monitoring the activation of an HIV-1 Gag-specific CTL clone. SAMHD1 depletion strongly enhanced productive infection of MDDC as well as endogenous HIV-1 antigen presentation. Time-lapse microscopy analysis demonstrated that in the absence of SAMHD1, the CTL rapidly killed infected MDDC. We also report that various transmitted/founder (T/F) HIV-1 strains poorly infected MDDC and, as a consequence, did not stimulate CTL. Vesicular stomatitis virus glycoprotein (VSV-G) pseudotyping of T/F alleviated a block in viral entry and induced antigen presentation only in the absence of SAMHD1. Furthermore, by using another CTL clone that mostly recognizes incoming HIV-1 antigens, we demonstrate that SAMHD1 does not influence exogenous viral antigen presentation. Altogether, our results demonstrate that the antiviral activity of SAMHD1 impacts antigen presentation by DC, highlighting the link that exists between restriction factors and adaptive immune responses. IMPORTANCE: Upon viral infection, DC may present antigens derived from incoming viral material in the absence of productive infection of DC or from newly synthesized viral proteins. In the case of HIV, productive infection of DC is blocked at an early postentry step. This is due to the presence of SAMHD1, a cellular enzyme that depletes intracellular levels of dNTPs and inhibits viral reverse transcription. We show that the depletion of SAMHD1 in DCs strongly stimulates the presentation of viral antigens derived from newly produced viral proteins, leading to the activation of HIV-1-specific cytotoxic T lymphocytes (CTL). We further show in real time that the enhanced activation of CTL leads to killing of infected DCs. Our results indicate that the antiviral activity of SAMHD1 not only impacts HIV replication but also impacts antigen presentation by DC. They highlight the link that exists between restriction factors and adaptive immune responses.

HIV-2 infects resting CD4+ T cells but not monocyte-derived dendritic cells.

BACKGROUND: Human Immunodeficiency Virus-type 2 (HIV-2) encodes Vpx that degrades SAMHD1, a cellular restriction factor active in non-dividing cells. HIV-2 replicates in lymphocytes but the susceptibility of monocyte-derived dendritic cells (MDDCs) to in vitro infection remains partly characterized. RESULTS: Here, we investigated HIV-2 replication in primary CD4+ T lymphocytes, both activated and non-activated, as well as in MDDCs. We focused on the requirement of Vpx for productive HIV-2 infection, using the reference HIV-2 ROD strain, the proviral clone GL-AN, as well as two primary HIV-2 isolates. All HIV-2 strains tested replicated in activated CD4+ T cells. Unstimulated CD4+ T cells were not productively infected by HIV-2, but viral replication was triggered upon lymphocyte activation in a Vpx-dependent manner. In contrast, MDDCs were poorly infected when exposed to HIV-2. HIV-2 particles did not potently fuse with MDDCs and did not lead to efficient viral DNA synthesis, even in the presence of Vpx. Moreover, the HIV-2 strains tested were not efficiently sensed by MDDCs, as evidenced by a lack of MxA induction upon viral exposure. Virion pseudotyping with VSV-G rescued fusion, productive infection and HIV-2 sensing by MDDCs. CONCLUSION: Vpx allows the non-productive infection of resting CD4+ T cells, but does not confer HIV-2 with the ability to efficiently infect MDDCs. In these cells, an entry defect prevents viral fusion and reverse transcription independently of SAMHD1. We propose that HIV-2, like HIV-1, does not productively infect MDDCs, possibly to avoid triggering an immune response mediated by these cells.

SAMHD1 restricts HIV-1 cell-to-cell transmission and limits immune detection in monocyte-derived dendritic cells.

SAMHD1 is a viral restriction factor expressed in dendritic cells and other cells, inhibiting infection by cell-free human immunodeficiency virus type 1 (HIV-1) particles. SAMHD1 depletes the intracellular pool of deoxynucleoside triphosphates, thus impairing HIV-1 reverse transcription and productive infection in noncycling cells. The Vpx protein from HIV-2 or simian immunodeficiency virus (SIVsm/SIVmac) antagonizes the effect of SAMHD1 by triggering its degradation. A large part of HIV-1 spread occurs through direct contacts between infected cells and bystander target cells. Here, we asked whether SAMHD1 impairs direct HIV-1 transmission from infected T lymphocytes to monocyte-derived dendritic cells (MDDCs). HIV-1-infected lymphocytes were cocultivated with MDDCs that have been pretreated or not with Vpx or with small interfering RNA against SAMHD1. We show that in the cocultures, SAMHD1 significantly inhibits productive cell-to-cell transmission to target MDDCs and prevents the type I interferon response and expression of the interferon-stimulated gene MxA. Therefore, SAMHD1, by controlling the sensitivity of MDDCs to HIV-1 infection during intercellular contacts, impacts their ability to sense the virus and to trigger an innate immune response.

Hyperthermia stimulates HIV-1 replication.

HIV-infected individuals may experience fever episodes. Fever is an elevation of the body temperature accompanied by inflammation. It is usually beneficial for the host through enhancement of immunological defenses. In cultures, transient non-physiological heat shock (42-45°C) and Heat Shock Proteins (HSPs) modulate HIV-1 replication, through poorly defined mechanisms. The effect of physiological hyperthermia (38-40°C) on HIV-1 infection has not been extensively investigated. Here, we show that culturing primary CD4+ T lymphocytes and cell lines at a fever-like temperature (39.5°C) increased the efficiency of HIV-1 replication by 2 to 7 fold. Hyperthermia did not facilitate viral entry nor reverse transcription, but increased Tat transactivation of the LTR viral promoter. Hyperthermia also boosted HIV-1 reactivation in a model of latently-infected cells. By imaging HIV-1 transcription, we further show that Hsp90 co-localized with actively transcribing provirus, and this phenomenon was enhanced at 39.5°C. The Hsp90 inhibitor 17-AAG abrogated the increase of HIV-1 replication in hyperthermic cells. Altogether, our results indicate that fever may directly stimulate HIV-1 replication, in a process involving Hsp90 and facilitation of Tat-mediated LTR activity.

Beta-variant recombinant booster vaccine elicits broad cross-reactive neutralization of SARS-CoV-2 including Omicron variants.

Background: SARS-CoV-2 Omicron lineage contains variants with multiple sequence mutations relative to the ancestral strain particularly in the viral spike gene. These mutations are associated inter alia with loss of neutralization sensitivity to sera generated by immunization with vaccines targeting ancestral strains or prior infection with circulating (non-Omicron) variants. Here we present a comparison of vaccine formulation elicited cross neutralization responses using two different assay readouts from a subpopulation of a Phase II/III clinical trial. Methods: Human sera from a Phase II/III trial (NCT04762680) was collected and evaluated for neutralizing responses to SARS-CoV-2 spike antigen protein vaccines formulated with AS03 adjuvant, following a primary series of two-doses of ancestral strain vaccine in individuals who were previously unvaccinated or as an ancestral or variant strain booster vaccine among individuals previously vaccinated with the mRNA BNT162b2 vaccine. Results: We report that a neutralizing response to Omicron BA.1 is induced by the two-dose primary series in 89% of SARS-CoV-2-seronegative individuals. A booster dose of each vaccine formulation raises neutralizing antibody titers that effectively neutralizes Omicron BA.1 and BA.4/5 variants. Responses are highest after the monovalent Beta variant booster and similar in magnitude to human convalescent plasma titers. Conclusion: The findings of this study suggest the possibility to generate greater breadth of cross-neutralization to more recently emerging viral variants through use of a diverged spike vaccine in the form of a Beta variant booster vaccine.