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Interferon restricts SARS-CoV-2 replication in cell culture, but only a handful of Interferon Stimulated Genes with antiviral activity against SARS-CoV-2 have been identified. Here, we describe a functional CRISPR/Cas9 screen aiming at identifying SARS-CoV-2 restriction factors. We identify DAXX, a scaffold protein residing in PML nuclear bodies known to limit the replication of DNA viruses and retroviruses, as a potent inhibitor of SARS-CoV-2 and SARS-CoV replication in human cells. Basal expression of DAXX is sufficient to limit the replication of SARS-CoV-2, and DAXX over-expression further restricts infection. DAXX restricts an early, post-entry step of the SARS-CoV-2 life cycle. DAXX-mediated restriction of SARS-CoV-2 is independent of the SUMOylation pathway but dependent on its D/E domain, also necessary for its protein-folding activity. SARS-CoV-2 infection triggers the re-localization of DAXX to cytoplasmic sites and promotes its degradation. Mechanistically, this process is mediated by the viral papain-like protease (PLpro) and the proteasome. Together, these results demonstrate that DAXX restricts SARS-CoV-2, which in turn has evolved a mechanism to counteract its action.

Live-attenuated influenza vaccines (LAIV) offer advantages over the commonly used inactivated split influenza vaccines. However, finding the optimal balance between sufficient attenuation and immunogenicity has remained a challenge. We recently developed an alternative LAIV based on the 2009 pandemic H1N1 virus with a truncated NS1 protein and lacking PA-X protein expression (NS1(1–126)-ΔPAX). This virus showed a blunted replication and elicited a strong innate immune response. In the present study, we evaluated the efficacy of this vaccine candidate in the porcine animal model as a pertinent in vivo system. Immunization of pigs via the nasal route with the novel NS1(1–126)-ΔPAX LAIV did not cause disease and elicited a strong mucosal immune response that completely blocked replication of the homologous challenge virus in the respiratory tract. However, we observed prolonged shedding of our vaccine candidate from the upper respiratory tract. To improve LAIV safety, we developed a novel prime/boost vaccination strategy combining primary intramuscular immunization with a haemagglutinin-encoding propagation-defective vesicular stomatitis virus (VSV) replicon, followed by a secondary immunization with the NS1(1–126)-ΔPAX LAIV via the nasal route. This two-step immunization procedure significantly reduced LAIV shedding, increased the production of specific serum IgG, neutralizing antibodies, and Th1 memory cells, and resulted in sterilizing immunity against homologous virus challenge. In conclusion, our novel intramuscular prime/intranasal boost regimen interferes with virus shedding and transmission, a feature that will help combat influenza epidemics and pandemics. Author summary: Nasally administered live-attenuated influenza vaccines induce a mucosal immune response that can effectively prevent primary infection and virus shedding. However, the development of alternative live vaccine strategies must consider the balance between eliciting a robust immune response and reducing the virulence of the vaccine candidate. To address this problem, we have developed a novel prime/boost vaccination strategy consisting of an initial intramuscular immunization with a propagation-defective RNA virus vector and subsequent nasal immunization with a modified attenuated influenza virus that has lost its ability to counteract the host innate immune response. Using the porcine model, we demonstrate that this novel strategy is capable of eliciting a robust immune response both systemically and at mucosal surfaces. Importantly, intranasal infection with homologous challenge virus remained undetectable in vaccinated animals. In conclusion, our innovative vaccination regimen represents a promising strategy to control influenza disease and virus spread in both humans and livestock. [ABSTRACT FROM AUTHOR]

Background and Aims: Severe acute respiratory syndrome coronavirus (SARS‐CoV‐2) preferentially infects the respiratory tract; however, several studies have implicated a multi‐organ involvement. Hepatic dysfunctions caused by SARS‐CoV‐2 infection have been increasingly recognized and described to correlate with disease severity. To elucidate molecular factors that could contribute towards hepatic infection, we concentrated on microRNAs (miRNAs), a class of small non‐coding RNAs that modulate various cellular processes and which are reported to be differentially regulated during liver injury. We aimed to study the infection of primary human hepatocytes (PHH) with SARS‐CoV‐2 and to evaluate the potential of miRNAs for modulating viral infection. Methods: We analysed liver autopsies from a coronavirus disease 19 (COVID‐19)‐positive cohort for the presence of viral RNA using Nanopore sequencing. PHH were used for the infection with SARS‐CoV‐2. The candidate miRNAs targeting angiotensin converting enzyme 2 (ACE2) and transmembrane serine protease 2 (TMPRSS2) were identified using in silico approaches. To discover the potential regulatory mechanism, transfection experiments, qRT‐PCRs, western blots and luciferase reporter assays were performed. Results: We could detect SARS‐CoV‐2 RNA in COVID‐19‐positive liver autopsies. We show that PHH express ACE2 and TMPRSS2 and can be readily infected with SARS‐CoV‐2, resulting in robust replication. Transfection of selected miRNA mimics reduced SARS‐CoV‐2 receptor expression and SARS‐CoV‐2 burden in PHH. In silico and biochemical analyses supported a potential direct binding of miR‐141‐3p to the SARS‐CoV‐2 genome. Conclusion: We confirm that PHH are susceptible to SARS‐CoV‐2 infection and demonstrate selected miRNAs targeting SARS‐CoV‐2 entry factors and/or the viral genome reduce viral loads. These data provide novel insights into hepatic susceptibility to SARS‐CoV‐2 and associated dysfunctions in COVID‐19. [ABSTRACT FROM AUTHOR]