Previous reports assumed that sphingomyelin (SM) is essential for HCV replication, but the mechanism was unclear. In this study, we showed for the first time that SM and ceramide transfer protein (CERT), which is in the SM biosynthesis pathway, are essential for the biosynthesis of double-membrane vesicles (DMVs), the sites of viral replication. Low numbers of DMVs were observed in CERT-KO cells transfected with replicon RNA or with constructs that drive HCV protein production in a replication-independent system. HCV replication was rescued by ectopic expression of the CERT protein, but not by CERT mutants, that abolishes the binding of CERT to vesicle-associated membrane protein-associated protein (VAP) or phosphatidylinositol 4-phosphate (PI4P), indicating new roles for VAP and PI4P in HCV replication. The biosynthesis of DMVs has great importance to replication by a variety of plus-stranded RNA viruses. Understanding of this process is expected to facilitate the development of diagnosis and antivirus.

The SARS-CoV-2 virus can utilize host cell proteases to facilitate cell entry, whereby the Spike (S) protein is cleaved at two specific sites to enable membrane fusion. Furin, transmembrane protease serine 2 (TMPRSS2), and cathepsin L (CatL) are the major proteases implicated, and are thus targets for anti-viral therapy. The human serpin (serine protease inhibitor) alpha-1 antitrypsin (A1AT) shows inhibitory activity for TMPRSS2, and has previously been found to suppress cell infection with SARS-CoV-2. Here, we have generated modified serpin inhibitors with increased specificity for these cellular proteases. Using SerpinB3 (SCCA-1), a cross-class inhibitor of CatL, as a scaffold, we have designed and produced reactive centre loop (RCL) variants to more specifically target both furin and TMPRSS2. Two further variants were generated by substituting the RCL P7–P1 with the spike protein S1/S2 cleavage site from either SARS-CoV-2 alpha or delta (P681R) sequences. Altered inhibitory specificity of purified recombinant proteins was verified in protease assays, with attenuated CatL inhibition and gain of furin or TMPRSS2 inhibition, as predicted, and modified serpins were shown to block S protein cleavage in vitro. Furthermore, the serpin variants were able to inhibit S-pseudoparticle entry into A549-ACE2-TMPRSS2 cells and suppress SARS-CoV-2 replication in Vero E6 cells expressing TMPRSS2. The construct designed to inhibit TMPRSS2 (B3-TMP) was most potent. It was more effective than A1AT for TMPRSS2 enzyme inhibition (with an eighteen-fold improvement in the second order inhibition rate constant) and for blocking SARS-CoV-2 viral replication. These findings advance the potential for serpin RCL mutagenesis to generate new inhibitors, and may lead to novel anti-viral biological molecules.

The host PYHIN (pyrin and HIN domain family) protein IFN-γ-inducible protein 16 (IFI16) was first discovered as a nuclear sensor of double-stranded DNA (dsDNA). Since then its roles in innate immunity have expanded to include restriction of infection of both DNA and RNA viruses. Mechanistically, IFI16 restricts DNA viruses through four principal mechanisms: (i) direct repression of viral gene expression by binding viral genomes and promoting epigenetic-mediated silencing; (ii) sequestration of host transcription factor Sp1; (iii) induction of interferons (IFNs) after sensing viral genomes in the nucleus and cytosol; and (iv) assembly of apoptosis-associated speck-like protein containing a CARD (ASC)-dependent inflammasomes that activates caspase-1 leading to maturation of interleukin-1 beta (IL-1β) and interleukin-18 (IL-18) and pyroptosis. These mechanisms have been reported across dsDNA virus families, including Herpesviridae, Papillomaviridae, Hepadnaviridae, Parvoviridae, Polyomaviridae, and Poxviridae. For RNA viruses, IFI16 can: (i) directly bind viral genomes or sequester Sp1; (ii) amplify antiviral signalling by promoting RIG-I transcription or activation or cooperating with cyclic GMP-AMP synthase (cGAS)- stimulator of IFN genes (STING), and (iii) in some settings activate inflammasomes and pyroptosis. These mechanisms were reported for RNA virus families including, Togaviridae, Flaviviridae, Picornaviridae, Caliciviridae, Arteriviridae, Orthomyxoviridae, Paramyxoviridae and Retroviridae. Consistent with these antiviral roles, many viruses have evolved both destructive (IFI16 degradation) and non-destructive mechanisms to evade IFI16. This review summarizes the current understanding of how IFI16 mediates broad antiviral restriction and how diverse viruses subvert this role to facilitate their replication.