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The antiviral activity of MARCH8 has been associated with the downregulation of envelope glycoproteins from a range of different viruses, resulting in reduced incorporation into nascent virions. Here, we show that MARCH8 restricts IAV at a late stage in virus replication, but this was not associated with reduced expression of IAV envelope glycoproteins on the surfaces of infected cells, pointing to a distinct mechanism of antiviral activity. Membrane-associated RING-CH8 (MARCH8) impairs the cell surface expression of envelope glycoproteins from different viruses, reducing their incorporation into virions. Using stable cell lines with inducible MARCH8 expression, we show that MARCH8 did not alter susceptibility to influenza A virus (IAV) infection, but virions released from infected cells were markedly less infectious. Knockdown of endogenous MARCH8 confirmed its effect on IAV infectivity. The expression of MARCH8 impaired the infectivity of both H3N2 and H1N1 strains and was dependent on its E3 ligase activity. Although virions released in the presence of MARCH8 expressed smaller amounts of viral hemagglutinin (HA) and neuraminidase (NA) proteins, there was no impact on levels of the viral HA, NA, or matrix 2 (M2) proteins detected on the surface of infected cells. Moreover, mutation of lysine residues in the cytoplasmic tails of HA, NA, and/or M2, or in the viral M1 protein, did not abrogate MARCH8-mediated restriction. While MARCH1 and -8 target similar immunological ligands and both restrict HIV-1, only MARCH8 inhibited IAV infectivity. Deletion of the N-terminal cytoplasmic (N-CT) domain of MARCH8 confirmed it to be a critical determinant of IAV inhibition. Of interest, deletion of the MARCH1 N-CT or its replacement with the MARCH8 N-CT resulted in acquisition of IAV restriction. Together, these data demonstrate that MARCH8 restricts a late stage in IAV replication by a mechanism distinct to its reported activity against other viruses. Moreover, we show that the N-CT of MARCH8 is essential for anti-IAV activity, whereas the MARCH1 N-CT inhibits its ability to restrict IAV. IMPORTANCE The antiviral activity of MARCH8 has been associated with the downregulation of envelope glycoproteins from a range of different viruses, resulting in reduced incorporation into nascent virions. Here, we show that MARCH8 restricts IAV at a late stage in virus replication, but this was not associated with reduced expression of IAV envelope glycoproteins on the surface of infected cells, pointing to a distinct mechanism of antiviral activity. Our studies also demonstrate the differential ability of MARCH1 and -8 to restrict IAV infectivity, highlighting the critical role of the N-CT domain of each protein in modulating IAV restriction. Overall, these studies provide novel insights regarding the mechanisms by which MARCH proteins contribute to cell-intrinsic immunity against IAV.

Andes virus (ANDV), a highly pathogenic orthohantavirus, enters host cells through low pH-triggered membrane fusion mediated by the Gc glycoprotein, a class II fusion protein containing a single C-terminal transmembrane domain (TMD). While the ectodomain has been extensively characterized, the role of the TMD in late-stage fusion remains unclear. Here, we investigated the minimal functional length and sequence requirements of the ANDV Gc TMD using site-directed mutagenesis. C-terminal deletion mutants and serine-to-alanine substitutions were evaluated for protein expression, virus-like particle production, cell-cell fusion, pseudotyped vector entry, and hemifusion activity. Deletion of the Gc cytoplasmic tail (CT) or a single C-terminal TMD residue was tolerated, whereas deletion of two or more residues impaired particle production and fusion, indicating that at least 21 of the 22 TMD residues are required for efficient membrane fusion and viral entry. Hemifusion assays showed that deletion of two or three residues, or substitution of the strictly conserved S1121, allowed lipid mixing but blocked progression to full fusion, while deletion of four residues also abolished hemifusion. In contrast, mutation of the less conserved S1126 had minimal effect. These results identify a precise TMD length and a conserved polar TMD residue as critical determinants of fusion pore formation in ANDV.

MARCH1 and MARCH8 are closely related E3 ubiquitin ligases that ubiquitinate an overlapping spectrum of host proteins and restrict replication of certain viruses. While the antiviral activity of MARCH8 has been intensively studied, less is known regarding virus inhibition by MARCH1. Isoforms 1 and 2 of MARCH1 are very similar in overall structure but show major differences in their N-terminal cytoplasmic domain (N-CT). Herein, we used a doxycycline-inducible overexpression system to demonstrate that MARCH1.1 reduces titres of influenza A virus (IAV) released from infected cells whereas MARCH1.2 does not. The deletion of the entire N-CT of MARCH1.2 restored its ability to restrict IAV infectivity and sequential deletions mapped the restoration of IAV inhibition to delete the 16 N-terminal residues within the N-CT of MARCH1.2. While only MARCH1.1 mediated anti-IAV activity, qPCR demonstrated the preferential expression of MARCH1.2 over MARCH1.1 mRNA in unstimulated human peripheral blood mononuclear cells and also in monocyte-derived macrophages. Together, these studies describe the differential ability of MARCH1 isoforms to restrict IAV infectivity for the first time. Moreover, as published immunological, virological and biochemical studies examining the ability of MARCH1 to target particular ligands generally use only one of the two isoforms, these findings have broader implications for our understanding of how MARCH1 isoforms might differ in their ability to modulate particular host and/or viral proteins.

Viral infection of different cell types induces a unique spectrum of host defence genes, including interferon-stimulated genes (ISGs) and genes encoding other proteins with antiviral potential. Although hundreds of ISGs have been described, the vast majority have not been functionally characterised. Cellular proteins with putative antiviral activity (hereafter referred to as “restriction factors”) can target various steps in the virus life-cycle. In the context of influenza virus infection, restriction factors have been described that target virus entry, genomic replication, translation and virus release. Genome wide analyses, in combination with ectopic overexpression and/or gene silencing studies, have accelerated the identification of restriction factors that are active against influenza and other viruses, as well as providing important insights regarding mechanisms of antiviral activity. Herein, we review current knowledge regarding restriction factors that mediate anti-influenza virus activity and consider the viral countermeasures that are known to limit their impact. Moreover, we consider the strengths and limitations of experimental approaches to study restriction factors, discrepancies between in vitro and in vivo studies, and the potential to exploit restriction factors to limit disease caused by influenza and other respiratory viruses.

RIG-I is an innate sensor of RNA virus infection and its activation induces interferon-stimulated genes (ISGs). In vitro studies using human cells have demonstrated the ability of synthetic RIG-I agonists (3pRNA) to inhibit IAV replication. However, in mouse models of IAV the effectiveness of 3pRNA reported to date differs markedly between studies. Myxoma resistance (Mx)1 is an ISG protein which mediates potent anti-IAV activity, however most inbred mouse strains do not express a functional Mx1. Herein, we utilised C57BL/6 mice that do (B6.A2G-Mx1) and do not (B6-WT) express functional Mx1 to assess the ability of prophylactic 3pRNA treatment to induce ISGs and to protect against subsequent IAV infection. In vitro, 3pRNA treatment of primary lung cells from B6-WT and B6.A2G-Mx1 mice resulted in ISG induction however inhibition of IAV infection was more potent in cells from B6.A2G-Mx1 mice. In vivo, a single intravenous injection of 3pRNA resulted in ISG induction in lungs of both B6-WT and B6.A2G-Mx1 mice, however potent and long-lasting protection against subsequent IAV challenge was only observed in B6.A2G-Mx1 mice. Thus, despite broad ISG induction, expression of a functional Mx1 is critical for potent and long-lasting RIG-I agonist-mediated protection in the mouse model of IAV infection.

Ectopic protein overexpression in immortalised cell lines is a commonly used method to screen host factors for their antiviral activity against different viruses. However, the question remains as to what extent such artificial protein overexpression recapitulates endogenous protein function. Previously, we used a doxycycline-inducible overexpression system, in conjunction with approaches to modulate the expression of endogenous protein, to demonstrate the antiviral activity of IFITM1, IFITM2, and IFITM3 against influenza A virus (IAV) but not parainfluenza virus-3 (PIV-3) in A549 cells. We now show that constitutive overexpression of the same IFITM constructs in A549 cells led to a significant restriction of PIV-3 infection by all three IFITM proteins. Variable IFITM mRNA and protein expression levels were detected in A549 cells with constitutive versus inducible overexpression of each IFITM. Our findings show that overexpression approaches can lead to levels of IFITM1, IFITM2, and IFITM3 that significantly exceed those achieved through interferon stimulation of endogenous protein. We propose that exceedingly high levels of overexpressed IFITMs may not accurately reflect the true function of endogenous protein, thus contributing to discrepancies when attributing the antiviral activity of individual IFITM proteins against different viruses. Our findings clearly highlight the caveats associated with overexpression approaches used to screen cellular host proteins for antiviral activity.

Hantaviruses can cause hantavirus pulmonary syndrome or hemorrhagic fever with renal syndrome in humans. To enter cells, hantaviruses fuse their envelope membrane with host cell membranes. Previously, we have shown that the Gc envelope glycoprotein is the viral fusion protein sharing characteristics with class II fusion proteins. The ectodomain of class II fusion proteins is composed of three domains connected by a stem region to a transmembrane anchor in the viral envelope. These fusion proteins can be inhibited through exogenous fusion protein fragments spanning domain III (DIII) and the stem region. Such fragments are thought to interact with the core of the fusion protein trimer during the transition from its pre-fusion to its post-fusion conformation. Based on our previous homology model structure for Gc from Andes hantavirus (ANDV), here we predicted and generated recombinant DIII and stem peptides to test whether these fragments inhibit hantavirus membrane fusion and cell entry. Recombinant ANDV DIII was soluble, presented disulfide bridges and beta-sheet secondary structure, supporting the in silico model. Using DIII and the C-terminal part of the stem region, the infection of cells by ANDV was blocked up to 60% when fusion of ANDV occurred within the endosomal route, and up to 95% when fusion occurred with the plasma membrane. Furthermore, the fragments impaired ANDV glycoprotein-mediated cell-cell fusion, and cross-inhibited the fusion mediated by the glycoproteins from Puumala virus (PUUV). The Gc fragments interfered in ANDV cell entry by preventing membrane hemifusion and pore formation, retaining Gc in a non-resistant homotrimer stage, as described for DIII and stem peptide inhibitors of class II fusion proteins. Collectively, our results demonstrate that hantavirus Gc shares not only structural, but also mechanistic similarity with class II viral fusion proteins, and will hopefully help in developing novel therapeutic strategies against hantaviruses.

Exosomes are extracellular vesicles secreted by cells that have an important biological function in intercellular communication by transferring biologically active proteins, lipids, and RNAs to neighboring or distant cells. While a role for exosomes in antimicrobial defense has recently emerged, currently very little is known regarding the nature and functional relevance of exosomes generated , particularly during an active viral infection. Here, we characterized exosomes released into the airways during influenza virus infection. We show that these vesicles dynamically change in protein composition over the course of infection, increasing expression of host proteins with known anti-influenza activity, and viral proteins with the potential to trigger host immune responses. We show that exosomes released into the airways during influenza virus infection trigger pulmonary inflammation and carry viral antigen that can be utilized by antigen presenting cells to drive the induction of a cellular immune response. Moreover, we show that attachment factors for influenza virus, namely α2,3 and α2,6-linked sialic acids, are present on the surface of airway exosomes and these vesicles have the ability to neutralize influenza virus, thereby preventing the virus from binding and entering target cells. These data reveal a novel role for airway exosomes in the antiviral innate immune defense against influenza virus infection.

Objectives Efforts to develop and deploy effective vaccines against severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) continue at pace. Here, we describe rational antigen design through to manufacturability and vaccine efficacy of a prefusion‐stabilised spike (S) protein, Sclamp, in combination with the licensed adjuvant MF59 ‘MF59C.1’ (Seqirus, Parkville, Australia). Methods A panel recombinant Sclamp proteins were produced in Chinese hamster ovary and screened in vitro to select a lead vaccine candidate. The structure of this antigen was determined by cryo‐electron microscopy and assessed in mouse immunogenicity studies, hamster challenge studies and safety and toxicology studies in rat. Results In mice, the Sclamp vaccine elicits high levels of neutralising antibodies, as well as broadly reactive and polyfunctional S‐specific CD4+ and cytotoxic CD8+ T cells in vivo. In the Syrian hamster challenge model (n = 70), vaccination results in reduced viral load within the lung, protection from pulmonary disease and decreased viral shedding in daily throat swabs which correlated strongly with the neutralising antibody level. Conclusion The SARS‐CoV‐2 Sclamp vaccine candidate is compatible with large‐scale commercial manufacture, stable at 2–8°C. When formulated with MF59 adjuvant, it elicits neutralising antibodies and T‐cell responses and provides protection in animal challenge models. We describe preclinical development of a prefusion stabilized spike protein subunit vaccine for SARS‐CoV‐2. Results include structural resolution by cryo‐electron microscopy, mouse immunogenicity and a hamster protection study.