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Dynamin-like GTPase proteins, including myxoma (Mx) and guanylate-binding proteins (GBPs), are among the many interferon stimulated genes induced following viral infections. While studies report that human (h)GBPs inhibit different viruses in vitro, few have convincingly demonstrated that mouse (m)GBPs mediate antiviral activity, although mGBP-deficient mice have been used extensively to define their importance in immunity to diverse intracellular bacteria and protozoa. Herein, we demonstrate that individual (overexpression) or collective (knockout (KO) mice) mGBPs of the chromosome 3 cluster (mGBPchr3) do not inhibit replication of five viruses from different virus families in vitro, nor do we observe differences in virus titres recovered from wild type versus mGBPchr3 KO mice after infection with three of these viruses (influenza A virus, herpes simplex virus type 1 or lymphocytic choriomeningitis virus). These data indicate that mGBPchr3 do not appear to be a major component of cell-intrinsic antiviral immunity against the diverse viruses tested in our studies., (© 2024. The Author(s).)

We determined the dengue virus (DENV) serotypes and genotypes in archived serum samples that were collected during the 2014-2016 and 2021 dengue outbreaks in Dire Dawa City and the Somali region in Ethiopia. DENV serotype 1 (DENV-1) was predominant followed by DENV serotype 2 (DENV-2). Thirteen of the DENV-1 strains were assigned to Genotype-I, while the remaining two were found to be Genotype-III. All three DENV-2 strains were assigned the Cosmopolitan Genotype. The DENV strains responsible for the outbreaks are genetically closely related to the DENV strains that circulated in neighboring and Asian countries. The findings also showed continued local transmission of a monophyletic lineage and a co-circulation of DENV-1 and DENV-2 during the outbreaks. There is a need to strengthen DENV genomic surveillance capacity for the early detection of circulating serotypes, and prevent devastating consequences of future outbreaks due to the co-circulation of different serotypes.

Many interferon (IFN)-stimulated genes are upregulated within host cells following infection with influenza and other viruses. While the antiviral activity of some IFN-stimulated genes, such as the IFN-inducible GTPase myxoma resistance (Mx)1 protein 1, has been well defined, less is known regarding the antiviral activities of related IFN-inducible GTPases of the guanylate-binding protein (GBP) family, particularly mouse GBPs, where mouse models can be used to assess their antiviral properties in vivo. Herein, we demonstrate that mouse GBP1 (mGBP1) was upregulated in a mouse airway epithelial cell line (LA-4 cells) following pretreatment with mouse IFNα or infection by influenza A virus (IAV). Whereas doxycycline-inducible expression of mouse Mx1 (mMx1) in LA-4 cells resulted in reduced susceptibility to IAV infection and reduced viral growth, inducible mGBP1 did not. Moreover, primary cells isolated from mGBP1-deficient mice (mGBP1 -/- ) showed no difference in susceptibility to IAV and mGBP1 -/- macrophages showed no defect in IAV-induced NLRP3 (NLR family pyrin domain containing 3) inflammasome activation. After intranasal IAV infection, mGBP1 -/- mice also showed no differences in virus replication or induction of inflammatory responses in the airways during infection. Thus, using complementary approaches such as mGBP1 overexpression, cells from mGBP1 -/- mice and intranasal infection of mGBP1 -/- we demonstrate that mGBP1 does not play a major role in modulating IAV infection in vitro or in vivo., (© 2023 The Authors. Immunology & Cell Biology published by John Wiley & Sons Australia, Ltd on behalf of the Australian and New Zealand Society for Immunology, Inc.)

Moraxella catarrhalis is an important human respiratory pathogen and a major causative agent of otitis media and chronic obstructive pulmonary disease. Toll-like receptors contribute to, but cannot fully account for, the complexity of the immune response seen in M. catarrhalis infection. Using primary mouse bone marrow-derived macrophages to examine the host response to M. catarrhalis infection, our global transcriptomic and targeted cytokine analyses revealed activation of immune signalling pathways by both membrane-bound and cytosolic pattern-recognition receptors. We show that M. catarrhalis and its outer membrane vesicles or lipooligosaccharide (LOS) can activate the cytosolic innate immune sensor caspase-4/11, gasdermin-D-dependent pyroptosis, and the NLRP3 inflammasome in human and mouse macrophages. This pathway is initiated by type I interferon signalling and guanylate-binding proteins (GBPs). We also show that inflammasomes and GBPs, particularly GBP2, are required for the host defence against M. catarrhalis in mice. Overall, our results reveal an essential role for the interferon-inflammasome axis in cytosolic recognition and immunity against M. catarrhalis, providing new molecular targets that may be used to mitigate pathological inflammation triggered by this pathogen., (© 2023 The Authors. Published under the terms of the CC BY 4.0 license.)

Inflammasomes are cytosolic signaling complexes capable of sensing microbial ligands to trigger inflammation and cell death responses. Here, we show that guanylate-binding proteins (GBPs) mediate pathogen-selective inflammasome activation. We show that mouse GBP1 and GBP3 are specifically required for inflammasome activation during infection with the cytosolic bacterium Francisella novicida. We show that the selectivity of mouse GBP1 and GBP3 derives from a region within the N-terminal domain containing charged and hydrophobic amino acids, which binds to and facilitates direct killing of F. novicida and Neisseria meningitidis, but not other bacteria or mammalian cells. This pathogen-selective recognition by this region of mouse GBP1 and GBP3 leads to pathogen membrane rupture and release of intracellular content for inflammasome sensing. Our results imply that GBPs discriminate between pathogens, confer activation of innate immunity, and provide a host-inspired roadmap for the design of synthetic antimicrobial peptides that may be of use against emerging and re-emerging pathogens., (© 2022. The Author(s).)

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.

Myxovirus resistance (Mx) proteins are dynamin-like GTPases that are inducible by interferons (IFNs) following virus infections. Most studies investigating Mx proteins have focused on their activity against influenza A viruses (IAV), although emerging evidence suggests that some Mx proteins may exhibit broader antiviral activity. Herein, we demonstrate that in addition to IAV, overexpression of mouse Mx1 (mMx1), but not mMx2, resulted in potent inhibition of growth of the human alphaherpesviruses herpes simplex virus 1 (HSV-1) and HSV-2, whereas neither inhibited the mouse betaherpesvirus murine cytomegalovirus (MCMV) in vitro . IFN induction of a functional endogenous mMx1 in primary mouse fibroblasts ex vivo was also associated with inhibition of HSV-1 growth. Using an in vitro overexpression approach, we demonstrate that mutations that result in redistribution of mMx1 from the nucleus to the cytoplasm or in loss of its combined GTP binding and GTPase activity also abrogated its ability to inhibit HSV-1 growth. Overexpressed mMx1 did not inhibit early HSV-1 gene expression but was shown to inhibit both replication of the HSV-1 genome as well as subsequent late gene expression. In a mouse model of cutaneous HSV-1 infection, mice expressing a functional endogenous mMx1 showed significant reductions in the severity of skin lesions as well as reduced HSV-1 titers in both the skin and dorsal root ganglia (DRG). Together, these data demonstrate that mMx1 mediates potent antiviral activity against human alphaherpesviruses by blocking replication of the viral genome and subsequent steps in virus replication. Moreover, endogenous mMx1 potently inhibited pathogenesis in the zosteriform mouse model of HSV-1 infection. IMPORTANCE While a number of studies have demonstrated that human Mx proteins can inhibit particular herpesviruses in vitro , we are the first to report the antiviral activity of mouse Mx1 (mMx1) against alphaherpesviruses both in vitro and in vivo . We demonstrate that both overexpressed mMx1 and endogenous mMx1 potently restrict HSV-1 growth in vitro . mMx1-mediated inhibition of HSV-1 was not associated with inhibition of virus entry and/or import of the viral genome into the nucleus, but rather with inhibition of HSV-1 genomic replication as well as subsequent late gene expression. Therefore, inhibition of human alphaherpesviruses by mMx1 occurs by a mechanism that is distinct from that reported for human Mx proteins against herpesviruses. Importantly, we also provide evidence that expression of a functional endogenous mMx1 can limit HSV-1 pathogenesis in a mouse model of infection.

Interferon-induced transmembrane (IFITM) proteins inhibit a broad range of enveloped viruses by blocking entry into host cells. We used an inducible overexpression system to investigate if IFITM1, IFITM2, and IFITM3 could modulate early and/or late stages of influenza A virus (IAV) or parainfluenza virus 3 (PIV-3) infection in human A549 airway epithelial cells. IAV and PIV-3 represent respiratory viruses which utilize distinct cellular entry pathways. We verify entry by endocytosis for IAV, whereas PIV-3 infection was consistent with fusion at the plasma membrane. Following induction prior to infection, all three IFITM proteins restricted the percentage of IAV-infected cells at 8 hours postinfection. In contrast, prior induction of IFITM1 and IFITM2 did not inhibit PIV-3 infection, although a modest reduction was observed with IFITM3. Small interfering RNA (siRNA)-mediated knockdown of endogenous IFITM1, IFITM2, and IFITM3 expression, in the presence or absence of pretreatment with type I interferon, resulted in increased IAV, but not PIV-3, infection. This finding suggests that while all three IFITMs display antiviral activity against IAV, they do not restrict the early stages of PIV-3 infection. IAV and PIV-3 infection culminates in viral egress through budding at the plasma membrane. Inducible expression of IFITM1, IFITM2, or IFITM3 immediately after infection did not impact titers of infectious virus released from IAV- or PIV-3-infected cells. Our findings show that IFITM proteins differentially restrict the early stages of infection of two respiratory viruses with distinct cellular entry pathways but do not influence the late stages of replication for either virus. IMPORTANCE Interferon-induced transmembrane (IFITM) proteins restrict the initial stages of infection for several respiratory viruses; however, their potential to modulate the later stages of virus replication has not been explored. In this study, we highlight the utility of an inducible overexpression system to assess the impact of IFITM proteins on either early- or late-stage replication of two respiratory viruses. We demonstrate antiviral activity by IFITM1, IFITM2, and IFITM3 against influenza A virus (IAV) but not parainfluenza virus 3 (PIV-3) during the early stages of cellular infection. Furthermore, IFITM induction following IAV or PIV-3 infection does not restrict the late stages of replication of either virus. Our findings show that IFITM proteins can differentially restrict the early stages of infection of two viruses with distinct cellular entry pathways and yet do not influence the late stages of replication for either virus.

Efflux pumps belonging to the ubiquitous resistance-nodulation-cell division (RND) superfamily transport substrates out of cells by coupling proton conduction across the membrane to a conformationally driven pumping cycle. The heavy metal-resistant bacteria Cupriavidus metallidurans CH34 relies notably on as many as 12 heavy metal efflux pumps of the RND superfamily. Here we show that C. metallidurans CH34 ZneA is a proton driven efflux pump specific for Zn(II), and that transport of substrates through the transmembrane domain may be electrogenic. We report two X-ray crystal structures of ZneA in intermediate transport conformations, at 3.0 and 3.7 Å resolution. The trimeric ZneA structures capture protomer conformations that differ in the spatial arrangement and Zn(II) occupancies at a proximal and a distal substrate binding site. Structural comparison shows that transport of substrates through a tunnel that links the two binding sites, toward an exit portal, is mediated by the conformation of a short 14-aa loop. Taken together, the ZneA structures presented here provide mechanistic insights into the conformational changes required for substrate efflux by RND superfamily transporters.