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Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccines provide essential tools for the control of the COVID-19 pandemic. A number of technologies have been employed to develop SARS-CoV-2 vaccines, including the inactivated SARS-CoV-2 particles, mRNA to express viral spike protein, recombinant spike proteins, and viral vectors. Here, we report the use of the vaccinia virus Tiantan strain as a vector to express the SARS-CoV-2 spike protein. When it was used to inoculate mice, robust SARS-CoV-2 spike protein-specific antibody response and T-cell response were detected. Sera from the vaccinated mice showed strong neutralizing activity against the ancestral Wuhan SARS-CoV-2, the variants of concern (VOCs) B.1.351, B.1.617.2, and the emerging B.1.1.529 (omicron). This finding supports the possibility of developing a new type of SARS-CoV-2 vaccine using the vaccinia virus vector.

Rationale: Respiratory syncytial virus (RSV) is the leading cause of childhood respiratory infections worldwide; however, no vaccine is available, and treatment options are limited. Identification ...

Interferon-regulated myxovirus resistance protein B (MxB) is an interferon-induced GTPase belonging to the dynamin superfamily. It inhibits infection with a wide range of different viruses, including HIV-1, by impairing viral DNA entry into the nucleus. Unlike the related antiviral GTPase MxA, MxB possesses an N-terminal region that contains a nuclear localization signal and is crucial for inhibiting HIV-1. Because MxB previously has been shown to reside in both the nuclear envelope and the cytoplasm, here we used bioinformatics and biochemical approaches to identify a nuclear export signal (NES) responsible for MxB's cytoplasmic location. Using the online computational tool LocNES (Locating Nuclear Export Signals or NESs), we identified five putative NES candidates in MxB and investigated whether their deletion caused nuclear localization of MxB. Our results revealed that none of the five deletion variants relocates to the nucleus, suggesting that these five predicted NES sequences do not confer NES activity. Interestingly, deletion of one sequence, encompassing amino acids 505-527, abrogated the anti-HIV-1 activity of MxB. Further mutation experiments disclosed that amino acids 515-519, and Pro-515 in particular, regulate MxB oligomerization and its binding to HIV-1 capsid, thereby playing an important role in MxB-mediated restriction of HIV-1 infection. In summary, our results indicate that none of the five predicted NES sequences in MxB appears to be required for its nuclear export. Our findings also reveal several residues in MxB, including Pro-515, critical for its oligomerization and anti-HIV-1 function.

SARS-CoV-2 has become a global threat to public health. Infected individuals can be asymptomatic or develop mild to severe symptoms, including pneumonia, respiratory distress, and death. This wide spectrum of clinical presentations of SARS-CoV-2 infection is believed in part due to the polymorphisms of key genetic factors in the population. In this study, we report that the interferon-induced antiviral factor IFITM3 inhibits SARS-CoV-2 infection by preventing SARS-CoV-2 spike-protein-mediated virus entry and cell-to-cell fusion. Analysis of a Chinese COVID-19 patient cohort demonstrates that the rs12252 CC genotype of IFITM3 is associated with SARS-CoV-2 infection risk in the studied cohort. These data suggest that individuals carrying the rs12252 C allele in the IFITM3 gene may be vulnerable to SARS-CoV-2 infection and thus may benefit from early medical intervention.

SERINC5 is a multi-span transmembrane protein that is incorporated into HIV-1 particles in producing cells and inhibits HIV-1 entry. Multiple retroviruses like HIV-1, equine infectious anemia virus and murine leukemia virus are subject to SERINC5 inhibition, while HIV-1 pseudotyped with envelope glycoproteins of vesicular stomatitis virus and Ebola virus are resistant to SERINC5. The antiviral spectrum and the underlying mechanisms of SERINC5 restriction are not completely understood. Here we show that SERINC5 inhibits influenza A virus infection by targeting virus-cell membrane fusion at an early step of infection. Further results show that different influenza hemagglutinin (HA) subtypes exhibit diverse sensitivities to SERINC5 restriction. Analysis of the amino acid sequences of influenza HA1 strains indicates that HA glycosylation sites correlate with the sensitivity of influenza HA to SERINC5, and the inhibitory effect of SERINC5 was lost when certain HA glycosylation sites were mutated. Our study not only expands the antiviral spectrum of SERINC5, but also reveals the role of viral envelope glycosylation in resisting SERINC5 restriction.

Long interspersed element type 1 (LINE-1, also L1 for short) is the only autonomously transposable element in the human genome. Its insertion into a new genomic site may disrupt the function of genes, potentially causing genetic diseases. Cells have thus evolved a battery of mechanisms to tightly control LINE-1 activity. Here, we report that a cellular antiviral protein, myxovirus resistance protein B (MxB), restricts the mobilization of LINE-1. This function of MxB requires the nuclear localization signal located at its N-terminus, its GTPase activity and its ability to form oligomers. We further found that MxB associates with LINE-1 protein ORF1p and promotes sequestration of ORF1p to G3BP1-containing cytoplasmic granules. Since knockdown of stress granule marker proteins G3BP1 or TIA1 abolishes MxB inhibition of LINE-1, we conclude that MxB engages stress granule components to effectively sequester LINE-1 proteins within the cytoplasmic granules, thus hindering LINE-1 from accessing the nucleus to complete retrotransposition. Thus, MxB protein provides one mechanism for cells to control the mobility of retroelements. Author summary Retrotransposons occupy more than 40% of human genome, and have co-evolved with humans for millions of years. Long interspersed element type 1 (LINE-1, or L1) is the only retrotransposon that is able to jump to a new locus. LINE-1 retrotransposition causes genome instability, and is associated with genetic diseases including autoimmune diseases and cancer. To suppress this genome toxicity caused by LINE-1, humans have developed multi-layered mechanisms to control LINE-1 activity. MxB has been previously shown to inhibit LINE-1 mobility, thus contributing to host restriction of LINE-1. Here, we further demonstrate that MxB effectively restricts LINE-1 retrotransposition by sequestering LINE-1 ribonucleoprotein (RNP) within the cytoplasmic stress granules, thus guards genome stability. Hence our data attribute the restriction function of MxB to sequestering LINE-1 RNP to stress granules.

Cyclic GMP-AMP synthase (cGAS) is a major sensor of cytosolic DNA from invading pathogens and damaged cellular organelles. Activation of cGAS promotes liquid-like phase separation and formation of membraneless cytoplasmic structures. Here, we found that cGAS bound G3BP1, a double-stranded nucleic acid helicase involved in the formation of stress granules. Loss of G3BP1 blocked subcellular cGAS condensation and suppressed the interferon response to intracellular DNA and DNA virus particles in cells. Furthermore, an RNA-dependent association with PKR promoted G3BP1 foci formation and cGAS-dependent interferon responses. Together, these results indicate that PKR promotes the formation of G3BP1-dependent, membraneless cytoplasmic structures necessary for the DNA-sensing function of cGAS in human cells. These data suggest that there is a previously unappreciated link between nucleic acid sensing pathways, which requires the formation of specialized subcellular structures.

Type I interferon (IFN) inhibits viruses by inducing the expression of antiviral proteins. The IFN-induced myxovirus resistance B (MxB) protein has been reported to inhibit a limited number of viruses, including HIV-1 and herpesviruses, but its antiviral coverage remains to be explored further. Here we show that MxB interferes with RNA replication of hepatitis C virus (HCV) and significantly inhibits viral replication in a cyclophilin A (CypA)-dependent manner. Our data further show that MxB interacts with the HCV protein NS5A, thereby impairing NS5A interaction with CypA and NS5A localization to the endoplasmic reticulum, two events essential for HCV RNA replication. Interestingly, we found that MxB significantly inhibits two additional CypA-dependent viruses of the Flaviviridae family, namely, Japanese encephalitis virus and dengue virus, suggesting a potential link between virus dependence on CypA and virus susceptibility to MxB inhibition. Collectively, these data have identified MxB as a key factor behind IFN-mediated suppression of HCV infection, and they suggest that other CypA-dependent viruses may also be subjected to MxB restriction. IMPORTANCE Viruses of the Flaviviridae family cause major illness and death around the world and thus pose a great threat to human health. Here we show that IFN-inducible MxB restricts several members of the Flaviviridae, including HCV, Japanese encephalitis virus, and dengue virus. This finding not only suggests an active role of MxB in combating these major pathogenic human viruses but also significantly expands the antiviral spectrum of MxB. Our study further strengthens the link between virus dependence on CypA and susceptibility to MxB restriction and also suggests that MxB may employ a common mechanism to inhibit different viruses. Elucidating the antiviral functions of MxB advances our understanding of IFN-mediated host antiviral defense and may open new avenues to the development of novel antiviral therapeutics.

MOV10 has emerged as an important host antiviral factor. MOV10 not only inhibits various viruses, including human immunodeficiency virus type 1, hepatitis C virus and vesicular stomatitis virus, but also restricts the activity of retroelements long interspersed nucleotide element-1, Alu, SVA and intracisternal A particles. Here, we report that MOV10 suppresses influenza A virus infection through interacting with viral nucleoprotein (NP), sequestering viral RNP in the cytoplasm and causing the degradation of viral vRNA. The antiviral activity of MOV10 depends on the integrity of P-bodies. We also found that the antiviral activity of MOV10 is partially countered by viral NS1 protein that interferes with the interaction of MOV10 with viral NP and causes MOV10 degradation through the lysosomal pathway. Moreover, NS1-defective influenza A virus is more susceptible to MOV10 restriction. Our data not only expand the antiviral spectrum of MOV10 but also reveal the NS1 protein as the first viral antagonist of MOV10.

BST-2 (tetherin, CD317, and HM1.24) is induced by interferon and restricts virus release by tethering the enveloped viruses to the cell surface. The effect of BST-2 on influenza A virus (IAV) infection has been inconclusive. In the present study, we report that BST-2 diminishes the production of IAV virus-like particles (VLPs) that are generated by viral neuraminidase and hemagglutinin proteins to a much greater degree than it inhibits the production of wild-type IAV particles. This relatively weaker inhibition of IAV is associated with reduction in BST-2 levels, which is caused by the M2 protein that interacts with BST-2 and leads to down-regulation of cell surface BST-2 via the proteasomal pathway. Similarly to the viral antagonist Vpu, M2 also rescues the production of human immunodeficiency virus-1 VLPs and IAV VLPs in the presence of BST-2. Replication of wild-type and the M2-deleted viruses were both inhibited by BST-2, with the M2-deleted IAV being more restricted. These data reveal one mechanism that IAV employs to counter restriction by BST-2.

Enterovirus 71 (EV71) infection causes hand-foot-and-mouth disease that leads to cardiopulmonary complications and death in young children. There is thus an urgent need to find new treatments to control EV71 infection. In this study, we report potent inhibition of EV71 by a polyene antibiotic Amphotericin B. Amphotericin B profoundly diminished the expression of EV71 RNA and viral proteins in the RD cells and the HEK293 cells. As a result, EV71 production was inhibited by Amphotericin B with an EC50 (50% effective concentration) of 1.75 μM in RD cells and 0.32 μM in 293 cells. In addition to EV71, EV68 was also strongly inhibited by Amphotericin B. Results of mechanistic studies revealed that Amphotericin B targeted the early stage of EV71 infection through impairing the attachment and internalization of EV71 by host cells. As an effective anti-fungi drug, Amphotericin B thus holds the promise of formulating a novel therapeutic to treat EV71 infection.

SAMHD1 (SAM domain- and HD domain-containing protein 1) inhibits HIV-1 infection of myeloid cells and resting CD4+ T-cells. Two lineages of primate lentiviruses, the sooty mangabey SIV (simian immunodeficiency virus) (SIVsm)/macaque SIV (SIVmac)/HIV-2 lineage and the red-capped mangabey SIV (SIVrcm) lineage, carry a SAMHD1 antagonist called Vpx. Vpx recognizes SAMHD1 and recruits a ubiquitin E3 ligase complex that is composed of CUL4 (Cullin4), DDB1 (damaged DNA-binding protein 1) and a member of the DCAF (DDB1/CUL4-associated factor) family called DCAF1. This E3 ligase complex polyubiquitinates SAMHD1, which leads to proteasomal degradation of SAMHD1. As opposed to the well-characterized interaction of SIVmac Vpx with human SAMHD1 and DCAF1, SIVrcm Vpx adopts a different mode of interaction with SAMHD1 of red-capped mangabeys. In the present study, we have characterized the interactions that are essential for SIVrcm Vpx-mediated degradation of rcmSAMHD1 (red-capped mangabey SAMHD1). Using mutagenesis and molecular modelling, we have determined the key role of the W23LHR26 peptide of SIVrcm Vpx in recognizing rcmSAMHD1. The amino acids Phe15, Leu36, Phe52, Arg55 and Arg56 at the N-terminal domain (NtD) of rcmSAMHD1 are involved in interaction with Vpxrcm (red-capped mangabey Vpx). The molecular model of rcmSAMHD1-NtD, Vpxrcm and C-terminal domain (CtD) of DCAF1 (DCAF1-CtD) complex reveals further that rcmSAMHD1-NtD and Vpxrcm utilize an interaction interface that is different from that used by human SAMHD1-CtD and Vpxsm. These findings provide further insights into the different modes of interaction between Vpx and SAMHD1 as the result of the ‘arms race’ of virus and host cell.

Members of the interferon-induced transmembrane (IFITM) protein family inhibit the entry of a wide range of viruses. Viruses often exploit the endocytosis pathways to invade host cells and escape from the endocytic vesicles often in response to low pH. Localization to these endocytic vesicles is essential for IFITM3 to interfere with the cytosolic entry of pH-dependent viruses. However, the nature of the sorting signal that targets IFITM3 to these vesicles is poorly defined. In this study, we report that IFITM3 possesses a YxxΦ sorting motif, i.e., 20-YEML-23, that enables IFITM3 to undergo endocytosis through binding to the μ2 subunit of the AP-2 complex. IFITM3 accumulates at the plasma membrane as a result of either mutating 20-YEML-23, depleting the μ2 subunit, or overexpressing μ2 mutants. Importantly, blocking endocytosis of IFITM3 abrogates its ability to inhibit pH-dependent viruses. We have therefore identified a critical sorting signal, namely 20-YEML-23, that controls both the endocytic trafficking and the antiviral action of IFITM3. This finding also reveals that as an endocytic protein, IFITM3 first arrives at the plasma membrane before it is endocytosed and further traffics to the late endosomes where it acts to impede virus entry.

Background BST-2 (bone marrow stromal cell antigen 2) is an interferon-inducible protein that inhibits virus release by tethering viral particles to the cell surface. This antiviral activity of BST-2 is antagonized by HIV-1 accessory protein Vpu. Vpu physically interacts with BST-2 through their mutual transmembrane (TM) domains. In this study, we utilized the BRET assay and molecular dynamics (MD) simulation method to further characterize the interaction of BST-2 and Vpu. Results Amino acids I34, L37, P40 and L41 in the TM domain of BST-2, and L11, A18 and W22 in the TM domain of Vpu were identified to be critical for the interaction between BST-2 and Vpu. The residues P40 in the TM domain of BST-2 and L11 in the TM domain of Vpu were shown, for the first time, to be important for their interaction. Furthermore, triple-amino-acid substitutions, 14–16 (AII to VAA) and 26–28 (IIE to AAA) in Vpu TM, not the single-residue mutation, profoundly disrupted BST-2/Vpu interaction. The results of MD simulation revealed significant conformational changes of the BST-2/Vpu complex as a result of mutating P40 of BST-2 and L11, 14–16 (AII to VAA) and 26–28 (IIE to AAA) of Vpu. In addition, disrupting the interaction between BST-2 and Vpu rendered BST-2 resistant to Vpu antagonization. Conclusions Through use of the BRET assay, we identified novel key residues P40 in the TM domain of BST-2 and L11 in the TM domain of Vpu that are important for their interaction. These results add new insights into the molecular mechanism behind BST-2 antagonization by HIV-1 Vpu.

Background Tetherin (also known as BST-2, CD317, and HM1.24) is an interferon- induced protein that blocks the release of a variety of enveloped viruses, such as retroviruses, filoviruses and herpesviruses. However, the relationship between tetherin and foamy viruses has not been clearly demonstrated. Results In this study, we found that tetherin of human, simian, bovine or canine origin inhibits the production of infectious prototypic foamy virus (PFV). The inhibition of PFV by human tetherin is counteracted by human immunodeficiency virus type 1 (HIV-1) Vpu. Furthermore, we generated human tetherin transmembrane domain deletion mutant (delTM), glycosyl phosphatidylinositol (GPI) anchor deletion mutant (delGPI), and dimerization and glycosylation deficient mutants. Compared with wild type tetherin, the delTM and delGPI mutants only moderately inhibited PFV production. In contrast, the dimerization and glycosylation deficient mutants inhibit PFV production as efficiently as the wild type tetherin. Conclusions These results demonstrate that tetherin inhibits the release and infectivity of PFV, and this inhibition is antagonized by HIV-1 Vpu. Both the transmembrane domain and the GPI anchor of tetherin are important for the inhibition of PFV, whereas the dimerization and the glycosylation of tetherin are dispensable.

Interferon-induced proteins (IFPs) exert multiple functions corresponding to diverse interferon signals. However, the intracellular functions of many IFPs are not fully characterized. Here, we report that IFP35, a member of the IFP family with a molecular mass of 35 kDa, can interact with the bovine Tas (BTas) regulatory protein of bovine foamy virus (BFV). The interaction involves NID2 (IFP35/Nmi homology domain) of IFP35 and the central domain of BTas. The overexpression of IFP35 disturbs the ability of BTas to activate viral-gene transcription and inhibits viral replication. The depletion of endogenous IFP35 by interfering RNA can promote the activation of BFV, suggesting an inhibitory function of IFP35 in viral-gene expression. In addition, IFP35 can interact with the homologous regulatory protein of prototype FV and arrest viral replication and repress viral transcription. Our study suggests that IFP35 may represent a novel pathway of interferon-mediated antiviral activity in host organisms that plays a role in the maintenance of FV latency.

The SAM domain and HD domain containing protein 1 (SAMHD1) inhibits retroviruses, DNA viruses and long interspersed element 1 (LINE-1). Given that in dividing cells, SAMHD1 loses its antiviral function yet still potently restricts LINE-1, we propose that, instead of blocking viral DNA synthesis by virtue of its dNTP triphosphohydrolase activity, SAMHD1 may exploit a different mechanism to control LINE-1. Here, we report a new activity of SAMHD1 in promoting cellular stress granule assembly, which correlates with increased phosphorylation of eIF2α and diminished eIF4A/eIF4G interaction. This function of SAMHD1 enhances sequestration of LINE-1 RNP in stress granules and consequent blockade to LINE-1 retrotransposition. In support of this new mechanism of action, depletion of stress granule marker proteins G3BP1 or TIA1 abrogates stress granule formation and overcomes SAMHD1 inhibition of LINE-1. Together, these data reveal a new mechanism for SAMHD1 to control LINE-1 by activating cellular stress granule pathway., Author Summary Long interspersed element 1 (LINE-1 or L1) comprises 17% of human genome, and has played a major role in shaping the evolution of human genome. Approximately 100 copies of LINE-1 are still active in an average individual genome. Movement of these LINE-1 sequences to new loci in the genome has the potential of causing sporadic cases of disease. Among the multi-layered mechanisms by which the host controls LINE-1 activity is a group of host restriction factors including APOBEC3 proteins. SAMHD1 was known for the association of its mutations with the Aicardi-Goutieres syndrome (AGS), a congenital autoimmune disease. SAMHD1 was recently reported as a host restriction factor that inhibits a number of retroviruses and DNA viruses including human immunodeficiency virus type 1 (HIV-1) and herpes simplex virus 1 (HSV-1). Here, we demonstrate that SAMHD1 inhibits LINE-1 retrotransposition through promoting the sequestration of LINE-1 RNP within the cytoplasmic stress granules. SAMHD1 promotes the formation of large stress granules by inducing phosphorylation of eIF2α and disrupting eIF4A/eIF4G interaction. This is the first report describing the role of SAMHD1 in modulating the formation of stress granules. We envision that this function of SAMHD1 not only contributes to the inhibition of LINE-1, but also the restriction of various viruses.

Cellular acetylation signaling is important for viral gene regulation, particularly during the transactivation of retroviruses. The regulatory protein of bovine foamy virus (BFV), BTas, is a transactivator that augments viral gene transcription from both the long terminal repeat (LTR) promoter and the internal promoter (IP). In this study, we report that the histone acetyltransferase (HAT), p300, specifically acetylates BTas both in vivo and in vitro. Further studies demonstrated that BTas acetylation markedly enhances its transactivation activity. Mutagenesis analysis identified three lysines at positions 66, 109 and 110 in BTas that are acetylated by p300. The K110R mutant lost its binding to BFV promoter as well as its ability to activate BFV promoter. The acetylation of K66 and K109 may contribute to increased BTas binding ability. These results suggest that the p300-acetylated lysines of BTas are important for transactivation of BFV promoters and therefore have an important role in BFV replication.

The BTas protein of bovine foamy virus (BFV) is a 249-amino-acid nuclear regulatory protein which transactivates viral gene expression directed by the long terminal repeat promoter (LTR) and the internal promoter (IP). Here, we demonstrate the BTas protein forms a dimeric complex in mammalian cells by using mammalian two hybrid systems and cross-linking assay. Functional analyses with deletion mutants reveal that the region of 46–62aa is essential for dimer formation. Furthermore, our results show that deleting the dimerization region of BTas did not affect the localization of BTas, but that it did result in the loss of its transactivational activity on the LTR and IP. Furthermore, BTas (Δ46–62aa) retained binding ability to the LTR and IP similar to that of the wild-type BTas. These data suggest the dimerization region is necessary for the transactivational function of BTas and is crucial to the replication of BFV.

Background Bovine herpesvirus 4 (BoHV-4) is a gammaherpesvirus whose genome was cloned as Bacterial Artificial Chromosome (BAC) and exploited as a gene delivery vector for vaccine purposes. Although BoHV-4 genome has been completely sequenced and its open reading frames (ORFs) structurally defined in silico, most of them are not functionally characterized. In BoHV-4 genome two major immediate early genes (IE) are present, IE1 and IE2. IE2 is an essential gene because its removal from the viral genome renders the virus unable to replicate, whereas for IE1 no many functional information are available. Results In this work, IE1 contribution in initiating and maintaining BoHV-4 lytic replication was assessed generating a recombinant BoHV-4 genome lacking of IE1 gene, BoHV-4ΔIE1. In contrast to BoHV-4IE2 deleted mutant, BoHV-4ΔIE1 infectious replicating viral particles (IRVPs) could be reconstituted following viral DNA electroporation in permissive cells. However the titer of BoHV-4ΔIE1 IRVPs produced into the cell supernatant and BoHV-4ΔIE1 plaques size were reduced respect to BoHV-4 undeleted control. Further the impaired BoHV-4ΔIE1 IRVPs produced into the cell supernatant could be rescued by expressing IE1 gene product in trans, confirming the implication of IE1 in BoHV-4 lytic replication. Next, the possible role of BoHV-4IE1 as bone marrow stromal cell antigen 2 (BST-2) counteracting factor, as hypothesized by IE1 amino-terminal gene product homology with Kaposi Sarcoma Associated Herpesvirus (KSHV) K5, was excluded too. Conclusions Although the real function of BoHV-4IE1 is still elusive, a new BoHV-4 genome gene locus as a target site for the insertion of foreign DNA and resulting in the attenuation of the virus has been revealed. These data can be considered of relevance to improve BoHV-4 gene delivery properties. Electronic supplementary material The online version of this article (doi:10.1186/s12917-015-0540-4) contains supplementary material, which is available to authorized users.