LY6E is a pan-coronavirus restriction factor in the respiratory tract.

LY6E is an antiviral restriction factor that inhibits coronavirus spike-mediated fusion, but the cell types in vivo that require LY6E for protection from respiratory coronavirus infection are unknown. Here we used a panel of seven conditional Ly6e knockout mice to define which Ly6e-expressing cells confer control of airway infection by murine coronavirus and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Loss of Ly6e in Lyz2-expressing cells, radioresistant Vav1-expressing cells and non-haematopoietic cells increased susceptibility to murine coronavirus. Global conditional loss of Ly6e expression resulted in clinical disease and higher viral burden after SARS-CoV-2 infection, but little evidence of immunopathology. We show that Ly6e expression protected secretory club and ciliated cells from SARS-CoV-2 infection and prevented virus-induced loss of an epithelial cell transcriptomic signature in the lung. Our study demonstrates that lineage confined rather than broad expression of Ly6e sufficiently confers resistance to disease caused by murine and human coronaviruses.

Swine IFI6 confers antiviral effects against Japanese encephalitis virus in vitro and in vivo.

Swine are considered to be an important intermediate host in the cycle of Japanese encephalitis virus (JEV) infection. Most existing antiviral studies of JEV mainly focus on the host factor of the dead-end hosts. However, little research has addressed this in swine. Here, we found that swine interferon alpha-inducible protein 6 (sIFI6) possessed antiviral activity against JEV. In vitro studies showed that overexpression of sIFI6 inhibited the infection of JEV, while sIFI6 knockdown enhanced the infection of JEV in PK-15 cells. In addition, we also found that the structural integrity of sIFI6 was required by anti-JEV activity and that sIFI6 interacted with JEV nonstructural protein 4A (NS4A), an integral membrane protein with a pivotal function in replication complex during JEV replication. The interaction domain was mapped to the fourth transmembrane domain (TMD), also known as the 2K peptide of NS4A. The antiviral activity of sIFI6 was regulated by endoplasmic reticulum (ER) stress-related protein, Bip. In vivo studies revealed that sIFI6 alleviated symptoms of JEV infection in C57BL/6 mice. In addition, the antiviral spectrum of sIFI6 showed that sIFI6 specifically inhibited JEV infection. In conclusion, this study identified sIFI6 as a host factor against JEV infection for the first time. Our findings provide a potential drug target against JEV infection.

FDA approved drugs with antiviral activity against SARS-CoV-2: From structure-based repurposing to host-specific mechanisms.

The continuing heavy toll of the COVID-19 pandemic necessitates development of therapeutic options. We adopted structure-based drug repurposing to screen FDA-approved drugs for inhibitory effects against main protease enzyme (Mpro) substrate-binding pocket of SARS-CoV-2 for non-covalent and covalent binding. Top candidates were screened against infectious SARS-CoV-2 in a cell-based viral replication assay. Promising candidates included atovaquone, mebendazole, ouabain, dronedarone, and entacapone, although atovaquone and mebendazole were the only two candidates with IC50s that fall within their therapeutic plasma concentration. Additionally, we performed Mpro assays on the top hits, which demonstrated inhibition of Mpro by dronedarone (IC50 18 µM), mebendazole (IC50 19 µM) and entacapone (IC50 9 µM). Atovaquone showed only modest Mpro inhibition, and thus we explored other potential mechanisms. Although atovaquone is Dihydroorotate dehydrogenase (DHODH) inhibitor, we did not observe inhibition of DHODH at the respective SARS-CoV-2 IC50. Metabolomic profiling of atovaquone treated cells showed dysregulation of purine metabolism pathway metabolite, where ecto-5'-nucleotidase (NT5E) was downregulated by atovaquone at concentrations equivalent to its antiviral IC50. Atovaquone and mebendazole are promising candidates with SARS-CoV-2 antiviral activity. While mebendazole does appear to target Mpro, atovaquone may inhibit SARS-CoV-2 viral replication by targeting host purine metabolism.

LY6E protects mice from pathogenic effects of murine coronavirus and SARS-CoV-2.

LY6E is an antiviral protein that inhibits coronavirus entry. Its expression in immune cells allows mice to control murine coronavirus infection. However, it is not known which immune cell subsets mediate this control or whether LY6E protects mice from SARS-CoV-2. In this study, we used tissue-specific Cre recombinase expression to ablate Ly6e in distinct immune compartments or in all epiblast-derived cells, and bone marrow chimeras to target Ly6e in a subset of radioresistant cells. Mice lacking Ly6e in Lyz2 -expressing cells and radioresistant Vav1 -expressing cells were more susceptible to lethal murine coronavirus infection. Mice lacking Ly6e globally developed clinical disease when challenged with the Gamma (P.1) variant of SARS-CoV-2. By contrast, wildtype mice and mice lacking type I and type III interferon signaling had no clinical symptoms after SARS-CoV-2 infection. Transcriptomic profiling of lungs from SARS-CoV-2-infected wildtype and Ly6e knockout mice revealed a striking reduction of secretory cell-associated genes in infected knockout mice, including Muc5b , an airway mucin-encoding gene that may protect against SARS-CoV-2-inflicted respiratory disease. Collectively, our study reveals distinct cellular compartments in which Ly6e confers cell intrinsic antiviral effects, thereby conferring resistance to disease caused by murine coronavirus and SARS-CoV-2.

Enterovirus 3C Protease Cleaves TRIM7 To Dampen Its Antiviral Activity.

Mammalian TRIM7 is an antiviral protein that inhibits multiple human enteroviruses by degrading the viral 2BC protein. Whether TRIM7 is reciprocally targeted by enteroviruses is not known. Here, we report that the 3C protease (3Cpro) from two enteroviruses, coxsackievirus B3 (CVB3) and poliovirus, targets TRIM7 for cleavage. CVB3 3Cpro cleaves TRIM7 at glutamine 24 (Q24), resulting in a truncated TRIM7 that fails to inhibit CVB3 due to dampened E3 ubiquitin ligase activity. TRIM7 Q24 is highly conserved across mammals, except in marsupials, which instead have a naturally occurring histidine (H24) that is not subject to 3Cpro cleavage. Marsupials also express two isoforms of TRIM7, and the two proteins from koalas have distinct antiviral activities. The longer isoform contains an additional exon due to alternate splice site usage. This additional exon contains a unique 3Cpro cleavage site, suggesting that certain enteroviruses may have evolved to target marsupial TRIM7 even if the canonical Q24 is missing. Combined with computational analyses indicating that TRIM7 is rapidly evolving, our data raise the possibility that TRIM7 may be targeted by enterovirus evasion strategies and that evolution of TRIM7 across mammals may have conferred unique antiviral properties. IMPORTANCE Enteroviruses are significant human pathogens that cause viral myocarditis, pancreatitis, and meningitis. Knowing how the host controls these viruses and how the viruses may evade host restriction is important for understanding fundamental concepts in antiviral immunity and for informing potential therapeutic interventions. In this study, we demonstrate that coxsackievirus B3 uses its virally encoded protease to target the host antiviral protein TRIM7 for cleavage, suggesting a potential mechanism of viral immune evasion. We additionally show that TRIM7 has evolved in certain mammalian lineages to express protein variants with distinct antiviral activities and susceptibilities to viral protease-mediated cleavage.

TRIM67 Suppresses TNFalpha-Triggered NF-kB Activation by Competitively Binding Beta-TrCP to IkBa.

The transcription factor NF-κB plays an important role in modulation of inflammatory pathways, which are associated with inflammatory diseases, neurodegeneration, apoptosis, immune responses, and cancer. Increasing evidence indicates that TRIM proteins are crucial role in the regulation of NF-κB signaling pathways. In this study, we identified TRIM67 as a negative regulator of TNFα-triggered NF-κB activation. Ectopic expression of TRIM67 significantly represses TNFα-induced NF-κB activation and the expression of pro-inflammatory cytokines TNFα and IL-6. In contrast, Trim67 depletion promotes TNFα-induced expression of TNFα, IL-6, and Mcp-1 in primary mouse embryonic fibroblasts. Mechanistically, we found that TRIM67 competitively binding ß-transducin repeat-containing protein (ß-TrCP) to IκBα results inhibition of ß-TrCP-mediated degradation of IκBα, which finally caused inhibition of TNFα-triggered NF-κB activation. In summary, our findings revealed that TRIM67 function as a novel negative regulator of NF-κB signaling pathway, implying TRIM67 might exert an important role in regulation of inflammation disease and pathogen infection caused inflammation.

Shiftless inhibits flavivirus replication in vitro and is neuroprotective in a mouse model of Zika virus pathogenesis.

Flaviviruses such as Zika virus and West Nile virus have the potential to cause severe neuropathology if they invade the central nervous system. The type I interferon response is well characterized as contributing to control of flavivirus-induced neuropathogenesis. However, the interferon-stimulated gene (ISG) effectors that confer these neuroprotective effects are less well studied. Here, we used an ISG expression screen to identify Shiftless (SHFL, C19orf66) as a potent inhibitor of diverse positive-stranded RNA viruses, including multiple members of the Flaviviridae (Zika, West Nile, dengue, yellow fever, and hepatitis C viruses). In cultured cells, SHFL functions as a viral RNA-binding protein that inhibits viral replication at a step after primary translation of the incoming genome. The murine ortholog, Shfl, is expressed constitutively in multiple tissues, including the central nervous system. In a mouse model of Zika virus infection, Shfl-/- knockout mice exhibit reduced survival, exacerbated neuropathological outcomes, and increased viral replication in the brain and spinal cord. These studies demonstrate that Shfl is an important antiviral effector that contributes to host protection from Zika virus infection and virus-induced neuropathological disease.

TRIM7 inhibits enterovirus replication and promotes emergence of a viral variant with increased pathogenicity.

To control viral infection, vertebrates rely on both inducible interferon responses and less well-characterized cell-intrinsic responses composed of "at the ready" antiviral effector proteins. Here, we show that E3 ubiquitin ligase TRIM7 is a cell-intrinsic antiviral effector that restricts multiple human enteroviruses by targeting viral 2BC, a membrane remodeling protein, for ubiquitination and proteasome-dependent degradation. Selective pressure exerted by TRIM7 results in emergence of a TRIM7-resistant coxsackievirus with a single point mutation in the viral 2C ATPase/helicase. In cultured cells, the mutation helps the virus evade TRIM7 but impairs optimal viral replication, and this correlates with a hyperactive and structurally plastic 2C ATPase. Unexpectedly, the TRIM7-resistant virus has a replication advantage in mice and causes lethal pancreatitis. These findings reveal a unique mechanism for targeting enterovirus replication and provide molecular insight into the benefits and trade-offs of viral evolution imposed by a host restriction factor.

LY6E impairs coronavirus fusion and confers immune control of viral disease.

Zoonotic coronaviruses (CoVs) are substantial threats to global health, as exemplified by the emergence of two severe acute respiratory syndrome CoVs (SARS-CoV and SARS-CoV-2) and Middle East respiratory syndrome CoV (MERS-CoV) within two decades1-3. Host immune responses to CoVs are complex and regulated in part through antiviral interferons. However, interferon-stimulated gene products that inhibit CoVs are not well characterized4. Here, we show that lymphocyte antigen 6 complex, locus E (LY6E) potently restricts infection by multiple CoVs, including SARS-CoV, SARS-CoV-2 and MERS-CoV. Mechanistic studies revealed that LY6E inhibits CoV entry into cells by interfering with spike protein-mediated membrane fusion. Importantly, mice lacking Ly6e in immune cells were highly susceptible to a murine CoV-mouse hepatitis virus. Exacerbated viral pathogenesis in Ly6e knockout mice was accompanied by loss of hepatic immune cells, higher splenic viral burden and reduction in global antiviral gene pathways. Accordingly, we found that constitutive Ly6e directly protects primary B cells from murine CoV infection. Our results show that LY6E is a critical antiviral immune effector that controls CoV infection and pathogenesis. These findings advance our understanding of immune-mediated control of CoV in vitro and in vivo-knowledge that could help inform strategies to combat infection by emerging CoVs.

LY6E impairs coronavirus fusion and confers immune control of viral disease.

Zoonotic coronaviruses (CoVs) are significant threats to global health, as exemplified by the recent emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) 1 . Host immune responses to CoV are complex and regulated in part through antiviral interferons. However, the interferon-stimulated gene products that inhibit CoV are not well characterized 2 . Here, we show that interferon-inducible lymphocyte antigen 6 complex, locus E (LY6E) potently restricts cellular infection by multiple CoVs, including SARS-CoV, SARS-CoV-2, and Middle East respiratory syndrome coronavirus (MERS-CoV). Mechanistic studies revealed that LY6E inhibits CoV entry into cells by interfering with spike protein-mediated membrane fusion. Importantly, mice lacking Ly6e in hematopoietic cells were highly susceptible to murine CoV infection. Exacerbated viral pathogenesis in Ly6e knockout mice was accompanied by loss of hepatic and splenic immune cells and reduction in global antiviral gene pathways. Accordingly, we found that Ly6e directly protects primary B cells and dendritic cells from murine CoV infection. Our results demonstrate that LY6E is a critical antiviral immune effector that controls CoV infection and pathogenesis. These findings advance our understanding of immune-mediated control of CoV in vitro and in vivo , knowledge that could help inform strategies to combat infection by emerging CoV.

Seneca Valley Virus Suppresses Host Type I Interferon Production by Targeting Adaptor Proteins MAVS, TRIF, and TANK for Cleavage.

Seneca Valley virus (SVV) is an oncolytic RNA virus belonging to the Picornaviridae family. Its nucleotide sequence is highly similar to those of members of the Cardiovirus genus. SVV is also a neuroendocrine cancer-selective oncolytic picornavirus that can be used for anticancer therapy. However, the interaction between SVV and its host is yet to be fully characterized. In this study, SVV inhibited antiviral type I interferon (IFN) responses by targeting different host adaptors, including mitochondrial antiviral signaling (MAVS), Toll/interleukin 1 (IL-1) receptor domain-containing adaptor inducing IFN-ß (TRIF), and TRAF family member-associated NF-κB activator (TANK), via viral 3C protease (3Cpro). SVV 3Cpro mediated the cleavage of MAVS, TRIF, and TANK at specific sites, which required its protease activity. The cleaved MAVS, TRIF, and TANK lost the ability to regulate pattern recognition receptor (PRR)-mediated IFN production. The cleavage of TANK also facilitated TRAF6-induced NF-κB activation. SVV was also found to be sensitive to IFN-ß. Therefore, SVV suppressed antiviral IFN production to escape host antiviral innate immune responses by cleaving host adaptor molecules.IMPORTANCE Host cells have developed various defenses against microbial pathogen infection. The production of IFN is the first line of defense against microbial infection. However, viruses have evolved many strategies to disrupt this host defense. SVV, a member of the Picornavirus genus, is an oncolytic virus that shows potential functions in anticancer therapy. It has been demonstrated that IFN can be used in anticancer therapy for certain tumors. However, the relationship between oncolytic virus and innate immune response in anticancer therapy is still not well known. In this study, we showed that SVV has evolved as an effective mechanism to inhibit host type I IFN production by using its 3Cpro to cleave the molecules MAVS, TRIF, and TANK directly. These molecules are crucial for the Toll-like receptor 3 (TLR3)-mediated and retinoic acid-inducible gene I (RIG-I)-like receptor (RLR)-mediated signaling pathway. We also found that SVV is sensitive to IFN-ß. These findings increase our understanding of the interaction between SVV and host innate immunity.

Integrin αvß3 promotes infection by Japanese encephalitis virus.

Japanese encephalitis virus (JEV) is a mosquito-borne flavivirus that is one of the major causes of viral encephalitis diseases worldwide. The JEV envelope protein facilitates viral entry, and its domain III contains an Arg-Gly-Asp (RGD) motif, that may modulate JEV entry through the RGD-binding integrin. In this study, the roles of integrin αv and ß3 on the infection of JEV were evaluated. Reduced expression of integrin αv/ß3 by special shRNA confers 2 to 4-fold inhibition of JEV replication in BHK-21 cells. Meanwhile, antibodies specific for integrin αv/ß3 displayed ~58% and ~33% inhibition of JEV infectivity and RGD-specific peptides produced ~36% of inhibition. Expression of E protein and JEV RNA loads were clearly increased in CHO cells transfected with cDNA encoding human integrin ß3. Moreover, integrin αv mediates JEV infection in viral binding stage of life cycle. Therefore, our study suggested that integrin αv and ß3 serve as a host factor associated with JEV entry into the target cells.

TRIM52: A nuclear TRIM protein that positively regulates the nuclear factor-kappa B signaling pathway.

Emerging evidence suggests that TRIM family proteins play a crucial role in regulating the NF-κB signaling pathway. TRIM52 is a novel noncanonical antiviral TRIM gene with a unique expanded RING domain. Information on the biological function of TRIM52 is limited. Herein, we demonstrated TRIM52 involvement in NF-κB activation. We found that TRIM52 overexpression specifically activated the NF-κB signal. TRIM52 overexpression can significantly induce TNFα and IL-6 expression. We also found that the RING domain of TRIM52 was essential for its activation of the NF-κB signal. Further study showed that TRIM52 overexpression did not affect the protein level of IκBα and phosphorylated p65 protein. We found that the pro-inflammatory cytokines TNFα and IL-6 could induce TRIM52 expression. Overall, these data suggested that TRIM52 was a positive regulator of the NF-κB pathway.

Isolation and full-genome sequencing of Seneca Valley virus in piglets from China, 2016.

BACKGROUND: Seneca valley virus (SVV), a member of the Picornaviridae family, is a small non-enveloped RNA virus, that is linked to porcine idiopathic vesicular disease (PIVD). SVV infection in swine results in vesicular disease and epidemic transient neonatal losses (ETNL). The first case of SVV infection was reported in Guangdong, South China in 2015. RESULTS: We isolated and characterized an SVV HB-CH-2016 strain from vesicular lesion tissue specimens from piglets with PIVD in Hubei, Central China. The complete genome sequence of SVV HB-CH-2016 strain shares high nucleotide identities (94 to 99 %) with all previously reported SVV genomes, moreover, the polyprotein accounts for 98-99 % of amino acid sequence identity. Therefore, the SVV HB-CH-2016 strain is closely related to the SVV CH-01-2015 strain. CONCLUSIONS: The case reported in this paper is the second SVV infection case in China. Our findings demonstrate that sporadic SVV infection has occurred in Central China, and therefore, active surveillance on the swine population is important. Moreover, veterinarians must pay attention to this vesicular disease and reinforce biosecurity measures and prevent SVV spread.

TRIM52 inhibits Japanese Encephalitis Virus replication by degrading the viral NS2A.

The members of tripartite-motif containing (TRIM) protein participate in various cellular processes and play an important role in host antiviral function. TRIM proteins exert their antiviral activity either directly by degrading viral proteins through their E3 ligase activity, or indirectly by promoting host innate immunity. This study demonstrated for the first time that TRIM52 is a novel antiviral TRIM protein against Japanese encephalitis virus (JEV) infection. Overexpression of TRIM52 restricted JEV replication in BHK-21 and 293T cells. In addition, JEV nonstructural protein 2A (NS2A) is a protein that interacts with TRIM52. Their interaction degraded NS2A in a proteasome-dependent manner via the E3 ligase activity of TRIM52. Thus, TRIM52 is a novel antiviral TRIM protein, and it exerted antiviral activity against JEV infection by targeting and degrading viral NS2A.

Antiviral activity of luteolin against Japanese encephalitis virus.

Japanese encephalitis virus (JEV), a member of family Flaviviridae, is a neurotropic flavivirus that causes Japanese encephalitis (JE). JEV is one of the most important causative agents of viral encephalitis in humans, and this disease leads to high fatality rates. Although effective vaccines are available, no effective antiviral therapy for JE has been developed. Hence, identifying effective antiviral agents against JEV infection is important. In this study, we found that luteolin was an antiviral bioflavonoid with potent antiviral activity against JEV replication in A549 cells with IC50=4.56µg/mL. Luteolin also showed extracellular virucidal activity on JEV. With a time-of-drug addition assay revealing that JEV replication was inhibited by luteolin after the entry stage. Overall, our results suggested that luteolin can be used to develop an antiviral drug against JEV.

Swine TRIM21 restricts FMDV infection via an intracellular neutralization mechanism.

The tripartite motif protein 21 (TRIM21) is a ubiquitously expressed E3 ubiquitin ligase and an intracellular antibody receptor. TRIM21 mediates antibody-dependent intracellular neutralization (ADIN) in cytosol and provides an intracellular immune response to protect host defense against pathogen infection. In this study, swine TRIM21 (sTRIM21) was cloned and its role in ADIN was investigated. The expression of sTRIM21 is induced by type I interferon in PK-15 cells. sTRIM21 restricts FMDV infection in the presence of FMDV specific antibodies. Furthermore, sTRIM21 interacts with Fc fragment of swine immunoglobulin G (sFc) fused VP1 of FMDV and thereby causing its degradation. Both the RING and SPRY domains are essential for sTRIM21 to degrade sFc-fused VP1. These results suggest that the intracellular neutralization features of FMDV contribute to the antiviral activity of sTRIM21. sTRIM21 provide another intracellular mechanism to inhibit FMDV infection in infected cells.

Apigenin restricts FMDV infection and inhibits viral IRES driven translational activity.

Foot-and-mouth disease (FMD) is a highly contagious disease of domestic and wild ruminants that is caused by FMD virus (FMDV). FMD outbreaks have occurred in livestock-containing regions worldwide. Apigenin, which is a flavonoid naturally existing in plant, possesses various pharmacological effects, including anti-inflammatory, anticancer, antioxidant and antiviral activities. Results show that apigenin can inhibit FMDV-mediated cytopathogenic effect and FMDV replication in vitro. Further studies demonstrate the following: (i) apigenin inhibits FMDV infection at the viral post-entry stage; (ii) apigenin does not exhibit direct extracellular virucidal activity; and (iii) apigenin interferes with the translational activity of FMDV driven by internal ribosome entry site. Studies on applying apigein in vivo are required for drug development and further identification of potential drug targets against FDMV infection.

Swine interferon-induced transmembrane protein, sIFITM3, inhibits foot-and-mouth disease virus infection in vitro and in vivo.

The interferon-induced transmembrane protein 3 (IFITM3) is a widely expressed potent antiviral effector of the host innate immune system. It restricts a diverse group of pathogenic, enveloped viruses, by interfering with endosomal fusion. In this report, the swine IFITM3 (sIFITM3) gene was cloned. It shares the functionally conserved CD225 domain and multiple critical amino acid residues (Y19, F74, F77, R86 and Y98) with its human ortholog, which are essential for antiviral activity. Ectopic expression of sIFITM3 significantly inhibited non-enveloped foot-and-mouth disease virus (FMDV) infection in BHK-21 cells. Furthermore, sIFITM3 blocked FMDV infection at early steps in the virus life cycle by disrupting viral attachment to the host cell surface. Importantly, inoculation of 2-day-old suckling mice with a plasmid expressing sIFITM3 conferred protection against lethal challenge with FMDV. These results suggest that sIFITM3 is a promising antiviral agent and that can safeguard the host from infection with FMDV.

Combined analysis of survivin autoantibody and carcinoembryonic antigen biomarkers for improved detection of colorectal cancer.

BACKGROUND: Survivin is a member of the family of inhibitor of apoptosis proteins that is overexpressed in several human tumors. Previous studies have found that overexpression of survivin in cancer cells induces an antibody response. METHODS: We compared 232 serum samples from colorectal cancer (CRC) patients and 365 samples from healthy volunteers using an in vitro enzyme-linked immunosorbent assay to evaluate the survivin autoantibody response in patients. RESULTS: The sensitivity of the anti-survivin response from patients with CRC was 56.9%, and the specificity was 64.1%. When a cut-off value of 5.0 ng/mL was chosen for carcinoembryonic antigen (CEA) in these same serum samples, the values for sensitivity and specificity were 40.9% and 86.6%, respectively. Combined detection using survivin autoantibodies and CEA produced better sensitivity (51.3%) and specificity (89.9%) compared to the sensitivity of CEA (40.9%) and the specificities of the individual markers (64.1% and 86.6%, respectively). The area under a receiver operating characteristic curve was 0.617 for survivin autoantibodies, 0.630 for CEA and 0.694 for both markers together. CONCLUSIONS: A positive association between autoantibodies against survivin and preoperative CEA concentrations in sera of patients with CRCs was established. Our results suggest that analysis of both parameters would assist in screening patients with CRC.