ANXA2 enhances virus replication through negatively regulating cGAS-STING and RLRs-mediated signal pathway

Host nucleic acid receptors can recognize the viral DNA or RNA upon virus infection, which further triggers multiple signal pathways to promote the translocation of the interferon regulatory factor 3 (IRF3) into nucleus and produce type I interferon (IFN), leading to the host antiviral response. Here, we report a novel negative regulator Annexin A2 (ANXA2) that regulates type I IFN production through multiple mechanisms. Ectopic expression of ANXA2 inhibited the production of type I IFN induced by DNA- and RNA viruses and enhanced virus replication, while knockout of ANXA2 expression enhanced the production of type I IFN and inhibited virus replication. Mechanistically, ANXA2 not only disrupted MDA5 recruiting MAVS, but also inhibited the interaction between MAVS and TRAF3 upon RNA virus infection. In addition, ANXA2 impacted the translocation of STING from endoplasmic reticulum to Golgi apparatus upon DNA virus infection. Interestingly, ANXA2 also inhibited IRF3 phosphorylation and nuclear translocation through competing with TANK-binds kinase 1 (TBK1) and inhibitor-κB kinase ε (IKKε) for binding to IRF3. Anxa2 deficiency in vivo increased the production of type I IFN, which resulted in suppression of encephalomyocarditis virus (EMCV) replication. Our findings reveal that ANXA2, as a negative regulator of type I IFN production, plays an important role in regulating the host antiviral responses.Author summaryAnnexin is a family of evolutionarily conserved multi-gene proteins, which are widely distributed in various tissues and cells of plants and animals. These proteins can reversibly bind to phospholipid membranes and to calcium ions (Ca2+). To date, several studies have confirmed that some members of the Annexin family regulate the antiviral innate immune response. Until now, regulation of the production of type I IFN by ANXA2 is not reported. In this study, ANXA2 were found to strongly inhibit the production of type I IFN, leading to increased virus replication while knockout of ANXA2 expression inhibited virus replication by increasing the amount of IFN. Compared with wild-type littermates, ANXA2 deficiency mice produced more type I IFN to inhibit virus replication. Our results provide methanistic insights into the novel role of ANXA2 in the antiviral innate immune responses.

HSP27 Protein Dampens Encephalomyocarditis Virus Replication by Stabilizing Melanoma Differentiation-Associated Gene 5

Heat shock proteins (HSPs) are a protein family that respond to physiological stress, such as heat, starvation, and infection. As cellular protein chaperones, they play an important role in protein folding, assembly, and degradation. Though it is well known that HSP27 is involved in a range of viral infections, its role during an encephalomyocarditis virus (EMCV) infection is not known. Here, we report that EMCV degrades HSP27 and that EMCV proteins 2Cpro and 3Apro are primarily responsible for its degradation. Consequently, loss of cellular HSP27 augmented EMCV proliferation, an effect that could be reversed upon HSP27 overexpression. Importantly, we found that HSP27 positively regulated EMCV-triggered type I interferon (IFN) production. Moreover, overexpression of 2Cpro and 3Apro significantly blocked type I IFN production. We also found for the first time that HSP27, as a molecular chaperone, can specifically interact with MDA5 and stabilize the expression of MDA5. Collectively, this study shows that HSP27 dampens EMCV infectivity by positively regulating EMCV-triggered retinoic acid-inducible gene (RIG)-I-like receptor (RLR)/melanoma differentiation-associated gene 5 (MDA5) signal pathway, while EMCV proteins 2Cpro and 3Apro interact with HSP27 and degrade HSP27 protein expression to allow EMCV proliferation. Our findings provide further mechanistic evidence for EMCV partaking in immune escape mechanisms, and that 2Cpro and 3Apro could serve as potential antiviral targets.

Development and Characterization of a Reverse Genetics System for a Human-Derived Severe Fever With Thrombocytopenia Syndrome Virus Isolate From South Korea

Severe fever with thrombocytopenia syndrome virus (SFTSV) is an emerging, tick-borne Bandavirus that causes lethal disease in humans. As there are no licensed vaccines and therapeutics for SFTSV, there is an urgent need to develop countermeasures against it. In this respect, a reverse genetics (RG) system is a powerful tool to help achieve this goal. Herein, we established a T7 RNA polymerase-driven RG system to rescue infectious clones of a Korean SFTSV human isolate entirely from complementary DNA (cDNA). To establish this system, we cloned cDNAs encoding the three antigenomic segments into transcription vectors, with each segment transcribed under the control of the T7 promoter and the hepatitis delta virus ribozyme (HdvRz) sequences. We also constructed two helper plasmids expressing the nucleoprotein (NP) or viral RNA-dependent RNA polymerase (RdRp) under the control of the T7 promoter and the encephalomyocarditis virus (EMCV) internal ribosome entry site (IRES). After co-transfection into BHK/T7-9 cells with three transcription and two helper plasmids, then passaging in Vero E6 or Huh-7 cells, we confirmed efficient rescue of the recombinant SFTSV. By evaluating the in vitro and in vivo virological properties of the parental and rescued SFTSVs, we show that the rescued virus exhibited biological properties similar to those of the parental virus. This system will be useful for identifying molecular viral determinants of SFTSV infection and pathogenesis and for facilitating the development of vaccine and antiviral approaches.

Curcumol inhibits encephalomyocarditis virus by promoting IFN-β secretion

Background Encephalomyocarditis virus (EMCV) infection can cause reproductive failure in sows and acute myocarditis and sudden death in piglets. It has caused huge economic losses to the global pig industry and that is why it is necessary to develop effective new treatment compounds. Zedoary turmeric oil has been used for treating myocarditis. Curcumol extracted from the roots of curcuma is one of the main active ingredient of zedoary turmeric oil. The anti-EMCV activity of curcumol along with the molecular mechanisms involved with a focus on IFN-β signaling pathway was investigated in this study. Method 3-(4,5-dimethyithiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was used to determine the maximum non-toxic concentration (MNTC), 50% cytotoxic concentration (CC50), maximum inhibition rate (MIR) and 50% effective concentration (EC50) against EMCV. Through EMCV load, the anti-viral effect of curcumol was quantitatively determined using real-time quantitative PCR (qPCR). The effect of curcumol on the expression of IFN-β was investigated using real-time quantitative PCR and ELISA. Western blot was used to determine the amounts of MDA5, MAVS, TANK, IRF3 and P-IRF3 proteins in human embryonic kidney 293 T (HEK-293 T) cells infected with EMCV. Results The results of MTT showed that compared with the ribavirin positive control group, the maximum inhibition ratio (MIR) of curcumol was greater but the selection index (SI) value was much smaller than that of ribavirin. The results of qPCR showed that curcumol and ribavirin significantly reduced the replication of EMCV in HEK-293 T cells. The curcumol (0.025 mg/mL) treatment has significantly increased IFN-β mRNA expression in the EMCV-infected HEK-293 T cells while ribavirin treatment did not. The results of ELISA showed that curcumol (0.025 mg/mL and 0.0125 mg/mL) has significantly increased the expression of IFN-β protein in EMCV-infected HEK-293 T cells. The results of Western blot showed that curcumol can inhibit the degradation of TANK protein mediated by EMCV and promote the expression of MDA5 and P-IRF3, while the protein expression level of MAVS and IRF3 remain unchanged. Conclusion Curcumol has biological activity against EMCV which we suggest that IFN-β signaling pathway is one of its mechanisms.

Genes for tRNA recycling are upregulated in response to infection with Theiler's mouse encephalitis virus

The concept of tRNA recycling has recently emerged from the studies of ribosome associated quality control. Therein tRNase ZS removes the 2', 3'>p from the ANKZF1-cleaved tRNA and the subsequent TRNT1 action re-generates the intact tRNA. To know the roles of the tRNA recycling in vivo, we investigated how viral infection affects the tRNA recycling system by analyzing the mRNA levels of tRNase ZS and TRNT1. We found that both genes in HeLa cells are upregulated in response to infection of Theiler's mouse encephalitis virus but not to that of an influenza A virus. Upregulation was also observed in cells infected with encephalomyocarditis virus with reduced efficiency. The levels of the IFN-β mRNA appeared to positively correlate with those of the tRNase ZS and TRNT1 mRNAs. The tRNase ZS gene may be regulated post-transcriptionally in the cells infected with Theiler's mouse encephalitis virus.

Rosin Soap Exhibits Virucidal Activity

Chemical methods of virus inactivation are used routinely to prevent viral transmission in both a personal hygiene capacity but also in at-risk environments like hospitals. Several virucidal products exist, including hand soaps, gels and surface disinfectants. Resin acids, which can be derived from Tall oil produced from trees, have been shown to exhibit anti-bacterial activity. However, whether these products or their derivatives have virucidal activity is unknown. Here, we assessed the capacity of Rosin soap to inactivate a panel of pathogenic mammalian viruses in vitro. We show that Rosin soap can inactivate the human enveloped viruses: influenza A virus (IAV), respiratory syncytial virus and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). For IAV, rosin soap could provide a 100,000-fold reduction in infectivity. However, Rosin soap failed to affect the non-enveloped encephalomyocarditis virus (EMCV). The inhibitory effect of Rosin soap against IAV infectivity was dependent on its concentration but not dependent on incubation time nor temperature. Together, we demonstrate a novel chemical inactivation method against enveloped viruses, which could be of use in preventing virus infections in certain settings.

USP18 positively regulates innate antiviral immunity by promoting K63-linked polyubiquitination of MAVS

Activation of MAVS, an adaptor molecule in Rig-I-like receptor (RLR) signaling, is indispensable for antiviral immunity, yet the molecular mechanisms modulating MAVS activation are not completely understood. Ubiquitination has a central function in regulating the activity of MAVS. Here, we demonstrate that a mitochondria-localized deubiquitinase USP18 specifically interacts with MAVS, promotes K63-linked polyubiquitination and subsequent aggregation of MAVS. USP18 upregulates the expression and production of type I interferon following infection with Sendai virus (SeV) or Encephalomyocarditis virus (EMCV). Mice with a deficiency of USP18 are more susceptible to RNA virus infection. USP18 functions as a scaffold protein to facilitate the re-localization of TRIM31 and enhances the interaction between TRIM31 and MAVS in mitochondria. Our results indicate that USP18 functions as a post-translational modulator of MAVS-mediated antiviral signaling.

Heat Shock Protein A6, a Novel HSP70, Is Induced During Enterovirus A71 Infection to Facilitate Internal Ribosomal Entry Site-Mediated Translation

Enterovirus A71 (EV-A71) is a human pathogen causing hand, foot, and mouth disease (HFMD) in children. Its infection can lead to severe neurological diseases or even death in some cases. While being produced in a large quantity during infection, viral proteins often require the assistance from cellular chaperones for proper folding. In this study, we found that heat shock protein A6 (HSPA6), whose function in viral life cycle is scarcely studied, was induced and functioned as a positive regulator for EV-A71 infection. Depletion of HSPA6 led to the reductions of EV-A71 viral proteins, viral RNA and virions as a result of the downregulation of internal ribosomal entry site (IRES)-mediated translation. Unlike other HSP70 isoforms such as HSPA1, HSPA8, and HSPA9, which regulate all phases of the EV-A71 life, HSPA6 was required for the IRES-mediated translation only. Unexpectedly, the importance of HSPA6 in the IRES activity could be observed in the absence of viral proteins, suggesting that HSPA6 facilitated IRES activity through cellular factor(s) instead of viral proteins. Intriguingly, the knockdown of HSPA6 also caused the reduction of luciferase activity driven by the IRES from coxsackievirus A16, echovirus 9, encephalomyocarditis virus, or hepatitis C virus, supporting that HSPA6 may assist the function of a cellular protein generally required for viral IRES activities.