Pseudorabies virus UL38 attenuates the cGAS-STING signaling pathway by recruiting Tollip to promote STING for autophagy degradation.

Natural immunity is the first defense line of the host immune system, which plays a significant role in combating foreign pathogenic microorganisms. The IFN-ß (interferon-beta) signaling pathway, being a typical example of innate immunity, plays a vital function. This study aimed to elucidate the function of pseudorabies virus (PRV) UL38 protein (unique long region 38) in suppressing the activation of the IFN-ß signaling pathway. The findings from our study indicate that the PRV UL38 protein effectively hampers the activation of IFN-ß by poly (dA: dT) (poly(deoxyadenylic-deoxythymidylic)) and 2'3'-cGAMP (2'-3'-cyclic GMP-AMP). Furthermore, UL38 exhibits spatial co-localization with STING (stimulator of interferon genes) and effectively hinders STING dimerization. Subsequently, STING was downgraded to suppress the production of IFN-ß and ISGs (interferon stimulated genes). Immunoprecipitation analysis revealed that the interaction between UL38 and STING, which subsequently initiated the degradation of STING via selective autophagy mediated by TOLLIP (toll interacting protein). To summarize, this research elucidates the function of UL38 in counteracting the cGAS (cGAMP synthase)-STING-induced IFN-ß pathway. The PRV UL38 protein may attenuate the activation of IFN-ß as a means of regulating the virus's persistence in the host.

Encephalomyocarditis Virus Abrogates the Interferon Beta Signaling Pathway via Its Structural Protein VP2.

Type I interferon (IFN)-mediated antiviral responses are critical for modulating host-virus responses, and indeed, viruses have evolved strategies to antagonize this pathway. Encephalomyocarditis virus (EMCV) is an important zoonotic pathogen, which causes myocarditis, encephalitis, neurological disease, reproductive disorders, and diabetes in pigs. This study aims to understand how EMCV interacts with the IFN pathway. EMCV circumvents the type I IFN response by expressing proteins that antagonize cellular innate immunity. Here, we show that EMCV VP2 is a negative regulator of the IFN-ß pathway. This occurs via the degradation of the MDA5-mediated cytoplasmic double-stranded RNA (dsRNA) antiviral sensing RIG-I-like receptor (RLR) pathway. We show that structural protein VP2 of EMCV interacts with MDA5, MAVS, and TBK1 through its C terminus. In addition, we found that EMCV VP2 could significantly degrade RLRs by the proteasomal and lysosomal pathways. For the first time, EMCV VP2 was shown to play an important role in EMCV evasion of the type I IFN signaling pathway. This study expands our understanding that EMCV utilizes its capsid protein VP2 to evade the host antiviral response.IMPORTANCE Encephalomyocarditis virus is an important pathogen that can cause encephalitis, myocarditis, neurological diseases, and reproductive disorders. It also causes huge economic losses for the swine industry worldwide. Innate immunity plays an important role in defending the host from pathogen infection. Understanding pathogen microorganisms evading the host immune system is of great importance. Currently, whether EMCV evades cytosolic RNA sensing and signaling is still poorly understood. In the present study, we found that viral protein VP2 antagonized the RLR signaling pathway by degrading MDA5, MAVS, and TBK1 protein expression to facilitate viral replication in HEK293 cells. The findings in this study identify a new mechanism for EMCV evading the host's innate immune response, which provide new insights into the virus-host interaction and help develop new antiviral approaches against EMCV.

Construction, expression, and characterization of a single-chain variable fragment (ScFv) antibody targeting to the encephalomyocarditis virus.

Encephalomyocarditis virus (EMCV) is as a potential zoonotic agent with a wide host range. Here, applying gene splicing by overlap extension PCR (SOE-PCR), we describe a simple method for producing single-chain variable fragment (scFv) antibody against EMCV that configurates in the orientation of VH-(GGGGS)4 -VL. DNA template was resverse transcribed by total RNA that derived from hyperimmunized antibody positive mice spleen after inoculation inactivated EMCV-PV21 as antigen. Using the degenerate primers designed for the variable regions of IgG of murine antibody, the 417 bp of gene encoding VH-linker (VHL) and 360 bp of gene encoding linker-VL (LVL) of the anti-EMCV was individually amplified from DNA template by PCR, repectively. The 762 bp gene encoding anti-EMCV scFv was constructed by SOE-PCR when the mixed VHL and LVL genes were used as the template. The amplified gene subcloned into pGEX-6P1 to yield pGEX-6P1/EMCV-scFv. Recombinant vector transformed into the Escherichia coli BL21 (DE3) and a 53 KDa GST-scFv fusion protein was obtained by SDS-PAGE electrophoresis. Animal experiment results showed that the pretective rate of mice in group A which challenged 500 µL 104 TCID50 EMCV per mouse for 7 d post-inoculation scFv 3 d (0.5 mg purified recombinant scFv per mouse) was 91.67% (11/12). The serum anti-EMCV antibody titer in group A mice was most significantly higher than that in positive control mouse (P < 0.01), coversely the serum relative mRNA copies were significantly lower than that in positive control mouse (P < 0.05). These findings indicated that recombinant anti-EMCV scFv has remarkable anti-EMCV effect in mice.

Transcriptional profiling of host cell responses to encephalomyocarditis virus (EMCV).

BACKGROUD: Encephalomyocarditis virus (EMCV) has been discovered on pig farms worldwide and can cause myocarditis in piglets and reproductive failure in sows. However, little is known about the host transcriptional responses to infection and host-pathogen interactions. METHODS: In this study, transcription profiling was performed by Illumina RNA-Sequencing (RNA-seq) to identify EMCV induced differentially expressed genes in BHK-21 cells at serial time points (12, 24, and 30 h post infection (hpi)), using mock infected cells as control. RESULTS: We identified 237, 241, and 207 differentially expressed genes (DEGs) respectively, majority of which were up-regulated. A large number of DEGs clustered into host defense, cellular signaling and metabolism categories. Moreover, short time series expression analysis revealed that 12 hpi was an important time point for expression change, indicating host virus resistance. CONCLUSIONS: This RNA-seq analysis provides the first data for understanding the network of virus host interactions under EMCV infection in vitro, and for identifying host components which involved in the virus infection course.

National serosurvey of encephalomyocarditis virus in healthy people and pigs in China.

Encephalomyocarditis virus (EMCV) is a zoonotic pathogen that has a wide spectrum of host range. The virus has been discovered on swine farms worldwide and can cause acute fatal myocarditis in piglets and reproductive disorders in sows. Although EMCV infection has been documented in farmed pigs in China, seroprevalence in humans has not been reported. In this study, we conducted nationwide serological surveys for EMCV in humans and farmed pigs in China in 2013, by the use of a double antigen sandwich ELISA method. A total of 3305 serum samples from healthy people were obtained from seven geographical regions in China, of which 1010 samples (30.56%) were positive for EMCV antibodies. The overall seroprevalence for EMCV in the age groups of 0-20, 21-40, 41-60 and >60 years were 13.5%, 30.25%, 36.83% and 38.71% respectively, showing a tendency of increasing with age (P = 0.000). A total of 3470 serum samples from farmed pigs were collected and tested for antibodies to EMCV. A high seroprevalence of 77% was recorded, and significant regional differences were observed. It was concluded that people and pigs in China were commonly infected by EMCV. In addition, in order to characterize changes of seroprevalence during natural EMCV infection in pigs, 240 serial serum samples were collected from 30 pigs (at 0, 15, 30, 60, 75, 90, 120, and 150 days of age) in a farrow-to-finish farm in China. The data showed that there were two EMCV antibody peaks: the first peak appeared at day 30, followed by a decrease in EMCV antibody titer, and the second occurred after day 75. Thus, the most susceptible period of pigs for EMCV infection was between day 30 and day 75 of age.

The complex interplay between encephalomyocarditis virus and the host defence system.

A variety of animals can be infected by encephalomyocarditis virus (EMCV). EMCV is the established causative agent of myocarditis and encephalitis in some animals. EMCV causes high fatality in suckling and weaning piglets, making pigs the most susceptible domestic animal species. Importantly, EMCV has zoonotic potential to infect the human population. The ability of the pathogen to avoid and undermine the initial defence mechanism of the host contributes to its virulence and pathogenicity. A large body of literature highlights the intricate strategies employed by EMCV to escape the innate immune machinery to suit its "pathogenic needs." Here, we also provide examples on how EMCV interacts with certain host proteins to dampen the infection process. Hence, this concise review aims to summarize these findings in a compendium of decades of research on this exciting yet underappreciated topic.

The interaction on HSA and the antibacterial activity from four polyoxometalate hybrids based on berberine.

Four organic-polyoxometalate hybrids BR4[SiW12O40] (BR-SiW), BR3[PMo12O40] (BR-PMo), BR4K[EuSiW11O40]·2H2O (BR-EuSiW) and BR6Na3[EuW10O36] (BR-EuW) were fabricated by the polyoxometalates (POMs) anions and berberine cations (BR) noted for the alkaloids in traditional Chinese herbal medicine. These hybrids have been characterized and confirmed. The interaction between hybrids and human serum albumin (HSA) was investigated in a buffer solution (pH 7.4) using ultraviolet-visible light absorption and fluorescence techniques. The classical Stern-Volmer equation was used to analyze the fluorescence quenching at three temperatures (296, 303 and 310 K), and the static quenching mechanism for interaction was proposed. The Thermodynamic parameters, enthalpy, entropy change, and Gibbs free energy of hybrids interacting on HSA were calculated by Scatchard equation. The results indicated that therewas one binding site on the protein and BR-POMs all showed stronger binding force than that of raw materials. Synchronous fluorescence results showed that the binding sites of BR-POMs and HSA were not effectively affected the surrounding microenvironment. The following antibacterial experiments implied that inhibitory effect of hybrids were synergistic effect from organic active ingredient and POMs but the simple combination. All these data were prepared for further research on biology.

New perspective of small-molecule antiviral drugs development for RNA viruses.

High variability and adaptability of RNA viruses allows them to spread between humans and animals, causing large-scale infectious diseases which seriously threat human and animal health and social development. At present, AIDS, viral hepatitis and other viral diseases with high incidence and low cure rate are still spreading around the world. The outbreaks of Ebola, Zika, dengue and in particular of the global pandemic of COVID-19 have presented serious challenges to the global public health system. The development of highly effective and broad-spectrum antiviral drugs is a substantial and urgent research subject to deal with the current RNA virus infection and the possible new viral infections in the future. In recent years, with the rapid development of modern disciplines such as artificial intelligence technology, bioinformatics, molecular biology, and structural biology, some new strategies and targets for antivirals development have emerged. Here we review the main strategies and new targets for developing small-molecule antiviral drugs against RNA viruses through the analysis of the new drug development progress against several highly pathogenic RNA viruses, to provide clues for development of future antivirals.

Encephalomyocarditis Virus Structural Protein VP3 Interacts with MAVS and Promotes its Autophagic Degradation to Interfere with the Type I Interferon Signaling Pathway.

BACKGROUND: Understanding the mechanisms through which interferon (IFN) signaling is negatively regulated is crucial for preserving the equilibrium of innate immune reactions, as the innate immune system functions, such as the original barrier, combat threats to the host. Although the function of the encephalomyocarditis virus (EMCV) viral proteins in antagonizing innate immunity has been related to earlier studies, the precise mechanism underlying the role of viral protein 3 (VP3) in type I IFN has yet to be fully illuminated. METHODS: VP3 expression and many other adaptor molecules belonging to type I IFN pathway expression levels were evaluated using Western blotting. The IFN and other antiviral genes, such as interferon-stimulated genes (ISGs) 15 and 56, were assessed by real-time quantitative polymerase chain reaction (RT-qPCR). A 50% tissue culture infectious dose (TCID50) assay was utilized to explore the effect of VP3 on EMCV proliferation in human embryonic kidney (HEK293) cells. Co-immunoprecipitation (Co-IP) assays and confocal microscope analysis were used to investigate the underlying mechanisms mediated by VP3. RESULTS: We discovered that the VP3 of EMCV acts as a suppressor of innate immune reactions. Increased levels of VP3 enhance viral reproduction through modulation of innate immune signaling pathways and suppression of antiviral responses. Additional information indicated that during viral infection, the VP3 of EMCV enhances autophagy and interacts specifically with mitochondrial antiviral signaling protein (MAVS), leading to its degradation in an autophagy pathway that relies on p62. CONCLUSIONS: Our findings showed that EMCV developed a tactic to combat host antiviral defenses by using autophagy to break down a protein that controls the innate immune response following a viral infection of the host. Notably, VP3 plays an important role in this process. Overall, these discoveries may provide a novel therapeutic target for EMCV.

Pseudorabies virus VHS protein abrogates interferon responses by blocking NF-κB and IRF3 nuclear translocation.

Herpesviruses antagonize host antiviral responses through a myriad of molecular strategies culminating in the death of the host cells. Pseudorabies virus (PRV) is a significant veterinary pathogen in pigs, causing neurological sequalae that ultimately lead to the animal's demise. PRV is known to trigger apoptotic cell death during the late stages of infection. The virion host shutdown protein (VHS) encoded by UL41 plays a crucial role in the PRV infection process. In this study, we demonstrate that UL41 inhibits PRV-induced activation of inflammatory cytokine and negatively regulates the cGAS-STING-mediated antiviral activity by targeting IRF3, thereby inhibiting the translocation and phosphorylation of IRF3. Notably, mutating the conserved amino acid sites (E192, D194, and D195) in the RNase domain of UL41 or knocking down UL41 inhibits the immune evasion of PRV, suggesting that UL41 may play a crucial role in PRV's evasion of the host immune response during infection. These results enhance our understanding of how PRV structural proteins assist the virus in evading the host immune response.

Histone H1.2 Inhibited EMCV Replication through Enhancing MDA5-Mediated IFN-ß Signaling Pathway.

Histone H1.2 is a member of the linker histone family, which plays extensive and crucial roles not only in the regulation of chromatin dynamics, cell cycle, and cell apoptosis, but also in viral diseases and innate immunity response. Recently, it was discovered that H1.2 regulates interferon-ß and inhibits influenza virus replication, whereas its role in other viral infections is poorly reported. Here, we first found the up-regulation of H1.2 during Encephalomyocarditis virus (EMCV) infection, implying that H1.2 was involved in EMCV infection. Overexpression of H1.2 inhibited EMCV proliferation, whereas knockdown of H1.2 showed a significant promotion of virus infection in HEK293T cells. Moreover, we demonstrated that overexpression of H1.2 remarkably enhanced the production of EMCV-induced type I interferon, which may be the crucial factor for H1.2 proliferation-inhibitory effects. We further found that H1.2 up-regulated the expression of the proteins of the MDA5 signaling pathway and interacted with MDA5 and IRF3 in EMCV infection. Further, we demonstrated that H1.2 facilitated EMCV-induced phosphorylation and nuclear translocation of IRF3. Briefly, our research uncovers the mechanism of H1.2 negatively regulating EMCV replication and provides new insight into antiviral targets for EMCV.

Mammalian reovirus µ1 protein attenuates RIG-I and MDA5-mediated signaling transduction by blocking IRF3 phosphorylation and nuclear translocation.

Mammalian reovirus (MRV) is a non-enveloped, gene segmented double-stranded RNA (dsRNA) virus. It is an important zoonotic pathogen that infects many mammals and vertebrates that act as natural hosts and causes respiratory and digestive tract diseases. Studies have reported that RIG-I and MDA5 in the innate immune cytoplasmic RNA-sensing RIG-like receptor (RLR) signaling pathway can recognize dsRNA from MRV and promote antiviral type I interferon (IFN) responses. However, the mechanism by which many MRV-encoded proteins evade the host innate immune response remains unclear. Here, we show that exogenous µ1 protein promoted the proliferation of MRV in vitro, while knockdown of MRV µ1 protein expression by shRNA could impair MRV proliferation. Specifically, µ1 protein inhibited MRV or poly(I:C)-induced IFN-ß expression, and attenuated RIG-I/MDA5-mediated signaling axis transduction during MRV infection. Importantly, we found that µ1 protein significantly decreased IFN-ß mRNA expression induced by MDA5, RIG-I, MAVS, TBK1, IRF3(5D), and degraded the protein expression of exogenous MDA5, RIG-I, MAVS, TBK1 and IRF3 via the proteasomal and lysosomal pathways. Additionally, we show that µ1 protein can physically interact with MDA5, RIG-I, MAVS, TBK1, and IRF3 and attenuate the RIG-I/MDA5-mediated signaling cascades by blocking the phosphorylation and nuclear translocation of IRF3. In conclusion, our findings reveal that MRV outer capsid protein µ1 is a key factor in antagonizing RLRs signaling cascades and provide new strategies for effective prevention and treatment of MRV infection.

Mammalian orthoreovirus capsid protein σ3 antagonizes RLR-mediated antiviral responses by degrading MAVS.

Mammalian orthoreovirus (MRV) outer capsid protein σ3 is a multifunctional protein containing a double-stranded RNA-binding domain, which facilitates viral entry and assembly. We reasoned that σ3 has an innate immune evasion function. Here, we show that σ3 protein localizes in the mitochondria and interacts with mitochondrial antiviral signaling protein (MAVS) to activate the intrinsic mitochondria-mediated apoptotic pathway. Consequently, σ3 protein promotes the degradation of MAVS through the intrinsic caspase-9/caspase-3 apoptotic pathway. Moreover, σ3 protein can also inhibit the expression of the components of the RNA-sensing retinoic acid-inducible gene (RIG)-like receptor (RLR) signaling pathway to block antiviral type I interferon responses. Mechanistically, σ3 inhibits RIG-I and melanoma differentiation-associated gene 5 expression is independent of its inhibitory effect on MAVS. Overall, we demonstrate that the MRV σ3 protein plays a vital role in negatively regulating the RLR signaling pathway to inhibit antiviral responses. This enables MRV to evade host defenses to facilitate its own replication providing a target for the development of effective antiviral drugs against MRV. IMPORTANCE: Mammalian orthoreovirus (MRV) is an important zoonotic pathogen, but the regulatory role of its viral proteins in retinoic acid-inducible gene-like receptor (RLR)-mediated antiviral responses is still poorly understood. Herein, we show that MRV σ3 protein co-localizes with mitochondrial antiviral signaling protein (MAVS) in the mitochondria and promotes the mitochondria-mediated intrinsic apoptotic pathway to cleave and consequently degrade MAVS. Furthermore, tryptophan at position 133 of σ3 protein plays a key role in the degradation of MAVS. Importantly, we show that MRV outer capsid protein σ3 is a key factor in antagonizing RLR-mediated antiviral responses, providing evidence to better unravel the infection and transmission mechanisms of MRV.

Construction, expression and assemble of EMCV VLPs and their potency evaluation.

Encephalomycarditis virus (EMCV) is an essential pathogen with a broad host range and causes enormous economic losses to the pig industry worldwide. Here, we constructed and assembled the EMCV virus-like particles (VLPs) in vitro and verified high efficiency of virus protection. Results showed that the proteins auto-assembled into VLPs successfully in vitro. The animal experiments revealed that high-titer antibody production is triggered by VLPs. Meanwhile, the mice challenged with EMCV were obviously protected. The protection rate of group VLPs with the adjuvant was 75%, while that of the VLPs group was 62.5% compared to the control. These findings indicate that recombinant EMCV VLPs have a remarkable anti-EMCV effect and could be a new vaccine candidate for the control of EMCV infection.

IFITM2 Presents Antiviral Response through Enhancing Type I IFN Signaling Pathway.

Interferon (IFN) helps cells fight viral infections by further inducing the expression of many downstream IFN-stimulated genes (ISGs). Human interferon-inducible transmembrane proteins (IFITM) are one of these ISGs. The antiviral function of human IFITM1, IFITM2, and IFITM3 are well known. In this study, we report that IFITM can significantly inhibit EMCV infectivity in HEK293 cells. Overexpression of IFITM proteins could promote IFN-ß production. Meanwhile, IFITMs facilitated type I IFN signaling pathway adaptor MDA5 expression. We detected the binding of IFITM2 to MDA5 in a co-immunoprecipitation assay. It was also found that the ability of IFITM2 to activate IFN-ß was significantly inhibited after interfering with MDA5 expression, suggesting that MDA5 may play an important role in the activation of the IFN-ß signaling pathway by IFITM2. Moreover, the N-terminal domain plays an active role in the antiviral activity and the activation of IFN-ß by IFITM2. These findings suggest that IFITM2 plays a vital role in antiviral signaling transduction. In addition, a positive feed-forward loop between IFITM2 and type I IFN establishes a key role for IFITM2 in enforcing innate immune responses.

Pseudorabies Virus ICP0 Abolishes Tumor Necrosis Factor Alpha-Induced NF-κB Activation by Degrading P65.

Nuclear factor κB (NF-κB) is involved in a wide range of innate immune activities in host cells and serves as an important component of a host's immunity system. To survive in infected cells, viruses have evolved intricate strategies to evade the host immune response. Pseudorabies virus (PRV) is a member of the alpha herpesvirus family and is capable of causing reproductive and neurological dysfunction in pigs. PRV has a large DNA genome and therefore has the ability to encode numerous proteins that modulate host innate immune responses. In the present study, we demonstrated that the PRV-encoded immediate early protein ICP0 inhibits the tumor necrosis factor alpha (TNF-α)-mediated NF-κB signaling pathway. An in-depth study showed that ICP0 protein was able to limit NF-κB activation and decreased the expression of inflammatory cytokines interleukin-6 (IL-6) and interleukin 8 (IL-8). In addition, ICP0 blocked the activation of NF-κB through interacting with p65, degrading its protein expression and limiting its phosphorylation. PRV protein ICP0 is shown for the first time to enable escape from innate immune response through the regulation of NF-κB during PRV infection. These results illustrate that PRV ICP0 is able to block NF-κB activation. This mechanism may represent a critical role in the early events leading to PRV infection.

DDX56 antagonizes IFN-ß production to enhance EMCV replication by inhibiting IRF3 nuclear translocation.

DEAD (Asp-Glu-Ala-Asp)-box RNA helicases (DDX) play important roles in viral infection, either as cytosolic viral nucleic acids sensors or as essential host factors for viral replication. In this study, we identified DDX56 as a positive regulator for encephalomyocarditis virus (EMCV) replication. EMCV infection promotes DDX56 expression via its viral proteins, VP3 and 3C. We showed that DDX56 overexpression promotes EMCV replication whereas its loss dampened EMCV replication. Consequently, knockdown of DDX56 increases type I interferon (IFN) expression during EMCV infection. We also showed that DDX56 interrupts IFN regulatory factor 3 (IRF3) phosphorylation and its nucleus translocation by directly targeting KPNA3 and KPNA4 in an EMCV-triggered MDA5 signaling activation cascade leading to the blockade of IFN-ß production. Overall, we showed that DDX56 is a novel negative regulator of EMCV-mediated IFN-ß responses and that DDX56 plays a critical role in EMCV replication. These findings reveal a novel strategy for EMCV to utilize a host factor to evade the host innate immune response and provide us new insight into the function of DDX56.

HSP27 Attenuates cGAS-Mediated IFN-ß Signaling through Ubiquitination of cGAS and Promotes PRV Infection.

Pseudorabies (PR) is a domestic and wild animal infectious disease caused by the pseudorabies virus (PRV) and is one of the major infectious diseases that endanger the global swine industry. Studies have reported that PRV may achieve cross-species transmission from pigs to humans in recent years. Therefore, in-depth exploration of the relationship between PRV and host proteins is of great significance for elucidating the pathogenic mechanism of PRV and anti-PRV infection. Here, we report that heat shock protein 27 (HSP27) ubiquitinates and degrades cyclic GMP-AMP synthase (cGAS) and attenuates cGAS-mediated antiviral responses, thereby promoting PRV infection. Overexpression of HSP27 promoted PRV proliferation in vitro, while knockdown of HSP27 inhibited PRV infection. Importantly, we found that HSP27 inhibited PRV infection or poly(dA:dT)-activated IFN-ß expression. Further studies found that HSP27 may inhibit cGAS-STING-mediated IFN-ß expression through targeting cGAS. In addition, we found that HSP27 can suppress the expression of endogenous cGAS in different cells at both gene transcription and protein expression levels, and that HSP27 interacts with and ubiquitinates cGAS. In conclusion, we reveal for the first time that HSP27 is a novel negative regulator of the cGAS-STING signaling pathway induced by PRV infection or poly(dA:dT) activation and demonstrate that HSP27 plays a crucial role in PRV infection.

DDX56 inhibits PRV replication through regulation of IFN-ß signaling pathway by targeting cGAS.

Pseudorabies virus (PRV) is an agent of Aujeszky's disease, and causes great economic losses to pig farming. Re-outburst of pseudorabies implies that new control measures are urgently needed. We show here that DDX56 possesses the ability to inhibit PRV replication in vitro, which may be an important factor for PRV infection. Overexpression of DDX56 inhibited PRV genomic DNA transcription and lower titers of PRV infection in PK15 cells, whereas down-regulation of the DDX56 expression had a promotion role on virus replication. Further study demonstrated that DDX56 exerted its proliferation-inhibitory effects of PRV through up-regulating cGAS-STING-induced IFN-ß expression. Moreover, we found that DDX56 could promote cGAS expression and direct interaction also existed between DDX56 and cGAS. Based on this, DDX56-regulated IFN-ß pathway may be targeted at cGAS. To verify this, down-regulated cGAS expression in DDX56 over-expression cells was performed. Results indicated that knockdown of cGAS expression could abrogate the inhibition role of DDX56 on PRV proliferation and weaken the effect of DDX56 on IFN-ß expression. In addition, DDX56 played a promotion role in IRF3 phosphorylation and nucleus translocation. Altogether, our results highlight DDX56's antiviral role in PRV infection, and our findings contribute to a better understanding of host factors controlling PRV replication.

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.