Description: Human herpesvirus 6 (HHV-6) is an important immunosuppressive and immunomodulatory virus. The mechanisms by which HHV-6 establishes latency and immunosuppression in its host are not well understood. Here we characterized HHV-6-specific T cells in peripheral blood mononuclear cells (PBMCs) from HHV-6-infected donors. Our results showed that HHV-6 infection could induce both CD4 + and CD8 + HHV-6-specific regulatory T (Treg) cells. These HHV-6-specific Treg cells had potent suppressive activity and expressed high levels of Treg-associated molecules CD25, FoxP3, and GITR. Both CD4 + and CD8 + Treg cells secreted gamma interferon (IFN-) and interleukin-10 (IL-10) but little or no IL-2, IL-4, or transforming growth factor β (TGF-β). Furthermore, HHV-6-specifc Treg cells not only could suppress naive and HHV-6-specific CD4 + effector T cell immune responses but also could impair dendritic cell (DC) maturation and functions. In addition, the suppressive effects mediated by HHV-6-specific Treg cells were mainly through a cell-to-cell contact-dependent mechanism but not through the identified cytokines. These results suggest that HHV-6 may utilize the induction of Treg cells as a strategy to escape antivirus immune responses and maintain the latency and immunosuppression in infected hosts.

Description: Porcine epidemic diarrhea virus (PEDV), an enteropathogenic Alphacoronavirus , has caused enormous economic losses in the pork industry. Nonstructural protein 1 (nsp1) is a characteristic feature of alpha- and betacoronaviruses, which exhibits both functional conservation and mechanistic diversity in inhibiting host gene expression and antiviral responses. However, the detailed structure and molecular mechanisms underlying the Alphacoronavirus nsp1 inhibition of host gene expression remain unclear. Here, we report the first full-length crystal structure of Alphacoronavirus nsp1 from PEDV. The structure displays a six-stranded β-barrel fold in the middle of two α-helices. The core structure of PEDV nsp1 shows high similarity to those of severe acute respiratory syndrome coronavirus (SARS-CoV) nsp1 and transmissible gastroenteritis virus (TGEV) nsp1, despite its low degree of sequence homology. Using ribopuromycylation and Renilla luciferase reporter assays, we showed that PEDV nsp1 can dramatically inhibit general host gene expression. Furthermore, three motifs (amino acids [aa] 67 to 71, 78 to 85, and 103 to 110) of PEDV nsp1 create a stable functional region for inhibiting protein synthesis, differing considerably from Betacoronavirus nsp1. These results elucidate the detailed structural basis through which PEDV nsp1 inhibits host gene expression, providing insight into the development of a new attenuated vaccine with nsp1 modifications. IMPORTANCE Porcine epidemic diarrhea virus (PEDV) has led to tremendous economic losses in the global swine industry. PEDV nsp1 plays a crucial role in inhibiting host gene expression, but its functional mechanism remains unclear. Here, we report the full-length structure of PEDV nsp1, the first among coronaviruses to be reported. The 1.25-Å resolution crystal structure of PEDV nsp1 shows high similarity to severe acute respiratory syndrome coronavirus (SARS-CoV) nsp1 13–128 and transmissible gastroenteritis virus (TGEV) nsp1 1–104 , despite a lack of sequence homology. Structural and biochemical characterization demonstrated that PEDV nsp1 possesses a stable functional region for inhibition of host protein synthesis, which is formed by loops at residues 67 to 71, 78 to 85, and 103 to 110. The different functional regions in PEDV nsp1 and SARS-CoV nsp1 may explain their distinct mechanisms. Importantly, our structural data are conducive to understanding the mechanism of PEDV nsp1 inhibition of the expression of host genes and may aid in the development of a new attenuated vaccine.

Description: Porcine deltacoronavirus (PDCoV) has recently emerged as an enteric pathogen that can cause serious vomiting and diarrhea in suckling piglets. The first outbreak of PDCoV occurred in the United States in 2014 and was followed by reports of PDCoV in South Korea, China, Thailand, Lao People's Democratic Republic, and Vietnam, leading to economic losses for pig farms and posing a considerable threat to the swine industry worldwide. Our previous studies have shown that PDCoV encodes three accessory proteins, NS6, NS7, and NS7a, but the functions of these proteins in viral replication, pathogenesis, and immune regulation remain unclear. Here, we found that ectopic expression of accessory protein NS6 significantly inhibits Sendai virus-induced interferon beta (IFN-β) production as well as the activation of transcription factors IRF3 and NF-B. Interestingly, NS6 does not impede the IFN-β promoter activation mediated via key molecules in the RIG-I-like receptor (RLR) signaling pathway, specifically RIG-I, MDA5, and their downstream molecules MAVS, TBK1, IKK, and IRF3. Further analyses revealed that NS6 is not an RNA-binding protein; however, it interacts with RIG-I/MDA5. This interaction attenuates the binding of double-stranded RNA by RIG-I/MDA5, resulting in the reduction of RLR-mediated IFN-β production. Taken together, our results demonstrate that ectopic expression of NS6 antagonizes IFN-β production by interfering with the binding of RIG-I/MDA5 to double-stranded RNA, revealing a new strategy employed by PDCoV accessory proteins to counteract the host innate antiviral immune response. IMPORTANCE Coronavirus accessory proteins are species specific, and they perform multiple functions in viral pathogenicity and immunity, such as acting as IFN antagonists and cell death inducers. Our previous studies have shown that PDCoV encodes three accessory proteins. Here, we demonstrated for the first time that PDCoV accessory protein NS6 antagonizes IFN-β production by interacting with RIG-I and MDA5 to impede their association with double-stranded RNA. This is an efficient strategy of antagonizing type I IFN production by disrupting the binding of host pattern recognition receptors (PRRs) and pathogen-associated molecular patterns (PAMPs). These findings deepen our understanding of the function of accessory protein NS6, and they may direct us toward novel therapeutic targets and lead to the development of more effective vaccines against PDCoV infection.

Description: Vancomycin is a preferred antibiotic for treating Clostridium difficile infection (CDI) and has been associated with a rate of recurrence of CDI of as high as 20% in treated patients. Recent studies have suggested that berberine, an alternative medical therapy for gastroenteritis and diarrhea, exhibits several beneficial effects, including induction of anti-inflammatory responses and restoration of the intestinal barrier function. This study investigated the therapeutic effects of berberine on preventing CDI relapse and restoring the gut microbiota in a mouse model. Berberine was administered through gavage to C57BL/6 mice with established CDI-induced intestinal injury and colitis. The disease activity index (DAI), mean relative weight, histopathology scores, and levels of toxins A and B in fecal samples were measured. An Illumina sequencing-based analysis of 16S rRNA genes was used to determine the overall structural change in the microbiota in the mouse ileocecum. Berberine administration significantly promoted the restoration of the intestinal microbiota by inhibiting the expansion of members of the family Enterobacteriaceae and counteracting the side effects of vancomycin treatment. Therapy consisting of vancomycin and berberine combined prevented weight loss, improved the DAI and the histopathology scores, and effectively decreased the mortality rate. Berberine prevented CDIs from relapsing and significantly improved survival in the mouse model of CDI. Our data indicate that a combination of berberine and vancomycin is more effective than vancomycin alone for treating CDI. One of the possible mechanisms by which berberine prevents a CDI relapse is through modulation of the gut microbiota. Although this conclusion was generated in the case of the mouse model, use of the combination of vancomycin and berberine and represent a novel therapeutic approach targeting CDI.

Description: Porcine epidemic diarrhea coronavirus (PEDV) has significantly damaged America's pork industry. Here we investigate the receptor usage and cell entry of PEDV. PEDV recognizes protein receptor aminopeptidase N from pig and human and sugar coreceptor N -acetylneuraminic acid. Moreover, PEDV infects cells from pig, human, monkey, and bat. These results support the idea of bats as an evolutionary origin for PEDV, implicate PEDV as a potential threat to other species, and suggest antiviral strategies to control its spread.

Description: Porcine epidemic diarrhea virus (PEDV) is an enteropathogenic coronavirus causing lethal watery diarrhea in piglets. Since 2010, a PEDV variant has spread rapidly in China, and it emerged in the United States in 2013, posing significant economic and public health concerns. The ability to circumvent the interferon (IFN) antiviral response, as suggested for PEDV, promotes viral survival and regulates pathogenesis of PEDV infections, but the underlying mechanisms remain obscure. Here, we show that PEDV-encoded 3C-like protease, nsp5, is an IFN antagonist that proteolytically cleaves the nuclear transcription factor kappa B (NF-B) essential modulator (NEMO), an essential adaptor bridging interferon-regulatory factor and NF-B activation. NEMO is cleaved at glutamine 231 (Q231) by PEDV, and this cleavage impaired the ability of NEMO to activate downstream IFN production and to act as a signaling adaptor of the RIG-I/MDA5 pathway. Mutations specifically disrupting the cysteine protease activity of PEDV nsp5 abrogated NEMO cleavage and the inhibition of IFN induction. Structural analysis suggests that several key residues outside the catalytic sites of PEDV nsp5 probably impact NEMO cleavage by modulating potential interactions of nsp5 with their substrates. These data show that PEDV nsp5 disrupts type I IFN signaling by cleaving NEMO. Previously, we and others demonstrated that NEMO is also cleaved by 3C or 3C-like proteinases of picornavirus and artertivirus. Thus, NEMO probably represents a prime target for 3C or 3C-like proteinases of different viruses. IMPORTANCE The continued emergence and reemergence of porcine epidemic diarrhea virus (PEDV) underscore the importance of studying how this virus manipulates the immune responses of its hosts. During coevolution with its hosts, PEDV has acquired mechanisms to subvert host innate immune responses for its survival advantage. At least two proteins encoded by PEDV have been identified as interferon (IFN) antagonists, papain-like protease (PLP) and N protein. Here, we report that the PEDV nsp5 gene, which encodes the 3C-like protease of PEDV, is another IFN antagonist. Mechanistically, the cysteine protease activity of PEDV nsp5 mediates proteolysis of NEMO, the key adaptor for IFN synthesis, and NEMO is cleaved at glutamine 231 (Q231). The new molecular details and determinants impacting NEMO scission by PEDV nsp5 delineated in this study are fundamental to our understanding of critical virus-host interactions that determine PEDV pathogenesis.

Description: Porcine reproductive and respiratory syndrome virus (PRRSV) RNA endoribonuclease nsp11 belongs to the XendoU superfamily and plays a crucial role in arterivirus replication. Here, we report the first crystal structure of the arterivirus nsp11 protein from PRRSV, which exhibits a unique structure and assembles into an asymmetric dimer whose structure is completely different from the hexameric structure of coronavirus nsp15. However, the structures of the PRRSV nsp11 and coronavirus nsp15 catalytic domains were perfectly superimposed, especially in the "active site loop" (His129 to His144) and "supporting loop" (Val162 to Thr179) regions. Importantly, our biochemical data demonstrated that PRRSV nsp11 exists mainly as a dimer in solution. Mutations of the major dimerization site determinants (Ser74 and Phe76) in the dimerization interface destabilized the dimer in solution and severely diminished endoribonuclease activity, indicating that the dimer is the biologically functional unit. In the dimeric structure, the active site loop and supporting loop are packed against one another and stabilized by monomer-monomer interactions. These findings may help elucidate the mechanism underlying arterivirus replication and may represent great potential for the development of antiviral drugs. IMPORTANCE Porcine reproductive and respiratory syndrome virus (PRRSV) is a member of the family Arteriviridae , order Nidovirales . PRRSV is a major agent of respiratory diseases in pigs, causing tremendous economic losses to the swine industry worldwide. The PRRSV nsp11 endoribonuclease plays a vital role in arterivirus replication, but its precise roles and mechanisms of action are poorly understood. Here, we report the first dimeric structure of the arterivirus nsp11 from PRRSV at 2.75-Å resolution. Structural and biochemical experiments demonstrated that nsp11 exists mainly as a dimer in solution and that nsp11 may be fully active as a dimer. Mutagenesis and structural analysis revealed NendoU active site residues, which are conserved throughout the order Nidovirales (families Arteriviridae and Coronaviridae ) and the major determinants of dimerization (Ser74 and Phe76) in Arteriviridae . Importantly, these findings may provide a new structural basis for antiviral drug development.

Description: Porcine deltacoronavirus (PDCoV) is an emerging swine enteropathogenic coronavirus. The first outbreak of PDCoV was announced from the United States in 2014, followed by reports in Asia. The nonstructural protein nsp5 is a 3C-like protease of coronavirus, and our previous study showed that PDCoV nsp5 inhibits type I interferon (IFN) production. In this study, we found that PDCoV nsp5 significantly inhibited IFN-stimulated response element (ISRE) promoter activity and transcription of IFN-stimulated genes (ISGs), suggesting that PDCoV nsp5 also suppresses IFN signaling. Detailed analysis showed that nsp5 cleaved signal transducer and activator of transcription 2 (STAT2) but not Janus kinase 1 (JAK1), tyrosine kinase 2 (TYK2), STAT1, and interferon regulatory factor 9 (IRF9), key molecules of the JAK-STAT pathway. STAT2 cleavage was dependent on the protease activity of nsp5. Interestingly, nsp5 cleaved STAT2 at two sites, glutamine 685 (Q685) and Q758, and similar cleavage was observed in PDCoV-infected cells. As expected, cleaved STAT2 impaired the ability to induce ISGs, demonstrating that STAT2 cleavage is an important mechanism utilized by PDCoV nsp5 to antagonize IFN signaling. We also discussed the substrate selection and binding mode of PDCoV nsp5 by homologous modeling of PDCoV nsp5 with the two cleaved peptide substrates. The results of our study demonstrate that PDCoV nsp5 antagonizes type I IFN signaling by cleaving STAT2 and provides structural insights for comprehending the cleavage mechanism of PDCoV nsp5, revealing a potential new function for PDCoV nsp5 in type I IFN signaling. IMPORTANCE The 3C-like protease encoded by nsp5 is a major protease of coronaviruses; thus, it is an attractive target for development of anticoronavirus drugs. Previous studies have revealed that the 3C-like protease of coronaviruses, including PDCoV and porcine epidemic diarrhea virus (PEDV), antagonizes type I IFN production by targeting the NF-B essential modulator (NEMO). Here, for the first time, we demonstrate that overexpression of PDCoV nsp5 also antagonizes IFN signaling by cleaving STAT2, an essential component of transcription factor complex ISGF3, and that PDCoV infection reduces the levels of STAT2, which may affect the innate immune response.