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Group A rotavirus (RVA), one of the leading pathogens causing severe acute gastroenteritis in children and a wide variety of young animals worldwide, induces apoptosis upon infecting cells. Though RVA-induced apoptosis mediated via the dual modulation of its NSP4 and NSP1 proteins is relatively well studied, the nature and signaling pathway(s) involved in RVA-induced necroptosis are yet to be fully elucidated. Here, we demonstrate the nature of RVA-induced necroptosis, the signaling cascade involved, and correlation with RVA-induced apoptosis. Infection with the bovine NCDV and human DS-1 RVA strains was shown to activate receptor-interacting protein kinase 1 (RIPK1), RIPK3, and mixed-lineage kinase domain-like protein (MLKL), the key necroptosis molecules in virus-infected cells. Using an immunoprecipitation assay, RIPK1 was found to bind phosphorylated RIPK3 (pRIPK3) and pMLKL. pMLKL, the major executioner molecule in the necroptotic pathway, was translocated to the plasma membrane of RVA-infected cells to puncture the cell membrane. Interestingly, transfection of RVA NSP4 also induced necroptosis through the RIPK1/RIPK3/MLKL necroptosis pathway. Blockage of each key necroptosis molecule in the RVA-infected or NSP4-transfected cells resulted in decreased necroptosis but increased cell viability and apoptosis, thereby resulting in decreased viral yields in the RVA-infected cells. In contrast, suppression of RVA-induced apoptosis increased necroptosis and virus yields. Our findings suggest that RVA NSP4 also induces necroptosis via the RIPK1/RIPK3/MLKL necroptosis pathway. Moreover, necroptosis and apoptosis—which have proviral and antiviral effects, respectively—exhibited cross talk in RVA-infected cells. These findings significantly increase our understanding of the nature of RVA-induced necroptosis and the cross talk between RVA-induced necroptosis and apoptosis. IMPORTANCE Viral infection usually culminates in cell death through apoptosis, necroptosis, and, rarely, pyroptosis. Necroptosis is a form of programmed necrosis that is mediated by signaling complexes of the receptor-interacting protein kinase 1 (RIPK1), RIPK3, and mixed-lineage kinase domain-like protein (MLKL). Although apoptosis induction by rotavirus and its NSP4 protein is well known, rotavirus-induced necroptosis is not fully understood. Here, we demonstrate that rotavirus and also its NSP4 protein can induce necroptosis in cultured cells through activation of the RIPK1/RIPK3/MLKL necroptosis pathway. Moreover, rotavirus-induced necroptosis and apoptosis have opposite effects on viral yield, i.e., they function as proviral and antiviral processes, respectively, and counterbalance each other in rotavirus-infected cells. Our findings provide important insights for understanding the nature of rotavirus-induced necroptosis and the development of novel therapeutic strategies against infection with rotavirus and other RNA viruses.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of the COVID-19 pandemic. One protein known to play a critical role in the coronavirus life cycle is nonstructural protein 1 (nsp1)., The periodic emergence of novel coronaviruses (CoVs) represents an ongoing public health concern with significant health and financial burdens worldwide. The most recent occurrence originated in the city of Wuhan, China, where a novel coronavirus (severe acute respiratory syndrome coronavirus 2 [SARS-CoV-2]) emerged causing severe respiratory illness and pneumonia. The continual emergence of novel coronaviruses underscores the importance of developing effective vaccines as well as novel therapeutic options that target either viral functions or host factors recruited to support coronavirus replication. The CoV nonstructural protein 1 (nsp1) has been shown to promote cellular mRNA degradation, block host cell translation, and inhibit the innate immune response to virus infection. Interestingly, deletion of the nsp1-coding region in infectious clones prevented the virus from productively infecting cultured cells. Because of nsp1’s importance in the CoV life cycle, it has been highlighted as a viable target for both antiviral therapy and vaccine development. However, the fundamental molecular and structural mechanisms that underlie nsp1 function remain poorly understood, despite its critical role in the viral life cycle. Here, we report the high-resolution crystal structure of the amino globular portion of SARS-CoV-2 nsp1 (residues 10 to 127) at 1.77-Å resolution. A comparison of our structure with the SARS-CoV-1 nsp1 structure reveals how mutations alter the conformation of flexible loops, inducing the formation of novel secondary structural elements and new surface features. Paired with the recently published structure of the carboxyl end of nsp1 (residues 148 to 180), our results provide the groundwork for future studies focusing on SARS-CoV-2 nsp1 structure and function during the viral life cycle. IMPORTANCE Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of the COVID-19 pandemic. One protein known to play a critical role in the coronavirus life cycle is nonstructural protein 1 (nsp1). As such, it has been highlighted in numerous studies as a target for both the development of antivirals and the design of live-attenuated vaccines. Here, we report the high-resolution crystal structure of nsp1 derived from SARS-CoV-2 at 1.77-Å resolution. This structure will facilitate future studies focusing on understanding the relationship between structure and function for nsp1. In turn, understanding these structure-function relationships will allow nsp1 to be fully exploited as a target for both antiviral development and vaccine design.

Group A rotavirus (RV) is a major cause of acute gastroenteritis in infants and young children worldwide. Recently, we established an entirely plasmid-based reverse genetics system for simian RV strain SA11. Although that system was robust enough to generate reassortant RVs, including human RV gene segments, and enabled better understanding of the biological differences between animal and human RV strains, a complete reverse genetics system for human RV strains is desirable. Here, we established a plasmid-based reverse genetics system for G4P[8] human RV strain Odelia. This technology was used to generate a panel of monoreassortant viruses between human and simian RV strains for all of the 11 gene segments demonstrating full compatibility between human and simian RV strains. Furthermore, we generated recombinant viruses lacking the C-terminal region of the viral nonstructural protein NSP1 and used it to define the biological function of the interaction between NSP1 and its target protein β-transducin repeat-containing protein (β-TrCP) during viral replication. While the NSP1 truncation mutant lacking the C-terminal 13 amino acids displayed lower β-TrCP degradation activity, it replicated as efficiently as the wild-type virus. In contrast, the truncation mutant lacking the C-terminal 166 amino acids of NSP1 replicated poorly, suggesting that the C-terminal region of NSP1 plays critical roles in viral replication. The system reported here will allow generation of engineered recombinant virus harboring desired mutations, increase our understanding of the molecular biology of human RV, and facilitate development of novel therapeutics and vaccines. IMPORTANCE Reverse genetics, an approach used to generate viruses from cloned cDNA, has increased our understanding of virus biology. Worldwide research led to the development of an entirely plasmid-based reverse genetics system for the simian RV laboratory strain. Although the technique allows generation of gene-modified recombinant RVs, biological differences between animal and human RVs mean that reverse genetics systems for human RV strains are still needed. Here, we describe a reverse genetics system for the high-yield human RV strain Odelia, which replicates efficiently and is suitable for in vitro molecular studies. Monoreassortant viruses between simian and human RV strains and NSP1 mutant viruses generated by the rescue system enabled study of the biological functions of viral gene segments. This human RV reverse genetics system will facilitate study of RV biology and development of vaccines and vectors.

Rotavirus is a segmented double-stranded (ds)RNA virus that causes severe gastroenteritis in young children. We have established an efficient simplified rotavirus reverse genetics (RG) system that uses eleven T7 plasmids, each expressing a unique simian SA11 (+)RNA, and a CMV support plasmid for the African swine fever virus NP868R capping enzyme. With the NP868R-based system, we generated recombinant rotavirus (rSA11/NSP3-FL-UnaG) with a genetically modified 1.5-kB segment 7 dsRNA that encodes full-length NSP3 fused to UnaG, a 139-aa green fluorescent protein (FP). Analysis of rSA11/NSP3-FL-UnaG showed that the virus replicated efficiently and was genetically stable over 10 rounds of serial passage. The NSP3-UnaG fusion product was well expressed in rSA11/NSP3-FL-UnaG-infected cells, reaching levels similar to NSP3 in wildtype rSA11-infected cells. Moreover, the NSP3-UnaG protein, like functional wildtype NSP3, formed dimersin vivo. Notably, NSP3-UnaG protein was readily detected in infected cells via live cell imaging, with intensity levels ~3-fold greater than that of the NSP1-UnaG fusion product of rSA11/NSP1-FL-UnaG. Our results indicate that FP-expressing recombinant rotaviruses can be made through manipulation of the segment 7 dsRNA without deleting or interrupting any of the twelve open reading frames of the virus. Because NSP3 is expressed at levels higher than NSP1 in infected cells, rotaviruses expressing NSP3-based FPs may be a more sensitive tool for studying rotavirus biology than rotaviruses expressing NSP1-based FPs. This is the first report of a recombinant rotavirus containing a genetically engineered segment 7 dsRNA.ImportancePrevious studies have generated recombinant rotaviruses that express fluorescent proteins (FPs) by inserting reporter genes into the NSP1 open reading frame (ORF) of genome segment 5. Unfortunately, NSP1 is expressed at low levels in infected cells, making viruses expressing FP-fused NSP1 less than ideal probes of rotavirus biology. Moreover, FPs were inserted into segment 5 in such a way as to compromise NSP1, an interferon antagonist affecting viral growth and pathogenesis. We have identified an alternative approach for generating rotaviruses expressing FPs, one relying on fusing the reporter gene to the NSP3 ORF of genome segment 7. This was accomplished without interrupting any of the viral ORFs, yielding recombinant viruses likely expressing the complete set of functional viral proteins. Given that NSP3 is made at moderate levels in infected cells, rotavirus encoding NSP3-based FPs should be more sensitive probes of viral infection than rotaviruses encoding NSP1-based FPs.

Rotavirus is an important cause of diarrheal disease in young mammals. Rotavirus species A (RVA) causes most human rotavirus diarrheal disease and primarily affects infants and young children. Rotavirus species B (RVB) has been associated with sporadic outbreaks of human adult diarrheal disease. RVA and RVB are predicted to encode mostly homologous proteins but differ significantly in the proteins encoded by the NSP1 gene. In the case of RVB, the NSP1 gene encodes two putative protein products of unknown function, NSP1-1 and NSP1-2. We demonstrate that human RVB NSP1-1 mediates syncytium formation in cultured human cells. Based on sequence alignment, NSP1-1 proteins from species B, G, and I contain features consistent with fusion-associated small transmembrane (FAST) proteins, which have previously been identified in other genera of the Reoviridae family. Like some other FAST proteins, RVB NSP1-1 is predicted to have an N-terminal myristoyl modification. Addition of an N-terminal FLAG peptide disrupts NSP1-1-mediated fusion. NSP1-1 from a human RVB mediates fusion of human cells but not hamster cells and, thus, may serve as a species tropism determinant. NSP1-1 also can enhance RVA replication in human cells, both in single-cycle infection studies and during a multicycle time course in the presence of fetal bovine serum, which inhibits rotavirus spread. These findings suggest potential yet untested roles for NSP1-1 in RVB species tropism, immune evasion, and pathogenesis. IMPORTANCE While species A rotavirus is commonly associated with diarrheal disease in young children, species B rotavirus has caused sporadic outbreaks of adult diarrheal disease. A major genetic difference between species A and B rotaviruses is the NSP1 gene, which encodes two proteins for species B rotavirus. We demonstrate that the smaller of these proteins, NSP1-1, can mediate fusion of cultured human cells. Comparison with viral proteins of similar function provides insight into NSP1-1 domain organization and fusion mechanism. These comparisons suggest that there is a fatty acid modification at the amino terminus of the protein, and our results show that an intact amino terminus is required for NSP1-1-mediated fusion. NSP1-1 from a human virus mediates fusion of human cells, but not hamster cells, and enhances species A rotavirus replication in culture. These findings suggest potential, but currently untested, roles for NSP1-1 in RVB host species tropism, immune evasion, and pathogenesis.

Alphaviruses have been the cause of both localized outbreaks and large epidemics of severe disease. Currently, there are no strategies or vaccines which are either safe or effective for preventing alphaviral infection or treating alphaviral disease. This deficit of viable therapeutics highlights the need to better understand the mechanisms behind alphaviral infection in order to develop novel antiviral strategies for treatment of alphaviral disease. In particular, this report details a previously uncharacterized aspect of the alphaviral life cycle: the importance of noncapped genomic viral RNAs for alphaviral infection. This offers new insights into the mechanisms of alphaviral replication and the impact of the noncapped genomic RNAs on viral packaging., Alphaviruses are arthropod-borne RNA viruses that are capable of causing severe disease and are a significant burden to public health. Alphaviral replication results in the production of both capped and noncapped viral genomic RNAs (ncgRNAs), which are packaged into virions during infections of vertebrate and invertebrate cells. However, the roles that the ncgRNAs play during alphaviral infection have yet to be exhaustively characterized. Here, the importance of the ncgRNAs to alphaviral infection was assessed by using mutations of the nsP1 protein of Sindbis virus (SINV), which altered the synthesis of the ncgRNAs during infection by modulating the protein’s capping efficiency. Specifically, point mutations at residues Y286A and N376A decreased capping efficiency whereas a point mutation at D355A increased the capping efficiency of the SINV genomic RNA during genuine viral infection. Viral growth kinetics levels were significantly reduced for the D355A mutant relative to wild-type infection, whereas the Y286A and N376A mutants showed modest decreases in growth kinetics. Overall genomic translation and nonstructural protein accumulation were found to correlate with increases and decreases in capping efficiency. However, genomic, minus-strand, and subgenomic viral RNA synthesis were largely unaffected by the modulation of alphaviral capping activity. In addition, translation of the subgenomic alphaviral RNA (vRNA) was found not to be impacted by changes in capping efficiency. The mechanism by which the decreased presence of ncgRNAs reduced viral growth kinetics levels operated through the impaired production of viral particles. Collectively, these data illustrate the importance of ncgRNAs to viral infection and suggest that they play an integral role in the production of viral progeny.

Middle East respiratory syndrome coronavirus (MERS-CoV) nsp1 suppresses host gene expression in expressed cells by inhibiting translation and inducing endonucleolytic cleavage of host mRNAs, the latter of which leads to mRNA decay. We examined the biological functions of nsp1 in infected cells and its role in virus replication by using wild-type MERS-CoV and two mutant viruses with specific mutations in the nsp1; one mutant lacked both biological functions, while the other lacked the RNA cleavage function but retained the translation inhibition function. In Vero cells, all three viruses replicated efficiently with similar replication kinetics, while wild-type virus induced stronger host translational suppression and host mRNA degradation than the mutants, demonstrating that nsp1 suppressed host gene expression in infected cells. The mutant viruses replicated less efficiently than wild-type virus in Huh-7 cells, HeLa-derived cells, and 293-derived cells, the latter two of which stably expressed a viral receptor protein. In 293-derived cells, the three viruses accumulated similar levels of nsp1 and major viral structural proteins and did not induce IFN-β and IFN-λ mRNAs; however, both mutants were unable to generate intracellular virus particles as efficiently as wild-type virus, leading to inefficient production of infectious viruses. These data strongly suggest that the endonucleolytic RNA cleavage function of the nsp1 promoted MERS-CoV assembly and/or budding in a 293-derived cell line. MERS-CoV nsp1 represents the first CoV gene 1 protein that plays an important role in virus assembly/budding and is the first identified viral protein whose RNA cleavage-inducing function promotes virus assembly/budding. IMPORTANCE MERS-CoV represents a high public health threat. Because CoV nsp1 is a major viral virulence factor, uncovering the biological functions of MERS-CoV nsp1 could contribute to our understanding of MERS-CoV pathogenicity and spur development of medical countermeasures. Expressed MERS-CoV nsp1 suppresses host gene expression, but its biological functions for virus replication and effects on host gene expression in infected cells are largely unexplored. We found that nsp1 suppressed host gene expression in infected cells. Our data further demonstrated that nsp1, which was not detected in virus particles, promoted virus assembly or budding in a 293-derived cell line, leading to efficient virus replication. These data suggest that nsp1 plays an important role in MERS-CoV replication and possibly affects virus-induced diseases by promoting virus particle production in infected hosts. Our data, which uncovered an unexpected novel biological function of nsp1 in virus replication, contribute to further understanding of the MERS-CoV replication strategies.

Infection with Ross River virus (RRV) causes debilitating polyarthritis and arthralgia in individuals. Alphaviruses are highly sensitive to type I interferon (IFN). Mutations at the conserved P3 position of the cleavage site between nonstructural protein 1 (nsP1) and nsP2 (1/2 site) modulate type I IFN induction for both RRV and Sindbis virus (SINV). We constructed and characterized RRV-T48A534V, a mutant harboring an A534V substitution in the P1 position of the 1/2 site, and compared it to parental RRV-T48 and to RRV-T48A532V, SINVI538 and SINVT538 harboring different substitutions in the same region. A534V substitution resulted in impaired processing of RRV nonstructural polyprotein and in elevated production of replicase-generated pathogen-associated molecular pattern (PAMP) RNAs that induce expression of type I IFN. Both A532V and A534V substitutions affected synthesis of viral RNAs, though the effects of these closely located mutations were drastically different affecting mostly either the viral negative-strand RNA or genomic and subgenomic RNA levels, respectively. Synthesis of PAMP RNAs was also observed for SINV replicase, and it was increased by I538T substitution. In comparison to RRV-T48, RRV-T48A534V was attenuated in vitro and in vivo. Interestingly, when type I IFN-deficient cells and type I IFN receptor-deficient mice were infected with RRV-T48 or RRV-T48A534V, differences between these viruses were no longer apparent. Compared to RRV-T48, RRV-T48A534V infection was associated with increased upregulation of type I IFN signaling proteins. We demonstrate novel mechanisms by which the A534V mutation affect viral nonstructural polyprotein processing that can impact PAMP RNA production, type I IFN induction/sensitivity, and disease., IMPORTANCE This study gives further insight into mechanisms of type I IFN modulation by the medically important alphaviruses Ross River virus (RRV) and Sindbis virus (SINV). By characterizing attenuated RRV mutants, the crucial role of amino acid residues in P1 and P3 positions (the first and third amino acid residues preceding the scissile bond) of the cleavage site between nsP1 and nsP2 regions was highlighted. The study uncovers a unique relationship between alphavirus nonstructural polyprotein processing, RNA replication, production of different types of pathogen-associated molecular pattern (PAMP) RNAs, type I IFN induction, and disease pathogenesis. This study also highlights the importance of the host innate immune response in RRV infections. The viral determinants of type I IFN modulation provide potential drug targets for clinical treatment of alphaviral disease and offer new approaches for rational attenuation of alphaviruses for construction of vaccine candidates.

The A7(74) strain of Semliki Forest virus (SFV; genus Alphavirus ) is avirulent in adult mice, while the L10 strain is virulent in mice of all ages. It has been previously demonstrated that this phenotypic difference is associated with nonstructural protein 3 (nsP3). Consensus clones of L10 (designated SFV6) and A7(74) (designated A774wt) were used to construct a panel of recombinant viruses. The insertion of nsP3 from A774wt into the SFV6 backbone had a minor effect on the virulence of the resulting recombinant virus. Conversely, insertion of nsP3 from SFV6 into the A774wt backbone or replacement of A774wt nsP3 with two copies of nsP3 from SFV6 resulted in virulent viruses. Unexpectedly, duplication of nsP3-encoding sequences also resulted in elevated levels of nsP4, revealing that nsP3 is involved in the stabilization of nsP4. Interestingly, replacement of nsP3 of SFV6 with that of A774wt resulted in a virulent virus; the virulence of this recombinant was strongly reduced by functionally coupled substitutions for amino acid residues 534 (P4 position of the cleavage site between nsP1 and nsP2) and 1052 (S4 subsite residue of nsP2 protease) in the nonstructural polyprotein. Pulse-chase experiments revealed that A774wt and avirulent recombinant virus were characterized by increased processing speed of the cleavage site between nsP1 and nsP2. A His534-to-Arg substitution specifically activated this cleavage, while a Val1052-to-Glu substitution compensated for this effect by reducing the basal protease activity of nsP2. These findings provide a link between nonstructural polyprotein processing and the virulence of SFV. IMPORTANCE SFV infection of mice provides a well-characterized model to study viral encephalitis. SFV also serves as a model for studies of alphavirus molecular biology and host-pathogen interactions. Thus far, the genetic basis of different properties of SFV strains has been studied using molecular clones, which often contain mistakes originating from standard cDNA synthesis and cloning procedures. Here, for the first time, consensus clones of SFV strains were used to map virulence determinants. Existing data on the importance of nsP3 for virulent phenotypes were confirmed, another determinant of neurovirulence and its molecular basis was characterized, and a novel function of nsP3 was identified. These findings provide links between the molecular biology of SFV and its biological properties and significantly increase our understanding of the basis of alphavirus-induced pathology. In addition, the usefulness of consensus clones as tools for studies of alphaviruses was demonstrated.

Mouse hepatitis virus (MHV) isolates JHM.WU and JHM.SD promote severe central nervous system disease. However, while JHM.WU replicates robustly and induces hepatitis, JHM.SD fails to replicate or induce pathology in the liver. These two JHM variants encode homologous proteins with few polymorphisms, and little is known about which viral proteins(s) is responsible for the liver tropism of JHM.WU. We constructed reverse genetic systems for JHM.SD and JHM.WU and, utilizing these full-length cDNA clones, constructed chimeric viruses and mapped the virulence factors involved in liver tropism. Exchanging the spike proteins of the two viruses neither increased replication of JHM.SD in the liver nor attenuated JHM.WU. By further mapping, we found that polymorphisms in JHM.WU structural protein M and nonstructural replicase proteins nsp1 and nsp13 are essential for liver pathogenesis. M protein and nsp13, the helicase, of JHM.WU are required for efficient replication in vitro and in the liver in vivo . The JHM.SD nsp1 protein contains a K194R substitution of Lys194, a residue conserved among all other MHV strains. The K194R polymorphism has no effect on in vitro replication but influences hepatotropism, and introduction of R194K into JHM.SD promotes replication in the liver. Conversely, a K194R substitution in nsp1 of JHM.WU or A59, another hepatotropic strain, significantly attenuates replication of each strain in the liver and increases IFN-β expression in macrophages in culture. Our data indicate that both structural and nonstructural proteins contribute to MHV liver pathogenesis and support previous reports that nsp1 is a Betacoronavirus virulence factor. IMPORTANCE The Betacoronavirus genus includes human pathogens, some of which cause severe respiratory disease. The spread of severe acute respiratory syndrome coronavirus (SARS-CoV) and Middle East respiratory syndrome coronavirus (MERS-CoV) into human populations demonstrates the zoonotic potential of emerging coronaviruses, and there are currently no vaccines or effective antivirals for human coronaviruses. Thus, it is important to understand the virus-host interaction that regulates coronavirus pathogenesis. Murine coronavirus infection of mice provides a useful model for the study of coronavirus-host interactions, including the determinants of tropism and virulence. We found that very small changes in coronavirus proteins can profoundly affect tropism and virulence. Furthermore, the hepatotropism of MHV-JHM depends not on the spike protein and viral entry but rather on a combination of the structural protein M and nonstructural replicase-associated proteins nsp1 and nsp13, which are conserved among betacoronaviruses. Understanding virulence determinants will aid in the design of vaccines and antiviral strategies.

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.

Stress granules (SGs) are protein-mRNA aggregates that are formed in response to environmental stresses, resulting in translational inhibition. SGs are generally believed to play an antiviral role and are manipulated by many viruses, including various alphaviruses. GTPase-activating protein (SH3 domain)-binding protein 1 (G3BP1) is a key component and commonly used marker of SGs. Its homolog G3BP2 is a less extensively studied SG component. Here, we demonstrate that Chikungunya virus (CHIKV) infection induces cytoplasmic G3BP1- and G3BP2-containing granules that differ from bona fide SGs in terms of morphology, composition, and behavior. For several Old World alphaviruses it has been shown that nonstructural protein 3 (nsP3) interacts with G3BPs, presumably to inhibit SG formation, and we have confirmed this interaction in CHIKV-infected cells. Surprisingly, CHIKV also relied on G3BPs for efficient replication, as simultaneous depletion of G3BP1 and G3BP2 reduced viral RNA levels, CHIKV protein expression, and viral progeny titers. The G3BPs colocalized with CHIKV nsP2 and nsP3 in cytoplasmic foci, but no colocalization with nsP1, nsP4, or dsRNA was observed. Furthermore, G3BPs could not be detected in a cellular fraction enriched for CHIKV replication/transcription complexes, suggesting that they are not directly involved in CHIKV RNA synthesis. Depletion of G3BPs did not affect viral entry, translation of incoming genomes, or nonstructural polyprotein processing but resulted in severely reduced levels of negative-stranded (and consequently also positive-stranded) RNA. This suggests a role for the G3BPs in the switch from translation to genome amplification, although the exact mechanism by which they act remains to be explored. IMPORTANCE Chikungunya virus (CHIKV) causes a severe polyarthritis that has affected millions of people since its reemergence in 2004. The lack of approved vaccines or therapeutic options and the ongoing explosive outbreak in the Caribbean underline the importance of better understanding CHIKV replication. Stress granules (SGs) are cytoplasmic protein-mRNA aggregates formed in response to various stresses, including viral infection. The RNA-binding proteins G3BP1 and G3BP2 are essential SG components. SG formation and the resulting translational inhibition are generally considered an antiviral response, and many viruses manipulate or block this process. Late in infection, we and others have observed CHIKV nonstructural protein 3 in cytoplasmic G3BP1- and G3BP2-containing granules. These virally induced foci differed from true SGs and did not appear to represent replication complexes. Surprisingly, we found that G3BP1 and G3BP2 were also needed for efficient CHIKV replication, likely by facilitating the switch from translation to genome amplification early in infection.

Here, we disclose the complete genomic sequence of a rare rotavirus group A G2P[4]-I2-R2-C2-M2-A15-N2-T2-E2-H2 strain detected in a fecal specimen from a rotaviral acute gastroenteritis patient who had previously received the Rotarix vaccine.

The use of overlapping open reading frames (ORFs) to synthesize more than one unique protein from a single mRNA has been described for several viruses. Segment 11 of the rotavirus genome encodes two nonstructural proteins, NSP5 and NSP6. The NSP6 ORF is present in the vast majority of rotavirus strains, and therefore the NSP6 protein would be expected to have a function in viral replication. However, there is no direct evidence of its function or requirement in the viral replication cycle yet. Here, taking advantage of a recently established plasmid-only-based reverse genetics system that allows rescue of recombinant rotaviruses entirely from cloned cDNAs, we generated NSP6-deficient viruses to directly address its significance in the viral replication cycle. Viable recombinant NSP6-deficient viruses could be engineered. Single-step growth curves and plaque formation of the NSP6-deficient viruses confirmed that NSP6 expression is of limited significance for RVA replication in cell culture, although the NSP6 protein seemed to promote efficient virus growth. IMPORTANCE Rotavirus is one of the most important pathogens of severe diarrhea in young children worldwide. The rotavirus genome, consisting of 11 segments of double-stranded RNA, encodes six structural proteins (VP1 to VP4, VP6, and VP7) and six nonstructural proteins (NSP1 to NSP6). Although specific functions have been ascribed to each of the 12 viral proteins, the role of NSP6 in the viral replication cycle remains unknown. In this study, we demonstrated that the NSP6 protein is not essential for viral replication in cell culture by using a recently developed plasmid-only-based reverse genetics system. This reverse genetics approach will be successfully applied to answer questions of great interest regarding the roles of rotaviral proteins in replication and pathogenicity, which can hardly be addressed by conventional approaches.

Porcine reproductive and respiratory syndrome, caused by porcine reproductive and respiratory syndrome virus (PRRSV), is a panzootic disease that is one of the most economically costly diseases to the swine industry. A key aspect of PRRSV virulence is that the virus suppresses the innate immune response and induces persistent infection, although the underlying mechanisms are not well understood. The dendritic cell (DC) marker CD83 belongs to the immunoglobulin superfamily and is associated with DC activation and immunosuppression of T cell proliferation when expressed as soluble CD83 (sCD83). In this study, we show that PRRSV infection strongly stimulates CD83 expression in porcine monocyte-derived DCs (MoDCs) and that the nucleocapsid (N) protein and nonstructural protein 10 (nsp10) of PRRSV enhance CD83 promoter activity via the NF-κB and Sp1 signaling pathways. R43A and K44A amino acid substitution mutants of the N protein suppress the N protein-mediated increase of CD83 promoter activity. Similarly, P192-5A and G214-3A mutants of nsp10 (with 5 and 3 alanine substitutions beginning at residues P192 and G214, respectively) abolish the nsp10-mediated induction of the CD83 promoter. Using reverse genetics, four mutant viruses (rR43A, rK44A, rP192-5A, and rG214-3A) and four revertants [rR43A(R), rK44A(R), rP192-5A(R), and rG214-3A(R)] were generated. Decreased induction of CD83 in MoDCs was observed after infection by mutants rR43A, rK44A, rP192-5A, and rG214-3A, in contrast to the results obtained using rR43A(R), rK44A(R), rP192-5A(R), and rG214-3A(R). These findings suggest that PRRSV N and nsp10 play important roles in modulating CD83 signaling and shed light on the mechanism by which PRRSV modulates host immunity. IMPORTANCE Porcine reproductive and respiratory syndrome virus (PRRSV) is one of the most economically costly pathogens affecting the swine industry. It is unclear how PRRSV inhibits the host's immune response and induces persistent infection. The dendritic cell (DC) marker CD83 belongs to the immunoglobulin superfamily and has previously been associated with DC activation and immunosuppression of T cell proliferation and differentiation when expressed as soluble CD83 (sCD83). In this study, we found that PRRSV infection induces sCD83 expression in porcine MoDCs via the NF-κB and Sp1 signaling pathways. The viral nucleocapsid protein, nonstructural protein 1 (nsp1), and nsp10 were shown to enhance CD83 promoter activity. Amino acids R43 and K44 of the N protein, as well as residues 192 to 196 (P192-5) and 214 to 216 (G214-3) of nsp10, play important roles in CD83 promoter activation. These findings provide new insights into the molecular mechanism of immune suppression by PRRSV.

The rotavirus nonstructural protein NSP1 repurposes cullin-RING E3 ubiquitin ligases (CRLs) to antagonize innate immune responses. By functioning as substrate adaptors of hijacked CRLs, NSP1 causes ubiquitination and proteasomal degradation of host proteins that are essential for expression of interferon (IFN) and IFN-stimulated gene products. The target of most human and porcine rotaviruses is the β-transducin repeat-containing protein (β-TrCP), a regulator of NF-κB activation. β-TrCP recognizes a phosphorylated degron (DSGΦXS) present in the inhibitor of NF-κB (IκB); phosphorylation of the IκB degron is mediated by IκB kinase (IKK). Because NSP1 contains a C-terminal IκB-like degron (ILD; DSGXS) that recruits β-TrCP, we investigated whether the NSP1 ILD is similarly activated by phosphorylation and whether this modification is required to trigger the incorporation of NSP1 into CRLs. Based on mutagenesis and phosphatase treatment studies, we found that both serine residues of the NSP1 ILD are phosphorylated, a pattern mimicking phosphorylation of IκB. A three-pronged approach using small-molecule inhibitors, small interfering RNAs, and mutagenesis demonstrated that NSP1 phosphorylation is mediated by the constitutively active casein kinase II (CKII), rather than IKK. In coimmunoprecipitation assays, we found that this modification was essential for NSP1 recruitment of β-TrCP and induced changes involving the NSP1 N-terminal RING motif that allowed formation of Cul3-NSP1 complexes. Taken together, our results indicate a highly regulated stepwise process in the formation of NSP1-Cul3 CRLs that is initiated by CKII phosphorylation of NSP1, followed by NSP1 recruitment of β-TrCP and ending with incorporation of the NSP1–β-TrCP complex into the CRL via interactions dependent on the highly conserved NSP1 RING motif. IMPORTANCE Rotavirus is a segmented double-stranded RNA virus that causes severe diarrhea in young children. A primary mechanism used by the virus to inhibit host innate immune responses is to hijack cellular cullin-RING E3 ubiquitin ligases (CRLs) and redirect their targeting activity to the degradation of cellular proteins crucial for interferon expression. This task is accomplished through the rotavirus nonstructural protein NSP1, which incorporates itself into a CRL and serves as a substrate recognition subunit. The substrate recognized by the NSP1 of many human and porcine rotaviruses is β-TrCP, a protein that regulates the transcription factor NF-κB. In this study, we show that formation of NSP1 CRLs is a highly regulated stepwise process initiated by CKII phosphorylation of the β-TrCP recognition motif in NSP1. This modification triggers recruitment of the β-TrCP substrate and induces subsequent changes in a highly conserved NSP1 RING domain that allow anchoring of the NSP1–β-TrCP complex to a cullin scaffold.

Rotavirus (RV) replicates efficiently in intestinal epithelial cells (IECs) in vivo despite the activation of a local host interferon (IFN) response. Previously, we demonstrated that homologous RV efficiently inhibits IFN induction in single infected and bystander villous IECs in vivo . Paradoxically, RV also induces significant type I IFN expression in the intestinal hematopoietic cell compartment in a relatively replication-independent manner. This suggests that RV replication and spread in IECs must occur despite exogenous stimulation of the STAT1-mediated IFN signaling pathway. Here we report that RV inhibits IFN-mediated STAT1 tyrosine 701 phosphorylation in human IECs in vitro and identify RV NSP1 as a direct inhibitor of the pathway. Infection of human HT29 IECs with simian (RRV) or porcine (SB1A or OSU) RV strains, which inhibit IFN induction by targeting either IFN regulatory factor 3 (IRF3) or NF-κB, respectively, resulted in similar regulation of IFN secretion. By flow cytometric analysis at early times during infection, neither RRV nor SB1A effectively inhibited the activation of Y701-STAT1 in response to exogenously added IFN. However, at later times during infection, both RV strains efficiently inhibited IFN-mediated STAT1 activation within virus-infected cells, indicating that RV encodes inhibitors of IFN signaling targeting STAT1 phosphorylation. Expression of RV NSP1 in the absence of other viral proteins resulted in blockage of exogenous IFN-mediated STAT1 phosphorylation, and this function was conserved in NSP1 from simian, bovine, and murine RV strains. Analysis of NSP1 determinants responsible for the inhibition of IFN induction and signaling pathways revealed that these determinants are encoded on discrete domains of NSP1. Finally, we observed that at later times during infection with SB1A, there was almost complete inhibition of IFN-mediated Y701-STAT1 in bystander cells staining negative for viral antigen. This property segregated with the NSP1 gene and was observed in a simian SA11 monoreassortant that encoded porcine OSU NSP1 but not in wild-type SA11 or a reassortant encoding simian RRV NSP1.

p53, a member of the innate immune system, is triggered under stress to induce cell growth arrest and apoptosis. Thus, p53 is an important target for viruses, as efficient infection depends on modulation of the host apoptotic machinery. This study focuses on how rotaviruses manipulate intricate p53 signaling for their advantage. Analysis of p53 expression revealed degradation of p53 during initial stages of rotavirus infection. However, in nonstructural protein-1 (NSP1) mutant strain A5-16, p53 degradation was not observed, suggesting a role of NSP1 in this process. This function of NSP1 was independent of its interferon or phosphatidylinositol 3-kinase (PI3K)/AKT modulation activity since p53 degradation was observed in Vero cells as well as in the presence of PI3K inhibitor. p53 transcript levels remained the same in SA11-infected cells (at 2 to 14 h postinfection), but p53 protein was stabilized only in the presence of MG132, suggesting a posttranslational process. NSP1 interacted with the DNA binding domain of p53, resulting in ubiquitination and proteasomal degradation of p53. Degradation of p53 during initial stages of infection inhibited apoptosis, as the proapoptotic genes PUMA and Bax were downregulated. During late viral infection, when progeny dissemination is the main objective, the NSP1-p53 interaction was diminished, resulting in restoration of the p53 level, with initiation of proapoptotic signaling ensuing. Overall results highlight the multiple strategies evolved by NSP1 to combat the host immune response.

Our previous study had reported on the interaction of rotavirus NSP1 with cellular phosphoinositide 3-kinase (PI3K) during activation of the PI3K pathway (P. Bagchi et al., J. Virol. 84:6834–6845, 2010). In this study, we have analyzed the molecular mechanism behind this interaction. Results showed that this interaction is direct and that both α and β isomers of the PI3K regulatory subunit p85 and full-length NSP1 are important for this interaction, which results in efficient activation of the PI3K/Akt pathway during rotavirus infection.

Rotavirus is a leading cause of death due to diarrhea among young children across the globe. Despite the limited coding capacity that is characteristic of RNA viruses, rotavirus dedicates substantial resources to avoiding the host innate immune response. Among these strategies is use of the interferon antagonist protein NSP1, which targets cellular proteins required for interferon production to be degraded by the proteasome. Although numerous cellular targets have been described, there remain many questions about the mechanism of NSP1 activity and its role in promoting replication in specific host species.

Four rotavirus SA11 temperature-sensitive ( ts ) mutants and seven rotavirus RRV ts mutants, isolated at the National Institutes of Health (NIH) and not genetically characterized, were assigned to reassortment groups by pairwise crosses with the SA11 mutant group prototypes isolated and characterized at Baylor College of Medicine (BCM). Among the NIH mutants, three of the RRV mutants and all four SA11 mutants contained mutations in single reassortment groups, and four RRV mutants contained mutations in multiple groups. One NIH mutant [RRV ts K(2)] identified the previously undefined 11th reassortment group (K) expected for rotavirus. Three NIH single mutant RRV viruses, RRV ts D(7), RRV ts J(5), and RRV ts K(2), were in reassortment groups not previously mapped to genome segments. These mutants were mapped using classical genetic methods, including backcrosses to demonstrate reversion or suppression in reassortants with incongruent genotype and temperature phenotype. Once located to specific genome segments by genetic means, the mutations responsible for the ts phenotype were identified by sequencing. The reassortment group K mutant RRV ts K(2) maps to genome segment 9 and has a Thr280Ileu mutation in the capsid surface glycoprotein VP7. The group D mutant RRV ts D(7) maps to segment 5 and has a Leu140Val mutation in the nonstructural interferon (IFN) antagonist protein NSP1. The group J mutant RRV ts J(5) maps to segment 11 and has an Ala182Gly mutation affecting only the NSP5 open reading frame. Rotavirus ts mutation groups are now mapped to 9 of the 11 rotavirus genome segments. Possible segment locations of the two remaining unmapped ts mutant groups are discussed.

Rotavirus replication and virulence are strongly influenced by virus strain and host species. The rotavirus proteins VP3, VP4, VP7, NSP1, and NSP4 have all been implicated in strain and species restriction of replication; however, the mechanisms have not been fully determined. Simian (RRV) and bovine (UK) rotaviruses have distinctive replication capacities in mouse extraintestinal organs such as the biliary tract. Using reassortants between UK and RRV, we previously demonstrated that the differential replication of these viruses in mouse embryonic fibroblasts is determined by the respective NSP1 proteins, which differ substantially in their abilities to degrade interferon (IFN) regulatory factor 3 (IRF3) and suppress the type I IFN response. In this study, we used an in vivo model of rotavirus infection of mouse gallbladder with UK × RRV reassortants to study the genetic and mechanistic basis of systemic rotavirus replication. We found that the low-replication phenotype of UK in biliary tissues was conferred by UK VP4 and that the high-replication phenotype of RRV was conferred by RRV VP4 and NSP1 . Viruses with RRV VP4 entered cultured mouse cholangiocytes more efficiently than did those with UK VP4 . Reassortants with RRV VP4 and UK NSP1 genes induced high levels of expression of IRF3-dependent p54 in biliary tissues, and their replication was increased 3-fold in IFN-α/β and -γ receptor or STAT1 knockout (KO) mice compared to wild-type mice. Our data indicate that systemic rotavirus strain-specific replication in the murine biliary tract is determined by both viral entry mediated by VP4 and viral antagonism of the host innate immune response mediated by NSP1 .

In the present investigation we molecularly characterized nontypeable rotavirus strains previously identified during surveillance in New Delhi, India. The majority of strains were demonstrated to belong to genotype G1 (54.5%) or P[8] (77.8%) on the basis of nucleotide sequencing of fragments from their VP7 and VP4 genes. The other genotypes detected included G2, G8, G9, G12, and P[4]. A G8P[6] strain, strain DS108, was detected for the first time in northern India. The VP7 gene of DS108 was most homologous with the VP7 gene of a bovine G8 strain, strain A5 (98.9%), indicating its bovine parentage. In contrast, the VP4 gene had a high degree of nucleotide sequence homology (92.9% to 99.1%) with the VP4 genes of human P[6] strains. The VP6 gene and nonstructural genes (NSP1 to NSP3 and NSP5) were most homologous with the VP6 gene and nonstructural genes of human rotaviruses belonging to the DS1 genogroup. Interestingly, the NSP4 gene of DS108 clustered within genotype E6 that until now had only two representative strains, both with G12P[6] specificity (strains RV176-00 and N26-02). Together, these results indicate that G8 strain DS108 belongs to the DS1 genogroup and could be the result of the acquisition of the VP7, VP4, and NSP4 genes by a human G2P[4] strain from more than one donor, similar to the evolution of G12P[6] strain RV176-00. The present study highlights the importance of characterizing multiple genes of nontypeable rotavirus strains to detect novel strains and get a more complete picture of rotavirus evolution.

Rotavirus host range restriction forms a basis for strain attenuation although the underlying mechanisms are unclear. In mouse fibroblasts, the inability of rotavirus NSP1 to mediate interferon (IFN) regulatory factor 3 (IRF3) degradation correlates with IFN-dependent restricted replication of the bovine UK strain but not the mouse EW and simian RRV strains. We found that UK NSP1 is unable to degrade IRF3 when expressed in murine NIH 3T3 cells in contrast to the EW and RRV NSP1 proteins. Surprisingly, UK NSP1 expression led to IRF3 degradation in simian COS7 cells, indicating that IRF3 degradation by NSP1 is host cell dependent, a finding further supported using adenovirus-expressed NSP1 from NCDV bovine rotavirus. By expressing heterologous IRF3 proteins in complementary host cells, we found that IRF3 is the minimal host factor constraining NSP1 IRF3-degradative ability. NSP1-mediated IRF3 degradation was enhanced by transfection of double-stranded RNA (dsRNA) in a host cell-specific manner, and in IRF3-dependent positive regulatory domain III reporter assays, NSP1 inhibited IRF3 function in response to pathway activation by dsRNA, TBK-1, IRF3, or constitutively activated IRF3-5D. An interesting observation arising from these experiments is the ability of transiently expressed UK NSP1 to inhibit poly(I:C)-directed IRF3 activity in NIH 3T3 cells in the absence of detectable IRF3 degradation, an unexpected finding since UK virus infection was unable to block IFN secretion, and UK NSP1 expression did not result in suppression of IRF3-directed activation of the pathway. RRV and EW but not UK NSP1 was proteasomally degraded, requiring E1 ligase activity, although NSP1 degradation was not required for IRF3 degradation. Using a chimeric RRV NSP1 protein containing the carboxyl 100 residues derived from UK NSP1, we found that the RRV NSP1 carboxyl 100 residues are critical for its IRF3 inhibition in murine cells but are not essential for NSP1 degradation. Thus, NSP1's ability to degrade IRF3 is host cell dependent and is independent of NSP1 proteasomal degradation.

To further characterize the humoral immune response of pigs to porcine reproductive and respiratory syndrome virus (PRRSV), direct enzyme-linked immunosorbent assays (ELISA) were used to study the kinetics of antibody responses directed against PRRSV nonstructural proteins in pigs experimentally exposed to the virus. The highest immunoreactivities were against nsp1, nsp2, and nsp7. Using the recombinant nsp7 as an antigen, we validated a dual ELISA for the simultaneous detection and differentiation of serum antibodies against type I and type II PRRSV. Receiver operating characteristic analysis based on 1,334 known-positive and 1,357 known-negative samples showed good specificity (98.3% to type I and 99.3% to type II) and sensitivity (97.4% for type I and 99.8% for type II). To differentiate type I and type II PRRSV, 470 sera originating from experimentally inoculated pigs were tested, and positive sera were correctly differentiated in 469 of 470 samples. The capability of the nsp7 dual ELISA to detect serum antibody responses from pigs infected with various genetically different field strains was determined. The nsp7 dual ELISA possessed 97.6% agreement with the Idexx HerdChek PRRS 2XR ELISA. In further testing of Idexx ELISA suspected false-positive samples, the nsp7 dual ELISA resolved 98% of the samples as negative. Taken together, these results indicate that the nsp7 dual ELISA can be used as a differential test for PRRSV serology with high levels of sensitivity and specificity. This ELISA offers an additional tool for routine or follow-up diagnostics, as well as having substantial value in epidemiological surveys and outbreak investigations.

nsp1 protein of severe acute respiratory syndrome coronavirus (SARS-CoV), a group 2b CoV, suppresses host gene expression by promoting host mRNA degradation and translation inhibition. The present study analyzed the activities of nsp1 proteins from the group 2 bat CoV strains Rm1, 133, and HKU9-1, belonging to groups 2b, 2c, and 2d, respectively. The host mRNA degradation and translational suppression activities of nsp1 of SARS-CoV and Rm1 nsp1 were similar and stronger than the activities of the nsp1 proteins of 133 and HKU9-1. Rm1 nsp1 expression in trans strongly inhibited the induction of type I interferon (IFN-I) and IFN-stimulated genes in cells infected with an IFN-inducing SARS-CoV mutant, while 133 and HKU9-1 nsp1 proteins had relatively moderate IFN-inhibitory activities. The results of our studies suggested a conserved function among nsp1 proteins of SARS-CoV and group 2 bat CoVs.

Alphavirus replicase protein nsP1 has multiple functions during viral RNA synthesis. It catalyzes methyltransferase and guanylyltransferase activities needed in viral mRNA capping, attaches the viral replication complex to cytoplasmic membranes, and is required for minus-strand RNA synthesis. Two temperature-sensitive ( ts ) mutations in Semliki Forest virus (SFV) were previously identified within nsP1: ts 10 (E529D) and ts 14 (D119N). Recombinant viruses containing these individual mutations reproduced the features of the original ts strains. We now find that the capping-associated enzymatic activities of recombinant nsP1, containing ts 10 or ts 14 lesions, were not ts . The mutant proteins and polyproteins also were membrane bound, mutant nsP1 interacted normally with the other nonstructural proteins, and there was no major defect in nonstructural polyprotein processing in the mutants, although ts 14 surprisingly displayed slightly retarded processing. The two mutant viruses were specifically defective in minus-strand RNA synthesis at the restrictive temperature. Integrating data from SFV and Sindbis virus, we discuss the domain structure of nsP1 and the relative positioning of and interactions between the replicase proteins. nsP1 is suggested to contain a specific subdomain involved in minus-strand synthesis and interaction with the polymerase nsP4 and the protease nsP2.