Expression Of NSP4 Research Articles

MERS-CoV nsp1 regulates autophagic flux via mTOR signalling and dysfunctional lysosomes

ABSTRACT Autophagy, a cellular surveillance mechanism, plays an important role in combating invading pathogens. However, viruses have evolved various strategies to disrupt autophagy and even hijack it for replication and release. Here, we demonstrated that Middle East respiratory syndrome coronavirus (MERS-CoV) non-structural protein 1(nsp1) induces autophagy but inhibits autophagic activity. MERS-CoV nsp1 expression increased ROS and reduced ATP levels in cells, which activated AMPK and inhibited the mTOR signalling pathway, resulting in autophagy induction. Meanwhile, as an endonuclease, MERS-CoV nsp1 downregulated the mRNA of lysosome-related genes that were enriched in nsp1-located granules, which diminished lysosomal biogenesis and acidification, and inhibited autophagic flux. Importantly, MERS-CoV nsp1-induced autophagy can lead to cell death in vitro and in vivo. These findings clarify the mechanism by which MERS-CoV nsp1-mediated autophagy regulation, providing new insights for the prevention and treatment of the coronavirus.

Characterization and subcellular localization of Alongshan virus proteins.

Alongshan virus (ALSV) in the Jingmenvirus group within the family Flaviviridae is a newly discovered tick-borne virus associated with human disease, whose genome includes four segments and encodes four structural proteins (VP1a, VP1b, VP2, VP3, and VP4) and two non-structural proteins (NSP1 and NSP2). Here, we characterized the subcellular distribution and potential function of ALSV proteins in host cells. We found that viral proteins exhibited diverse subcellular distribution in multiple tissue-deriving cells and induced various morphological changes in the endoplasmic reticulum (ER), and NSP2, VP1b, VP2, and VP4 were all co-localized in the ER. The nuclear transfer and co-localization of VP4 and calnexin (a marker protein of ER), which were independent of their interaction, were unique to HepG2 cells. Expression of NSP1 could significantly reduce mitochondria quantity by inducing mitophagy. These findings would contribute to better understanding of the pathogenesis of emerging segmented flaviviruses.

Cap-independent translation and a precisely located RNA sequence enable SARS-CoV-2 to control host translation and escape anti-viral response.

Translation of SARS-CoV-2-encoded mRNAs by the host ribosomes is essential for its propagation. Following infection, the early expressed viral protein NSP1 binds the ribosome, represses translation, and induces mRNA degradation, while the host elicits an anti-viral response. The mechanisms enabling viral mRNAs to escape this multifaceted repression remain obscure. Here we show that expression of NSP1 leads to destabilization of multi-exon cellular mRNAs, while intron-less transcripts, such as viral mRNAs and anti-viral interferon genes, remain relatively stable. We identified a conserved and precisely located cap-proximal RNA element devoid of guanosines that confers resistance to NSP1-mediated translation inhibition. Importantly, the primary sequence rather than the secondary structure is critical for protection. We further show that the genomic 5′UTR of SARS-CoV-2 drives cap-independent translation and promotes expression of NSP1 in an eIF4E-independent and Torin1-resistant manner. Upon expression, NSP1 further enhances cap-independent translation. However, the sub-genomic 5′UTRs are highly sensitive to eIF4E availability, rendering viral propagation partially sensitive to Torin1. We conclude that the combined NSP1-mediated degradation of spliced mRNAs and translation inhibition of single-exon genes, along with the unique features present in the viral 5′UTRs, ensure robust expression of viral mRNAs. These features can be exploited as potential therapeutic targets.

Genes with 5' terminal oligopyrimidine tracts preferentially escape global suppression of translation by the SARS-CoV-2 Nsp1 protein.

Viruses rely on the host translation machinery to synthesize their own proteins. Consequently, they have evolved varied mechanisms to co-opt host translation for their survival. SARS-CoV-2 relies on a nonstructural protein, Nsp1, for shutting down host translation. However, it is currently unknown how viral proteins and host factors critical for viral replication can escape a global shutdown of host translation. Here, using a novel FACS-based assay called MeTAFlow, we report a dose-dependent reduction in both nascent protein synthesis and mRNA abundance in cells expressing Nsp1. We perform RNA-seq and matched ribosome profiling experiments to identify gene-specific changes both at the mRNA expression and translation levels. We discover that a functionally coherent subset of human genes is preferentially translated in the context of Nsp1 expression. These genes include the translation machinery components, RNA binding proteins, and others important for viral pathogenicity. Importantly, we uncovered a remarkable enrichment of 5′ terminal oligo-pyrimidine (TOP) tracts among preferentially translated genes. Using reporter assays, we validated that 5′ UTRs from TOP transcripts can drive preferential expression in the presence of Nsp1. Finally, we found that LARP1, a key effector protein in the mTOR pathway, may contribute to preferential translation of TOP transcripts in response to Nsp1 expression. Collectively, our study suggests fine-tuning of host gene expression and translation by Nsp1 despite its global repressive effect on host protein synthesis.

Effect of porcine epidemic diarrhea virus nsp1 on type Ⅰ interferon response

Porcine epidemic diarrhea virus (PEDV) inhibits the host typeⅠinterferon and cellular antiviral response, but its inhibition mechanism is unclear, and the roles of PEDV nonstructural proteins in regulating typeⅠinterferon responses have been seldom studied. To study the effect of nsp1 on typeⅠinterferon response, nsp1 gene was cloned into a eukaryotic expression vector pCAGGS. The expression of nsp1 in transfected cells was determined by Western blot and indirect immunofluorescence assay. The effects of nsp1 on the induction of typeⅠinterferon were evaluated by dual luciferase reporter gene assay, ELISA and VSV bioassay. Western blot and indirect immunofluorescence assay showed that nsp1 was highly expressed in transfected cells and PEDV-infected cells. Dual luciferase reporter gene assay results indicated that nsp1 strongly inhibited the IFN-β promoter activity, and the inhibitory effect was nsp1 dose-dependent. ELISA results showed that nsp1 significantly inhibited the expression of IFN-β in protein level. And VSV replication-inhibition bioassay revealed that nsp1 significantly inhibited typeⅠIFN antiviral activities induced by poly(I:C). Our results implied that nsp1 was a highly conserved protein of PEDV and exhibited antagonistic function on interferon promoter activity. The results have laid a foundation for further understanding the immune evasion mechanism of PEDV and for developing new effective vaccine against PEDV.

A Study of Nonstructural Protein 1 of SARS Coronavirus

Severe acute respiratory syndrome coronavirus (SARS‐CoV) encoded nonstructural protein 1 (nsp1) orchestrates a multi‐pronged mechanism to down regulate host gene expression, also known as host shutoff. Nsp1 is a 180 amino acid long protein with primarily flexible structure that allows nsp1 to interact with various cellular factors during host shutoff to inhibit translation and promote mRNA decay. Mutations of the surface residues of nsp1 either attenuates or augments its host shutoff function prompting the hypothesis that these residues bind cellular proteins that facilitate nsp1 during host shutoff. To identify cellular proteins that interact with nsp1 and assist its function, we conducted a quantitative proteomics experiment by comparing protein complexes associated with nsp1 and its inactive mutant (K164AH165A) and computer modeling to predict structural changes. By comparing protein complexes, we identified a previously undetected mechanism by which nsp1 exploits the nuclear pore complex and disrupts nuclear‐cytoplasmic transport of biomolecules. Expression of nsp1 in 293 cells disrupts Nup93 localization around the nuclear envelope without triggering proteolytic degradation of the protein while other nucleoporins and the nuclear lamina remain unperturbed, suggesting a significant alteration of the nuclear pore complex in the presence of nsp1. However, expression of mutant nsp1, which does not bind to Nup93, does not alter the nuclear pore complex. To better understand the structural basis of this protein’s function, we have utilized computational modeling tools, more specifically, I‐TASSER (Iterative Threading ASSEmbly Refinement). Structural models of various mutants of nsp1 were obtained (by using I‐TASSER) in order to explore the structural impact of different mutations. Our preliminary explorations show significant topological changes on nsp1’s surface for specific mutations. These perturbations of surface properties can provide a steric‐based mechanism of interfering with nsp1’s binding or recognition function therefore, aggravating its host shutoff function.

SARS coronavirus protein nsp1 disrupts localization of Nup93 from the nuclear pore complex

Severe acute respiratory syndrome coronavirus nonstructural protein 1 (nsp1) is a key factor in virus-induced down-regulation of host gene expression. In infected cells, nsp1 engages in a multipronged mechanism to inhibit host gene expression by binding to the 40S ribosome to block the assembly of translationally competent ribosome, and then inducing endonucleolytic cleavage and the degradation of host mRNAs. Here, we report a previously undetected mechanism by which nsp1 exploits the nuclear pore complex and disrupts the nuclear-cytoplasmic transport of biomolecules. We identified members of the nuclear pore complex from the nsp1-associated protein assembly and found that the expression of nsp1 in HEK cells disrupts Nup93 localization around the nuclear envelope without triggering proteolytic degradation, while the nuclear lamina remains unperturbed. Consistent with its role in host shutoff, nsp1 alters the nuclear-cytoplasmic distribution of an RNA binding protein, nucleolin. Our results suggest that nsp1, alone, can regulate multiple steps of gene expression including nuclear-cytoplasmic transport.

Dissecting the mechanism of host shutoff by SARS coronavirus

Down regulation of host gene expression during viral infection, known as host shutoff, involves destabilization of host mRNA. Several viruses, including Kaposi's sarcoma virus, Epstein B virus, severe acute respiratory syndrome coronavirus (SARS CoV) and Middle Eastern respiratory syndrome coronavirus inhibit host gene expression by stalling the ribosome on mRNA and subsequently cleaving and degrading host mRNA. In SARS‐CoV, nsp1 is the key factor in virus‐induced down‐regulation of host gene expression. In infected cells, nsp1 engages in a multi‐pronged mechanism to inhibit host gene expression by binding to the 40S ribosomal subunit to stall further ribosome assembly and inducing endonucleolytic cleavage and degradation of host mRNAs. We identified a previously undetected mechanism by which nsp1 exploits the nuclear pore complex and disrupts nuclear‐cytoplasmic transport of biomolecules. Using quantitative proteomics we identified members of the nuclear pore complex from nsp1‐associated protein assembly. Expression of nsp1 in HEK cells disrupts Nup93 localization around the nuclear envelope without triggering proteolytic degradation of the protein while other nucleoporins and the nuclear lamina remain unperturbed, suggesting significant alteration of the nuclear pore complex in the presence of nsp1. Furthermore, Nup93 reconvenes to the nuclear pore when nsp1 is removed. Consistent with its role in host shutoff, nsp1 alters the nuclear‐cytoplasmic distribution of a RNA binding protein, nucleolin. Our results suggest that nsp1 alone can regulate multiple steps of gene expression including nuclear‐cytoplasmic transport.Support or Funding InformationSC INBRE Bioinformatics Pilot Project AwardSCICUThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Mechanisms of antidiarrhoeal effects by diosmectite in human intestinal cells

BackgroundRotavirus (RV) induces diarrhoea through a sequence of enterotoxic and cytotoxic effects. The former are NSP4-dependent, induce calcium-dependent chloride secretion and involve oxidative stress. Diosmectite (DS) is a natural clay that has been recommended as an active therapy for diarrhoea, but the mechanism of its effect is not clear. Electrical parameters may be used to measure the direct enterotoxic and cytotoxic effects in polar epithelial intestinal cells. To investigate the effects of DS on RV-induced enterotoxic and cytotoxic damage. Caco-2 cells were used as a model of RV infection to evaluate chloride secretion, epithelial integrity, oxidative stress and viral infectivity in Ussing chambers.ResultsDiosmectite reduced the expression of NSP4 and oxidative stress, resulting in a strong inhibition of chloride secretion. Preincubating RV with DS reduced the cytotoxic effect. Finally, the viral load was reduced by DS but not by control clay. This result suggests that DS specifically affects the early events of RV infection protecting the enterocyte, whereas it does not restore already-established cell damage.ConclusionThese findings indicate that DS exerts an anti-diarrhoeal effect by inhibiting viral replication and the expression of NSP4. Both ion secretion and cell damage induced by RV are strongly inhibited consequent to the antiviral effect, which explains its clinical efficacy.

Nonstructural Protein 4 of Porcine Reproductive and Respiratory Syndrome Virus Modulates Cell Surface Swine Leukocyte Antigen Class I Expression by Downregulating β2-Microglobulin Transcription.

Porcine reproductive and respiratory syndrome virus (PRRSV) is the causative agent of PRRS, which has important impacts on the pig industry. PRRSV infection results in disruption of the swine leukocyte antigen class I (SLA-I) antigen presentation pathway. In this study, highly pathogenic PRRSV (HP-PRRSV) infection inhibited transcription of the β2-microglobulin (β2M) gene (B2M) and reduced cellular levels of β2M, which forms a heterotrimeric complex with the SLA-I heavy chain and a variable peptide and plays a critical role in SLA-I antigen presentation. HP-PRRSV nonstructural protein 4 (Nsp4) was involved in the downregulation of β2M expression. Exogenous expression of Nsp4 downregulated β2M expression at both the mRNA and the protein level and reduced SLA-I expression on the cell surface. Nsp4 bound to the porcine B2M promoter and inhibited its transcriptional activity. Domain III of Nsp4 and the enhancer PAM element of the porcine B2M promoter were identified as essential for the interaction between Nsp4 and B2M These findings demonstrate a novel mechanism whereby HP-PRRSV may modulate the SLA-I antigen presentation pathway and provide new insights into the functions of HP-PRRSV Nsp4. IMPORTANCE PRRSV modulates the host response by disrupting the SLA-I antigen presentation pathway. We show that HP-PRRSV downregulates SLA-I expression on the cell surface via transcriptional inhibition of B2M expression by viral Nsp4. The interaction between domain III of Nsp4 and the enhancer PAM element of the porcine B2M promoter is essential for inhibiting B2M transcription. These observations reveal a novel mechanism whereby HP-PRRSV may modulate SLA-I antigen presentation and provide new insights into the functions of viral Nsp4.

Inhibition of host protein synthesis by Sindbis virus: correlation with viral RNA replication and release of nuclear proteins to the cytoplasm.

SummaryInfection of mammalian cells by Sindbis virus (SINV) profoundly blocks cellular mRNA translation. Experimental evidence points to viral non‐structural proteins (nsPs), in particular nsP2, as the mediator of this inhibition. However, individual expression of nsP1, nsP2, nsP3 or nsP1‐4 does not block cellular protein synthesis in BHK cells. Trans‐complementation of a defective SINV replicon lacking most of the coding region for nsPs by the co‐expression of nsP1‐4 propitiates viral RNA replication at low levels, and inhibition of cellular translation is not observed. Exit of nuclear proteins including T‐cell intracellular antigen and polypyrimidine tract‐binding protein is clearly detected in SINV‐infected cells, but not upon the expression of nsPs, even when the defective replicon was complemented. Analysis of a SINV variant with a point mutation in nsP2, exhibiting defects in the shut‐off of host protein synthesis, indicates that both viral RNA replication and the release of nuclear proteins to the cytoplasm are greatly inhibited. Furthermore, nucleoside analogues that inhibit cellular and viral RNA synthesis impede the blockade of host mRNA translation, in addition to the release of nuclear proteins. Prevention of the shut‐off of host mRNA translation by nucleoside analogues is not due to the inhibition of eIF2α phosphorylation, as this prevention is also observed in PKR −/− mouse embryonic fibroblasts that do not phosphorylate eIF2α after SINV infection. Collectively, our observations are consistent with the concept that for the inhibition of cellular protein synthesis to occur, viral RNA replication must take place at control levels, leading to the release of nuclear proteins to the cytoplasm.

CERBERUS and NSP1 of Lotus japonicus are Common Symbiosis Genes that Modulate Arbuscular Mycorrhiza Development

Arbuscular mycorrhizal symbiosis (AMS) and root nodule symbiosis (RNS) are mutualistic plant-microbe interactions that confer nutritional benefits to both partners. Leguminous plants possess a common genetic system for intracellular symbiosis with AM fungi and with rhizobia. Here we show that CERBERUS and NSP1, which respectively encode an E3 ubiquitin ligase and a GRAS transcriptional regulator and which have previously only been implicated in RNS, are involved in AM fungal infection in Lotus japonicus. Hyphal elongation along the longitudinal axis of the root was reduced in the cerberus mutant, giving rise to a lower colonization level. Knockout of NSP1 decreased the frequency of plants colonized by AM fungi or rhizobia. CERBERUS and NSP1 showed different patterns of expression in response to infection with symbiotic microbes. A low constitutive level of CERBERUS expression was observed in the root and an increased level of NSP1 expression was detected in arbuscule-containing cells. Induction of AM marker gene was triggered in both cerberus and nsp1 mutants by infection with symbiotic microbes; however, the mutants showed a weaker induction of marker gene expression than the wild type, mirroring their lower level of colonization. The common symbiosis genes are believed to act in an early signaling pathway for recognition of symbionts and for triggering early symbiotic responses. Our quantitative analysis of symbiotic phenotypes revealed developmental defects of the novel common symbiosis mutants in both symbioses, which demonstrates that common symbiosis mechanisms also contribute to a range of functions at later or different stages of symbiont infection.

Down-Regulation of NSP2 Expression in Developmentally Young Regions of Lotus japonicus Roots in Response to Rhizobial Inoculation

During the early 1980s, Bauer and associates reported that nodulation potential in primary roots of soybean seedlings following inoculation with rhizobia was significantly reduced in developmentally younger regions. They suggested that this phenomenon might be due to a fast-acting regulatory mechanism in the host that prevented excessive nodulation. However, the molecular mechanism of this fast-acting regulatory response remains uncertain. Here, we sought to elucidate components of this regulatory mechanism by investigating the expression of the NSP1 and NSP2 genes that encode a GRAS transcription factor required for nodule initiation. First, we confirmed that younger regions of Lotus japonicus roots also show a reduction in nodule numbers in response to Mesorhizobium loti. Then, we compared the expression levels of NSP1 and NSP2 in developmentally younger regions of primary roots. After inoculation with M. loti, expression of NSP1 was transiently induced whereas that of NSP2 was significantly down-regulated 1 d after inoculation. This result implicates that down-regulation of NSP2 might cause a fast-acting regulatory mechanism to prevent further nodulation. Next we overexpressed NSP2 in wild-type plants. Overexpression resulted in the clustering of nodules in the upper region of the root but strong suppression of nodulation in the lower region. In contrast, overexpression of NSP2 in har1 hypernodulating mutants resulted in an increased number of nodule primordia even in the root tip region. These results indicate that HAR1 negatively regulates NSP2-induced excessive nodule formation in the developmentally younger regions of roots.

Rotaviral Enterotoxin Nonstructural Protein 4 Targets Mitochondria for Activation of Apoptosis during Infection

Viruses have evolved to encode multifunctional proteins to control the intricate cellular signaling pathways by using very few viral proteins. Rotavirus is known to express six nonstructural and six structural proteins. Among them, NSP4 is the enterotoxin, known to disrupt cellular Ca(2+) homeostasis by translocating to endoplasmic reticulum. In this study, we have observed translocation of NSP4 to mitochondria resulting in dissipation of mitochondrial membrane potential during virus infection and NSP4 overexpression. Furthermore, transfection of the N- and C-terminal truncated NSP4 mutants followed by analyzing NSP4 localization by immunofluorescence microscopy identified the 61-83-amino acid region as the shortest mitochondrial targeting signal. NSP4 exerts its proapoptotic effect by interacting with mitochondrial proteins adenine nucleotide translocator and voltage-dependent anion channel, resulting in dissipation of mitochondrial potential, release of cytochrome c from mitochondria, and caspase activation. During early infection, apoptosis activation by NSP4 was inhibited by the activation of cellular survival pathways (PI3K/AKT), because PI3K inhibitor results in early induction of apoptosis. However, in the presence of both PI3K inhibitor and NSP4 siRNA, apoptosis was delayed suggesting that the early apoptotic signal is initiated by NSP4 expression. This proapoptotic function of NSP4 is balanced by another virus-encoded protein, NSP1, which is implicated in PI3K/AKT activation because overexpression of both NSP4 and NSP1 in cells resulted in reduced apoptosis compared with only NSP4-expressing cells. Overall, this study reports on the mechanism by which enterotoxin NSP4 exerts cytotoxicity and the mechanism by which virus counteracts it at the early stage for efficient infection.

Dissecting the Ca2+ entry pathways induced by rotavirus infection and NSP4-EGFP expression in Cos-7 cells

Rotavirus infection modifies Ca2+ homeostasis provoking an increase in Ca2+ permeation, cytoplasmic Ca2+ concentration ([Ca2+]cyto), total Ca2+ pools and, a decrease of Ca2+ response to agonists. These effects are mediated by NSP4. The mechanism by which NSP4 deranges Ca2+ homeostasis is not yet known. It has been proposed that the increase in [Ca2+]cyto is the result of Ca2+ release from intracellular stores, thereby activating store-operated Ca2+ entry (SOCE). We studied the mechanisms involved in the changes of Ca2+ permeability of the plasma membrane elicited by rotavirus infection and NSP4 expression in Cos-7 cells loaded with fura-2 or fluo-4, using inhibitors and activators of different pathways. Total depletion of ER Ca2+ stores induced by thapsigargin or ATP was not able to elicit Ca2+ entry in mock-infected cells to the level attained with infection or NSP4-EGFP expression. The pathway induced by NSP4-EGFP expression or infection shows properties shared by SOCE: it can be inactivated by high [Ca2+]cyto, is permeable to Mn2+ and inhibited by La3+ and the SOC inhibitor 2-aminoethoxydiphenyl borate (2-APB). Contribution of the agonist-operated channels (AOCs) to Ca2+ entry is small and not modified by infection. The plasma membrane permeability to Ca2+ in rotavirus infected or NSP4-EGFP expressing cells is also blocked by KB-R7943, an inhibitor of the plasma membrane Na+/Ca2+ exchanger (NCX), operating in its reverse mode. In conclusion, the expression of NSP4 in infected Cos-7 cells appears to activate the NCX in reverse mode and the SOCE pathway to induce increased Ca2+ entry.

Genetic Divergence of Rotavirus Nonstructural Protein 4 Results in Distinct Serogroup-Specific Viroporin Activity and Intracellular Punctate Structure Morphologies

Nonstructural protein 4 (NSP4) viroporin activity is critical for the replication and assembly of serogroup A rotavirus (RVA); however, the dramatic primary sequence divergence of NSP4s across serogroups raises the possibility that viroporin activity is not a common feature among RVs. We tested for NSP4 viroporin activity from divergent strains, including RVA (EC and Ty-1), RVB (IDIR), and RVC (Cowden). Canonical viroporin motifs were identified in RVA, RVB, and RVC NSP4s, but the arrangement of basic residues and the amphipathic α-helices was substantially different between serogroups. Using Escherichia coli and mammalian cell expression, we showed that each NSP4 tested had viroporin activity, but serogroup-specific viroporin phenotypes were identified. Only mammalian RVA and RVC NSP4s induced BL21-pLysS E. coli cell lysis, a classical viroporin activity assay. In contrast, RVA, RVB, and RVC NSP4 expression was universally cytotoxic to E. coli and disrupted reduction-oxidation activities, as measured by a new redox dye assay. In mammalian cells, RVB and RVC NSP4s were initially localized in the endoplasmic reticulum (ER) and trafficked into punctate structures that were mutually exclusive with RVA NSP4. The punctate structures partially localized to the ER-Golgi intermediate compartment (ERGIC) but primarily colocalized with punctate LC3, a marker for autophagosomes. Similar to RVA NSP4, expression of RVB and RVC NSP4s significantly elevated cytosolic calcium levels, demonstrating that despite strong primary sequence divergence, RV NSP4 has maintained viroporin activity across serogroups A to C. These data suggest that elevated cytosolic calcium is a common critical process for all rotavirus strains.

The rotavirus enterotoxin (NSP4) promotes re-modeling of the intracellular microtubule network

Expression of the rotavirus enterotoxin (NSP4) in transfected monkey kidney cells was found to result in a dramatic re-modeling of the microtubule (MT) network. This important centrosome organized cytoskeletal element was dissolved by expression of NSP4 and re-formed in a ring array at the periphery of the cell, similar to that seen following normal virus infection. Site directed mutagenesis of the N-linked glycosylation sites in NSP4 was employed to show that glycosylation of NSP4 was not required for it to promote changes in the MT network. This result together with experiments using conventional inhibitors indicated that NSP4's ability to cause elevation of intracellular calcium levels was also not necessary to effect the changes in the MT network. Use of the centrosome function inhibitor nocodazole demonstrated that NSP4 based remodeling of the MT network was dominant over the normal organizational role of the centrosome. Finally the remodeling of the MT network was shown not to be linked to cellular apoptosis or necrosis. The potential importance of this newly recognised role for NSP4 in the overall process of intracellular pathogenesis by rotaviruses is discussed.

Murine Hepatitis Virus Nonstructural Protein 4 Regulates Virus-Induced Membrane Modifications and Replication Complex Function

Positive-strand RNA viruses induce modifications of cytoplasmic membranes to form replication complexes. For coronaviruses, replicase nonstructural protein 4 (nsp4) has been proposed to function in the formation and organization of replication complexes. Murine hepatitis virus (MHV) nsp4 is glycosylated at residues Asn176 (N176) and N237 during plasmid expression of nsp4 in cells. To test if MHV nsp4 residues N176 and N237 are glycosylated during virus replication and to determine the effects of N176 and N237 on nsp4 function and MHV replication, alanine substitutions of nsp4 N176, N237, or both were engineered into the MHV-A59 genome. The N176A, N237A, and N176A/N237A mutant viruses were viable, and N176 and N237 were glycosylated during infection of wild-type (wt) and mutant viruses. The nsp4 glycosylation mutants exhibited impaired virus growth and RNA synthesis, with the N237A and N176A/N237A mutant viruses demonstrating more profound defects in virus growth and RNA synthesis. Electron microscopic analysis of ultrastructure from infected cells demonstrated that the nsp4 mutants had aberrant morphology of virus-induced double-membrane vesicles (DMVs) compared to those infected with wt virus. The degree of altered DMV morphology directly correlated with the extent of impairment in viral RNA synthesis and virus growth of the nsp4 mutant viruses. The results indicate that nsp4 plays a critical role in the organization and stability of DMVs. The results also support the conclusion that the structure of DMVs is essential for efficient RNA synthesis and optimal replication of coronaviruses.

IRF3 Inhibition by Rotavirus NSP1 Is Host Cell and Virus Strain Dependent but Independent of NSP1 Proteasomal Degradation

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.

Bovine coronavirus nonstructural protein 1 (p28) is an RNA binding protein that binds terminal genomic cis-replication elements.

Nonstructural protein 1 (nsp1), a 28-kDa protein in the bovine coronavirus (BCoV) and closely related mouse hepatitis coronavirus, is the first protein cleaved from the open reading frame 1 (ORF 1) polyprotein product of genome translation. Recently, a 30-nucleotide (nt) cis-replication stem-loop VI (SLVI) has been mapped at nt 101 to 130 within a 288-nt 5'-terminal segment of the 738-nt nsp1 cistron in a BCoV defective interfering (DI) RNA. Since a similar nsp1 coding region appears in all characterized groups 1 and 2 coronavirus DI RNAs and must be translated in cis for BCoV DI RNA replication, we hypothesized that nsp1 might regulate ORF 1 expression by binding this intra-nsp1 cistronic element. Here, we (i) establish by mutation analysis that the 72-nt intracistronic SLV immediately upstream of SLVI is also a DI RNA cis-replication signal, (ii) show by gel shift and UV-cross-linking analyses that cellular proteins of approximately 60 and 100 kDa, but not viral proteins, bind SLV and SLVI, (SLV-VI) and (iii) demonstrate by gel shift analysis that nsp1 purified from Escherichia coli does not bind SLV-VI but does bind three 5' untranslated region (UTR)- and one 3' UTR-located cis-replication SLs. Notably, nsp1 specifically binds SLIII and its flanking sequences in the 5' UTR with approximately 2.5 muM affinity. Additionally, under conditions enabling expression of nsp1 from DI RNA-encoded subgenomic mRNA, DI RNA levels were greatly reduced, but there was only a slight transient reduction in viral RNA levels. These results together indicate that nsp1 is an RNA-binding protein that may function to regulate viral genome translation or replication but not by binding SLV-VI within its own coding region.