Differential induction of isolated lymphoid follicles in the gut by 18ß-glycyrrhetinic acid.

18ß-glycyrrhetinic acid (GRA) is a pharmacologically active component of licorice root with documented immunomodulatory properties. We reported that GRA administered orally to mice induces B cell recruitment to isolated lymphoid follicles (ILF) in the small intestine and shortens the duration of rotavirus antigen shedding. ILF are dynamic lymphoid tissues in the gut acquired post-natally upon colonization with commensal bacteria and mature through B cell recruitment to the follicles, resulting in up-regulation of IgA synthesis in response to changes in the composition of microbiota. In this study, we investigated potential mechanisms by which GRA induces ILF maturation in the ileum and the colon using mice depleted of enteric bacteria and a select group of mice genetically deficient in pattern recognition receptors. The data show GRA was unable to induce ILF maturation in ileums of mice devoid of commensal bacteria, MyD88-/- or NOD2-/- mice, but differentially induced ILF in colons. Increased expression of chemokine and chemokine receptor genes that modulate B and T cell recruitment to the mucosa were in part dependent on NOD2, TLR, and signaling adaptor protein MyD88. Together the results suggest GRA induces ILF through cooperative signals provided by bacterial ligands under normal conditions to induce B cell recruitment to ILF to the gut, but that the relative contribution of these signals differ between ileum and colon.

18ß-Glycyrrhetinic acid inhibits methicillin-resistant Staphylococcus aureus survival and attenuates virulence gene expression.

Methicillin-resistant Staphylococcus aureus (MRSA) has become a major source of infection in hospitals and in the community. Increasing antibiotic resistance in S. aureus strains has created a need for alternative therapies to treat disease. A component of the licorice root Glycyrrhiza spp., 18ß-glycyrrhetinic acid (GRA), has been shown to have antiviral, antitumor, and antibacterial activity. This investigation explores the in vitro and in vivo effects of GRA on MRSA pulsed-field gel electrophoresis (PFGE) type USA300. GRA exhibited bactericidal activity at concentrations exceeding 0.223 µM. Upon exposure of S. aureus to sublytic concentrations of GRA, we observed a reduction in expression of key virulence genes, including saeR and hla. In murine models of skin and soft tissue infection, topical GRA treatment significantly reduced skin lesion size and decreased the expression of saeR and hla genes. Our investigation demonstrates that at high concentrations GRA is bactericidal to MRSA and at sublethal doses it reduces virulence gene expression in S. aureus both in vitro and in vivo.

18ß-glycyrrhetinic acid delivered orally induces isolated lymphoid follicle maturation at the intestinal mucosa and attenuates rotavirus shedding.

Glycyrrhizin, an abundant bioactive component of the medicinal licorice root is rapidly metabolized by gut commensal bacteria into 18ß-glycyrrhetinic acid (GRA). Either or both of these compounds have been shown to have antiviral, anti-hepatotoxic, anti-ulcerative, anti-tumor, anti-allergenic and anti-inflammatory activity in vitro or in vivo. In this study, the ability of GRA to modulate immune responses at the small intestinal mucosa when delivered orally was investigated. Analysis of cytokine transcription in duodenal and ileal tissue in response to GRA treatment revealed a pattern of chemokine and chemokine receptor gene expression predictive of B cell recruitment to the gut. Consistent with this finding, GRA induced increases in CD19(+) B cells in the lamina propria and B220(+) B cell aggregates framed by CD11c(+) dendritic cells in structures resembling isolated lymphoid follicles (ILF). Using a mouse model of rotavirus infection, GRA reduced the duration of viral antigen shedding, and endpoint serum antibody titers were higher in GRA-treated animals. Together the data suggest GRA delivered orally augments lymphocyte recruitment to the intestinal mucosa and induces maturation of B cell-rich ILF independently of ectopic antigenic stimulus. These results provide further support a role for dietary ligands in modulation of dynamic intestinal lymphoid tissue.

18ß-glycyrrhetinic acid inhibits rotavirus replication in culture.

BACKGROUND: Glycyrrhizin (GA) and primary metabolite 18ß-glycyrrhetinic acid (GRA) are pharmacologically active components of the medicinal licorice root, and both have been shown to have antiviral and immunomodulatory properties. Although these properties are well established, the mechanisms of action are not completely understood. In this study, GA and GRA were tested for the ability to inhibit rotavirus replication in cell culture, toward a long term goal of discovering natural compounds that may complement existing vaccines. METHODS: Epithelial cells were treated with GA or GRA various times pre- or post-infection and virus yields were measured by immunofluorescent focus assay. Levels of viral proteins VP2, VP6, and NSP2 in GRA treated cells were measured by immunoblot to determine if there was an effect of GRA treatment on the accumulation of viral protein. RESULTS: GRA treatment reduced rotavirus yields by 99% when added to infected cultures post-- virus adsorption, whereas virus yields in GA treated cultures were similar to mock treated controls. Time of addition experiments indicated that GRA-mediated replication inhibition likely occurs at a step or steps subsequent to virus entry. The amounts of VP2, VP6 and NSP2 were substantially reduced when GRA was added to cultures up to two hours post-entry. CONCLUSIONS: GRA, but not GA, has significant antiviral activity against rotavirus replication in vitro, and studies to determine whether GRA attenuates rotavirus replication in vivo are underway.

Rotavirus infection activates the UPR but modulates its activity.

BACKGROUND: Rotaviruses are known to modulate the innate antiviral defense response driven by IFN. The purpose of this study was to identify changes in the cellular proteome in response to rotavirus infection in the context of the IFN response. We also sought to identify proteins outside the IFN induction and signaling pathway that were modulated by rotavirus infection. METHODS: 2D-DIGE and image analysis were used to identify cellular proteins that changed in levels of expression in response to rotavirus infection, IFN treatment, or IFN treatment prior to infection. Immunofluorescence microscopy was used to determine the subcellular localization of proteins associated with the unfolded protein response (UPR). RESULTS: The data show changes in the levels of multiple proteins associated with cellular stress in infected cells, including levels of ER chaperones GRP78 and GRP94. Further investigations showed that GRP78, GRP94 and other proteins with roles in the ER-initiated UPR including PERK, CHOP and GADD34, were localized to viroplasms in infected cells. CONCLUSIONS: Together the results suggest rotavirus infection activates the UPR, but modulates its effects by sequestering sensor, transcription factor, and effector proteins in viroplasms. The data consequently also suggest that viroplasms may directly or indirectly play a fundamental role in regulating signaling pathways associated with cellular defense responses.

Cysteine protease activation and apoptosis in Murine norovirus infection.

BACKGROUND: Noroviruses are the leading cause of viral gastroenteritis. Because a suitable in vitro culture system for the human virus has yet to be developed, many basic details of the infection process are unknown. Murine norovirus (MNV) serves as a model system for the study of norovirus infection. Recently it was shown that infection of RAW 264.7 cells involved a novel apoptotic pathway involving survivin. RESULTS: Using a different set of approaches, the up-regulation of caspases, DNA condensation/fragmentation, and membrane blebbing, all of which are markers of apoptosis, were confirmed. Live cell imaging and activity-based protein profiling showed that activation of caspase-like proteases occurred within two hours of infection, followed by morphological changes to the cells. MNV infection in the presence of caspase inhibitors proceeded via a distinct pathway of rapid cellular necrosis and reduced viral production. Affinity purification of activity-based protein profiling targets and identification by peptide mass fingerprinting showed that the cysteine protease cathepsin B was activated early in infection, establishing this protein as an upstream activator of the intrinsic apoptotic pathway. CONCLUSION: This work adds cathepsin B to the noncanonical programmed cell death induced by MNV, and provides data suggesting that the virus may induce apoptosis to expand the window of time for viral replication. This work also highlights the significant power of activity-based protein profiling in the study of viral pathogenesis.

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.

Rotavirus NSP1 inhibits NFkappaB activation by inducing proteasome-dependent degradation of beta-TrCP: a novel mechanism of IFN antagonism.

Mechanisms by which viruses counter innate host defense responses generally involve inhibition of one or more components of the interferon (IFN) system. Multiple steps in the induction and amplification of IFN signaling are targeted for inhibition by viral proteins, and many of the IFN antagonists have direct or indirect effects on activation of latent cytoplasmic transcription factors. Rotavirus nonstructural protein NSP1 blocks transcription of type I IFNalpha/beta by inducing proteasome-dependent degradation of IFN-regulatory factors 3 (IRF3), IRF5, and IRF7. In this study, we show that rotavirus NSP1 also inhibits activation of NFkappaB and does so by a novel mechanism. Proteasome-mediated degradation of inhibitor of kappaB (IkappaBalpha) is required for NFkappaB activation. Phosphorylated IkappaBalpha is a substrate for polyubiquitination by a multisubunit E3 ubiquitin ligase complex, Skp1/Cul1/F-box, in which the F-box substrate recognition protein is beta-transducin repeat containing protein (beta-TrCP). The data presented show that phosphorylated IkappaBalpha is stable in rotavirus-infected cells because infection induces proteasome-dependent degradation of beta-TrCP. NSP1 expressed in isolation in transiently transfected cells is sufficient to induce this effect. Targeted degradation of an F-box protein of an E3 ligase complex with a prominent role in modulation of innate immune signaling and cell proliferation pathways is a unique mechanism of IFN antagonism and defines a second strategy of immune evasion used by rotaviruses.

Recombinant norovirus-specific scFv inhibit virus-like particle binding to cellular ligands.

BACKGROUND: Noroviruses cause epidemic outbreaks of gastrointestinal illness in all age-groups. The rapid onset and ease of person-to-person transmission suggest that inhibitors of the initial steps of virus binding to susceptible cells have value in limiting spread and outbreak persistence. We previously generated a monoclonal antibody (mAb) 54.6 that blocks binding of recombinant norovirus-like particles (VLP) to Caco-2 intestinal cells and inhibits VLP-mediated hemagglutination. In this study, we engineered the antigen binding domains of mAb 54.6 into a single chain variable fragment (scFv) and tested whether these scFv could function as cell binding inhibitors, similar to the parent mAb. RESULTS: The scFv54.6 construct was engineered to encode the light (VL) and heavy (VH) variable domains of mAb 54.6 separated by a flexible peptide linker, and this recombinant protein was expressed in Pichia pastoris. Purified scFv54.6 recognized native VLPs by immunoblot, inhibited VLP-mediated hemagglutination, and blocked VLP binding to H carbohydrate antigen expressed on the surface of a CHO cell line stably transfected to express alpha 1,2-fucosyltransferase. CONCLUSION: scFv54.6 retained the functional properties of the parent mAb with respect to inhibiting norovirus particle interactions with cells. With further engineering into a form deliverable to the gut mucosa, norovirus neutralizing antibodies represent a prophylactic strategy that would be valuable in outbreak settings.

A single-amino-acid substitution in the P2 domain of VP1 of murine norovirus is sufficient for escape from antibody neutralization.

Noroviruses cause epidemic outbreaks of acute viral gastroenteritis worldwide, and the number of reported outbreaks is increasing. Human norovirus strains do not grow in cell culture. However, murine norovirus (MNV) replicates in the RAW 264.7 macrophage cell line and thus provides a tractable model to investigate norovirus interactions with host cells. Epitopes recognized by monoclonal antibodies (MAbs) against the human norovirus strains Norwalk virus and Snow Mountain virus (SMV) identified regions in the P domain of major capsid protein VP1 important for interactions with putative cellular receptors. To determine if there was a relationship between domains of MNV VP1 and VP1 of human norovirus strains involved in cell binding, epitope mapping by phage display was performed with an MNV-1-neutralizing MAb, A6.2.1. A consensus peptide, GWWEDHGQL, was derived from 20 third-round phage clones. A synthetic peptide containing this sequence and constrained through a disulfide linkage reacted strongly with the A6.2.1 MAb, whereas the linear sequence did not. Four residues in the A6.2.1-selected peptide, G327, G333, Q334, and L335, aligned with amino acid residues in the P2 domain of MNV-1 VP1. This sequence is immediately adjacent to the epitope recognized by anti-SMV MAb 61.21. Neutralization escape mutants selected with MAb A6.2.1 contained a leucine-to-phenylalanine substitution at position 386 in the P2 domain. The predicted location of these residues on VP1 suggests that the phage peptide and the mutation in the neutralization-resistant viruses may be in close proximity to each other and to residues reported to be important for carbohydrate binding to VP1 of human norovirus strains.

Zinc-binding domain of rotavirus NSP1 is required for proteasome-dependent degradation of IRF3 and autoregulatory NSP1 stability.

Interferon regulatory factor 3 (IRF3) is a key transcription factor involved in the induction of interferon (IFN) in response to viral infection. Rotavirus non-structural protein NSP1 binds to and targets IRF3 for proteasome degradation early post-infection. Mutational analysis of cysteine and histidine residues within the conserved N-terminal zinc-binding domain in NSP1 of bovine rotavirus strain B641 abolished IRF3 degradation in transfected cells. Thus, the integrity of the zinc-binding domain in NSP1 is important for degradation of IRF3. In contrast to bovine strain B641, IRF3 was stable in cells infected with porcine rotavirus strain OSU and OSU NSP1 bound only weakly to IRF3. Both B641 NSP1 and OSU NSP1 were stabilized in cells or cell-free extracts in the presence of the proteasome inhibitor MG132 and when the zinc-binding domain was disrupted by site-directed mutagenesis. Data from the B641 analyses that show IRF3 degradation is dependent on the presence of NSP1 and the integrity of the N-terminal zinc-binding domain, coupled with the regulated stability of IRF3 and NSP1 by the proteasome, collectively support the hypothesis that NSP1 is an E3 ubiquitin ligase.

Natural products that reduce rotavirus infectivity identified by a cell-based moderate-throughput screening assay.

BACKGROUND: There is widespread interest in the use of innate immune modulators as a defense strategy against infectious pathogens. Using rotavirus as a model system, we developed a cell-based, moderate-throughput screening (MTS) assay to identify compounds that reduce rotavirus infectivity in vitro, toward a long-term goal of discovering immunomodulatory agents that enhance innate responses to viral infection. RESULTS: A natural product library consisting of 280 compounds was screened in the assay and 15 compounds that significantly reduced infectivity without cytotoxicity were identified. Time course analysis of four compounds with previously characterized effects on inflammatory gene expression inhibited replication with pre-treatment times as minimal as 2 hours. Two of these four compounds, alpha-mangostin and 18-beta-glycyrrhetinic acid, activated NFkappaB and induced IL-8 secretion. The assay is adaptable to other virus systems, and amenable to full automation and adaptation to a high-throughput format. CONCLUSION: Identification of several compounds with known effects on inflammatory and antiviral gene expression that confer resistance to rotavirus infection in vitro suggests the assay is an appropriate platform for discovery of compounds with potential to amplify innate antiviral responses.

VPg of murine norovirus binds translation initiation factors in infected cells.

BACKGROUND: Norovirus genomic and subgenomic RNAs are covalently linked at the 5' nucleotide to a 15 kD protein called VPg. VPg of two human norovirus strains binds translation initiation factor eIF3 and other eIFs in vitro, suggesting VPg functions in initiation of protein synthesis on viral RNA. Human norovirus strains are not cultivable, and thus experimental evidence of interactions between VPg and eIFs in infected cells has been lacking. We used the cultivable murine norovirus MNV-1 as a model to study interactions between VPg and eIFs in infected cells. RESULTS: As shown previously for human norovirus VPg, MNV-1 VPg bound eIF3, eIF4GI, eIF4E, and S6 ribosomal protein in cell extracts by GST pull-down assay. Importantly, MNV-1 VPg co-precipitated eIF4GI and eIF4E from infected macrophages, providing evidence that VPg interacts with components of the translation machinery in norovirus infected cells. CONCLUSION: The interactions between MNV-1 VPg and eIFs completely mimic those reported for the human norovirus VPg, illustrating the utility of MNV-1 as a relevant molecular model to study mechanisms of human norovirus replication.

Epitopes in the P2 domain of norovirus VP1 recognized by monoclonal antibodies that block cell interactions.

Noroviruses cause the majority of epidemic outbreaks of acute viral gastroenteritis worldwide. Human norovirus strains do not grow in cell culture, but recent carbohydrate binding, sequence and structural analyses have begun to define functional domains in the norovirus capsid that may be conserved among multiple antigenic types. The purpose of this study was to localize domains and define sequences in the major capsid protein VP1 that are important for cell interactions. Monoclonal antibodies to genogroups GI.1 and GII.2 reference strains Norwalk virus and Snow Mountain virus, respectively, were generated that blocked binding of recombinant virus-like particles to Caco-2 intestinal cells and inhibited haemagglutination. Peptides that mimicked the mAb binding epitopes were selected from a phage-displayed random nonapeptide library. Anti-recombinant Norwalk virus mAb 54.6 and anti-recombinant Snow Mountain virus mAb 61.21 recognized epitopes located in the protruding P2 domain of VP1. The epitope recognized by mAb 61.21 contained amino acids that are completely conserved among norovirus strains across genogroups, including strains isolated from swine, bovine and murine species. This study identifies the first epitope involved in inhibition of norovirus-cell interactions and supports increasing evidence that interactions between noroviruses and host cells rely on structures in the P2 domain of VP1.

Norovirus protein structure and function.

Noroviruses are positive strand RNA viruses that have received increased attention in recent years because their role as etiologic agents in acute gastroenteritis outbreaks is now clearly established. Much has been learned about the epidemiology of these viruses and the extent of genetic diversity among circulating strains. In contrast, progress on understanding the basic mechanisms of virus replication has been far slower due to the inability to cultivate virus in the laboratory. Despite this limitation, significant progress has been made in defining some basic functions of the norovirus proteins, and the structures of two have been solved to near atomic resolution. This minireview summarizes these recent advances in understanding the structure and function of the norovirus proteins and provides speculation about what roles they may play in the virus replication cycle.

Norwalk virus nonstructural protein p48 forms a complex with the SNARE regulator VAP-A and prevents cell surface expression of vesicular stomatitis virus G protein.

Norwalk virus (NV), a reference strain of human calicivirus in the Norovirus genus of the family Caliciviridae, contains a positive-strand RNA genome with three open reading frames. ORF1 encodes a 1,789-amino-acid polyprotein that is processed into nonstructural proteins that include an NTPase, VPg, protease, and RNA-dependent RNA polymerase. The N-terminal protein p48 of ORF1 shows no significant sequence similarity to viral or cellular proteins, and its function in the human calicivirus replication cycle is not known. The lack of sequence similarity to any protein in the public databases suggested that p48 may have a unique function in the NV replication cycle or, alternatively, may perform a characterized function in replication by a unique mechanism. In this report, it is shown that p48 displays a vesicular localization pattern in transfected cells when fused to the fluorescent reporter EYFP. A predicted transmembrane domain at the C terminus of p48 was not necessary for the observed localization pattern, but this domain was sufficient to redirect localization of EYFP to a fluorescent pattern consistent with the Golgi apparatus. A yeast two-hybrid screen identified the SNARE regulator vesicle-associated membrane protein-associated protein A (VAP-A) as a binding partner of p48. Biochemical assays confirmed that p48 and VAP-A interact and form a stable complex in mammalian cells. Furthermore, expression of the vesicular stomatitis virus G glcyoprotein on the cell surface was inhibited when cells coexpressed p48, suggesting that p48 disrupts intracellular protein trafficking.

The genome-linked protein VPg of the Norwalk virus binds eIF3, suggesting its role in translation initiation complex recruitment.

The positive-strand RNA genomes of caliciviruses are not capped, but are instead covalently linked at their 5' ends to a viral protein called VPg. The lack of a cap structure typical of eukaryotic mRNA and absence of an internal ribosomal entry site suggest that VPg may function in translation initiation on calicivirus RNA. This hypothesis was tested by analyzing binding of Norwalk virus VPg to translation initiation factors. The eIF3d subunit of eIF3 was identified as a binding partner of VPg by yeast two-hybrid analysis. VPg bound to purified mammalian eIF3 and to eIF3 in mammalian cell lysates. To test the effects of the VPg- eIF3 interaction on translation, VPg was added to cell-free translation reactions programmed with either capped reporter RNA, an RNA containing an EMCV internal ribosomal entry site (IRES) or an RNA with a cricket paralysis virus IRES. VPg inhibited translation of all reporter RNAs in a dose-dependent manner. Together, the data suggest that VPg may play a role in initiating translation on calicivirus RNA through unique protein-protein interactions with the translation machinery.

Snow Mountain virus genome sequence and virus-like particle assembly.

Snow Mountain virus (SMV) belongs to the Norovirus genus of the Caliciviridae family. SMV is a genogroup II (GII) reference strain of human enteric caliciviruses associated with epidemic gastroenteritis. In this study, the positive sense RNA genome sequence of SMV was determined to be 7,537 nucleotides in length excluding the 3' polyadenylated tract. The genome is organized into three open reading frames typical of caliciviruses in the Norovirus genus. Pairwise sequence alignments showed SMV ORF1 is highly conserved with other genogroup II noroviruses, and most closely related to GII strains Melksham and Hawaii virus. In addition, comparative sequence analyses indicated that SMV is likely a recombinant norovirus. VP1/VP2 proteins self-assembled into virus-like particles (VLPs) when expressed in insect cells by a recombinant baculovirus. Characterization of one clone that expressed VP1, but failed to assemble into VLPs, identified histidine residue 91 as important for particle assembly under standard conditions of expression.

Translational regulation of rotavirus gene expression.

Rotavirus mRNAs are transcribed from 11 genomic dsRNA segments within a subviral particle. The mRNAs are extruded into the cytoplasm where they serve as mRNA for protein synthesis and as templates for packaging and replication into dsRNA. The molecular steps in the replication pathway that regulate the levels of viral gene expression are not well defined. We have investigated potential mechanisms of regulation of rotavirus gene expression by functional evaluation of two differentially expressed viral mRNAs. NSP1 (gene 5) and VP6 (gene 6) are expressed early in infection, and VP6 is expressed in excess over NSP1. We formulated the hypothesis that the amounts of NSP1 and VP6 were regulated by the translational efficiencies of the respective mRNAs. We measured the levels of gene 5 and gene 6 mRNA and showed that they were not significantly different, and protein analysis indicated no difference in stability of NSP1 compared with VP6. Polyribosome analysis showed that the majority of gene 6 mRNA was present on large polysomes. In contrast, sedimentation of more than half of the gene 5 mRNA was subpolysomal. The change in distribution of gene 5 mRNA in polyribosome gradients in response to treatment with low concentrations of cycloheximide suggested that gene 5 is a poor translation initiation template compared with gene 6 mRNA. These data define a regulatory mechanism for the difference in amounts of VP6 and NSP1 and provide evidence for post-transcriptional control of rotavirus gene expression mediated by the translational efficiency of individual viral mRNAs.

Substrate specificity of the Norwalk virus 3C-like proteinase.

The Norwalk Virus (NV) is the prototype strain of human caliciviruses that cause epidemic outbreaks of foodborne and waterborne gastroenteritis. These viruses do not grow in cell culture and the mechanisms of virus replication are obscure. The NV genome is a 7.7 kb ssRNA molecule that encodes three open reading frames (ORFs). The first ORF is a 1789 amino acid polyprotein that is processed into nonstructural proteins by a viral protease similar to the picornavirus 3C protease. Primary cleavage sites in the ORF1 polyprotein of two Norwalk-like viruses have been identified as QG dipeptides. We studied primary cleavage sites in the NV polyprotein and residues surrounding the scissile bond that are important in substrate recognition. A series of mutations were made at amino acids occupying positions implicated as important in cleavage site recognition for chymotrypsin-like viral proteases. We determined that effective processing at amino acid 398 to release the N-terminal p48 protein is necessary for proteolytic release of the p41 protein, that the P4 position N-terminal to the scissile bond is important for efficient processing, and that substitution of large hydrophobic residues were tolerated at this position. Finally, we defined the acidic residue of the 3CL(pro) catalytic site.