Processing of Norwalk virus nonstructural proteins by a 3C-like cysteine proteinase

Expression of Norwalk virus nonstructural polyprotein precursor in vitro resulted in rapid cotranslational cleavage at specific sites. The cleavage products were similar to those previously identified for Southampton virus, a highly related virus. We inactivated the virally encoded proteinase responsible for cleavage of the nonstructural polyprotein by mutation of the putative catalytic cysteine residue, which resulted in production of full-length polyprotein precursor. NV proteinase was expressed in Escherichia coli as a glutathione S-transferase fusion and purified by GST-affinity chromatography. Activity of the purified proteinase was demonstrated by incubation with the full-length precursor protein. trans cleavage of the nonstructural protein precursor resulted in cleavage products similar to those observed during cotranslational cleavage, however, at lesser efficiency. NV proteinase displayed sensitivities to cysteine and serine protease inhibitors similar to poliovirus 3C proteinase, suggesting that NV proteinase is a member of the viral cysteine proteinase family. We propose that the proteinase may play a regulatory role in viral replication.     

Activity of a purified His-tagged 3C-like proteinase from the coronavirus infectious bronchitis virus

Previous studies in vitro of the processing of cloned polyprotein fragments from the coronavirus infectious bronchitis virus (IBV) large open reading frame (ORF1), confirmed the activity of a predicted 3C-like proteinase (3CLP) domain and suggested that the proteinase is released autocatalytically from the polyprotein in the form of a 35 kDa protein, 3CLpro, capable of further cleavages in trans. In order to identify such cleavages within the ORF1 polyprotein mediated by 3CLpro, the proteinase was expressed in bacteria, purified and used in trans cleavage assays with polyprotein fragments lacking the 3CLP domain as targets. The proteinase was expressed as a polyprotein fragment which was able to process during expression in bacterial cells, releasing mature 3CLpro. A histidine (His₆) tag was introduced close to the C-terminus of the proteinase to aid purification. Processing demonstrated by the tagged proteinase was indistinguishable from that of the wild-type enzyme indicating that the site chosen for the tag was permissive. From these studies we were able to demonstrate trans cleavages consistent with the use of most of the previously predicted or identified sites within the open reading frame of gene 1. This tentatively completes the processing map for the ORF1 region with respect to 3CLpro.     

Further characterization of the coronavirus infectious bronchitis virus 3C-like proteinase and determination of a new cleavage site

Coronavirus infectious bronchitis virus (IBV) encodes a trypsin-like proteinase (3C-like proteinase) by ORF 1a, which has been demonstrated to play a pivotal role in proteolytic processing of gene 1-encoded polyproteins. In our previous studies, the proteinase was identified as a 33-kDa protein in IBV-infected cells, and its catalytic center was shown to consist of H(2820) and C(2922) residues. It is released from the 1a and 1a/1b polyproteins by autoprocessing at two Q-S dipeptide bonds (Q(2779)-S(2780) and Q(3086)-S(3087)). In this report, further characterization of the two cleavage sites demonstrates that the N-terminal Q(2779)-S(2780) site is tolerant to mutations at the P1 position. Deletion of the C-terminal region of the proteinase shows that a significant amount of the enzymatic activity is maintained upon deletion of up to 67 amino acids, suggesting that the extreme C-terminal region may be dispensable for the proteolytic activity of the proteinase. Analysis of the autoprocessing kinetics in vitro reveals that proteolysis at the Q(2779)-S(2780) site is the first cleavage event mediated by this proteinase. This is followed by cleavage at the Q(3086)-S(3087) site. The occurrence of both cleavage events in intact cells is potentially rapid and efficient, as no intermediate cleavage products covering the proteinase were detected in either IBV-infected or transfected cells. Immunofluorescence microscopy and subcellular fractionation studies further show differential subcellular localization of the proteinase in IBV-infected cells and in cells expressing the 3C-like proteinase alone, indicating that additional roles in viral replication might be played by this protein. Finally, a Q-A (Q(3379)-A(3380)) dipeptide bond encoded by nucleotides 10,663 to 10,668 was demonstrated to be a cleavage site of the proteinase.     

Substrate specificity of the neuraminidase of different mumps virus strains

The hydrolysis of neuraminlactose, fetuin, ovomucoid, kappa-caseinglycopeptide and mucine from the bovine submaxillary gland with the neuraminidase (NA) of mumps virus strains Jeryl Lynn, Enders and Berlin 9/76 was investigated to determine the pH-dependence of the reaction with the different substrates. The corresponding curves showed splitting into several peaks with reaction maxima ranging from pH 4.7 to pH 6.7. This occurred probably due to the heterogeneity of the virus samples. The NA of each virus strain had the best reaction affinity to neuraminlactose followed by decreasing affinities to ovomucoid, fetuin and kappa-caseinglycopeptide. The mucine from bovine submaxillary gland was not digested at all. The highest hydrolysis of each substrate was found with the Jeryl Lynn strain, which also possessed the lowest substrate specificity. It was followed by that of strain Enders and finally by isolate Berlin 9/76 which, in turn, had the highest substrate specificity. The lower substrate specificity of mumps virus NA seemed to correlate with a lower degree of its cytopathogenicity for hamster ependyma cells.     

Expression and partial purification of recombinant tomato ringspot nepovirus 3C-like proteinase: comparison of the activity of the mature proteinase and the VPg-proteinase precursor

The 3C-like proteinase (Pro) from Tomato ringspot virus (genus Nepovirus) is responsible for the processing of the RNA1-encoded (P1) and RNA2-encoded (P2) polyproteins. Cleavage between the VPg and Pro domains is inefficient in vitro and in E. coli, resulting in the accumulation of the VPg-Pro. In this study, we have compared the trans-activity of the Pro and VPg-Pro on various P1- and P2-derived precursors. Recombinant Pro and VPg-Pro were partially purified using an E. coli expression system. A mutation of the VPg-Pro cleavage site was introduced into the VPg-Pro to prevent slow release of the Pro. The Pro was five to ten times more active than the VPg-Pro on two P2 cleavage sites (at the N- and C-termini of the movement protein domain) and was approximately two times more active than the VPg-Pro on the third P2 cleavage site (between the X3 and X4 domains). Neither the Pro nor the VPg-Pro could cleave in trans P1-derived substrates containing the three cleavage sites delineating the X1, X2, putative NTP-binding protein and VPg domains. These results are discussed in light of the possible regulation of the proteinase activity during virus replication.     

Completion of the Norwalk virus genome sequence

Norwalk virus (NV) is the prototype human calicivirus, and causes epidemic outbreaks of acute gastroenteritis. The sequence and predicted genome organization of NV and a NV-like virus [Southampton virus (SHV)] suggested they are similar viruses at the nucleotide and amino acid level, although SHV was reported to be antigenically distinct from NV. A recent review described the discovery of an additional 12 nucleotides at the 5 end of SHV and prompted us to investigate the possibility of additional nucleotides at the 5 end of the NV genome. The results obtained by homopolymeric tailing of NV cDNA with dCTP and dATP showed 12 additional nucleotides also are present on the NV genomic RNA. These data are important with respect to the biology of the virus, and make the genome sequence of NV complete.     

Substrate specificity of Xenopus matrix metalloproteinase stromelysin-3

The matrix metalloproteinase stromelysin-3 (ST3 or MMP11) was initially identified as a breast carcinoma associated protease and has since been shown to be highly expressed in diverse carcinomas and in developmental processes that involve extensive cell death (apoptosis) and tissue remodeling. Unlike other MMPs, purified ST3 has little activity toward known extracellular matrix (ECM) proteins in vitro but cleaves strongly a few non-ECM, extracellular proteins, including human alpha1-proteinase inhibitor (alpha1-PI). To investigate the possibility of alpha1-PI as a conserved physiological substrate for ST3 during vertebrate development, we analyzed the ability of Xenopus laevis ST3 catalytic domain to cleave frog alpha1-PI. Surprisingly, we found the ST3 failed to recognize the site in alpha1-PI equivalent to the major cleavage site in human alpha1-PI by mammalian ST3. Sequence and mutagenic analysis revealed that multiple substitutions at P2-P3' positions between human and Xenopus alpha1-PI contributed to the inability of Xenopus alpha1-PI to be cleaved by ST3. Our studies showed that (A)(G/A)(A)(M)(F/A)(L) (P3-P3') as a preferred cleavage site for ST3. We further demonstrated that mutations in the cleavage sites affected cleavage by ST3 differently from several other MMPs. These findings, together with earlier reports on ST3, showed that ST3 has distinct substrate specificities compared to other MMPs. Our results further suggest that alpha1-PI is unlikely to be a physiological substrate for ST3, at least with regard to evolutionarily conserved developmental processes.     

Substrate specificity of the herpes simplex virus type 2 UL13 protein kinase

The UL13 protein kinase is conserved among many herpesviruses but HSV-2 UL13 specificity is not known. Here, we found that HSV-2 UL13 is a phosphoprotein that autophosphorylates, and that serines within ERK and Cdc2 motifs were important for autophosphorylation but not for UL13 phosphorylation of exogenous substrates. HSV-2 UL13 phosphorylated a peptide also recognized by ERK and Cdc2. However, mutation of substrate residues critical for Cdc2 or Erk phosphorylation did not alter HSV-2 UL13 phosphorylation of the peptide, and HSV-2 UL13 did not phosphorylate standard Cdc2 or Erk peptide substrates. Mutation of prolines surrounding the peptide phosphoacceptor site reduced phosphorylation by HSV-2 UL13, and a peptide containing serine-proline amid alanines and glycines was phosphorylated. Thus, HSV-2 UL13 does not mimic ERK or Cdc2 substrate recognition and its minimal recognition motif can be serine-proline. This motif's simplicity indicates that distal sequence or protein structure contributes to HSV-2 UL13 substrate specificity.     

Characterization of the norovirus 3C-like protease

The recombinant 3C-like protease of Chiba virus, a Norovirus, expressed in Escherichia coli cells was purified and characterized as to effects of pH, temperature, salt contents, and SH reagents on its proteolytic activity. The optimal pH and temperature of the 3C-like protease for the proteolytic activity were 8.6 and 37 °C, respectively. Increased concentration (100 mM) of monovalent cations such as Na⁺ and K⁺ was inhibitory to the activity. Hg²⁺ and Zn²⁺ remarkably inhibited the protease activity, while Mg²⁺ and Ca²⁺ had no virtual effect. Several sulfhydryl reagents such as p-chloromercuribenzoic acid, methyl methanethiosulfonate, N-ethylmaleimide and N-phenylmaleimide also blocked the activity, confirming the previous result that cysteine residue(s) were responsible for the proteolysis.     

Substrate specificity of adenovirus protease

The adenovirus protease, adenain is functionally required for virion uncoating and virion maturation and release from the infected cell. In addition to hydrolysis of precursor proteins at specific consensus sites, adenain has also been observed to cleave viral proteins at other sites. Here we re-examine the sequences in the consensus sites and also the phenomena of cleavage at other sites on viral proteins II, 100K, V, VI and VII. An examination of the eight residues flanking the scissile bond in 274 consensus sites from 36 different adenovirus serotypes in the DNA sequence databanks provided the following main conclusions: (1) two types of consensus sites, type 1, (M,I,L)XGX-G and type 2, (M,I,L)XGG-X, (2) the variant positions P(3) and P(1) never contained C,P,D,H,W,Y and C,P,G,M amino acids, respectively in type 1, (3) the variant positions P(3) and P(1)' never contained C,D,L,W and C,P,D,Q,H,Y,W amino acids, respectively in type 2, and (4) the thiol forming C residue occurred only twice within the eight residues flanking the scissile bond and that in the P(4)' position. Six unusual serotypes had (M,L,I)XAT-G as the PVII consensus site. Adenain has been proposed to cleave protein VI at an unknown site in the course of virion uncoating. The cleavage of capsid protein VI in the absence of a consensus site is confirmed here in vitro using recombinant adenain. Virion proteins II, V and VII and the nonstructural protein 100K were also digested in vitro into discrete fragments by recombinant adenain. We conclude that adenain preferentially cleaves viral proteins at their consensus sites, but is capable, in vitro of cleavages at other discrete sites which resemble the consensus cleavage sites.     

Immunoglobulin M responses to the Norwalk virus of gastroenteritis.

Eighty-seven serum specimens from 20 human subjects experimentally inoculated one or more times with Norwalk virus were quantitatively examined for virus-specific immunoglobulin M (IgM). A sensitive and specific radioimmunoassay for anti-Norwalk virus blocking activity was applied to whole serum and to separate IgM and IgG fractions obtained by sucrose density gradient ultracentrifugation. The peak IgM response occurred at about 2 weeks after illness, but IgM was detectable at lower titers for up to 21 weeks after infection. The IgM response was seen in volunteers who became ill, whether or not prechallenge total serum antibody was present. On long-term (27 to 42 months) rechallenge, volunteers who were previously ill and had produced IgM antibody again developed illness, and a secondary IgM response greater than the first was detected. Inoculated volunteers who did not develop illness, as well as previously ill volunteers on short-term rechallenge (4 to 14 weeks), usually failed to generate an IgM response, whether or not an IgG response had occurred. In ill subjects, the rise in IgM and IgG occurred concomitantly. Virus-specific IgM is not necessarily indicative of primary infection with Norwalk agent inasmuch as reinfection produces an enhancement of the IgM response. Furthermore, Norwalk-specific IgM responses do not appear to be associated with subclinical illness.     

Further requirements for cleavage by the murine coronavirus 3C-like proteinase: identification of a cleavage site within ORF1b.

The coronavirus mouse hepatitis virus strain A59 (MHV-A59) encodes a 3C-like proteinase (3CLpro) that is proposed to be responsible for the majority of the processing events that take place within the replicase polyproteins pp1a and pp1ab. In this study we demonstrate that the Q939/S940 peptide bond, located between the polymerase and Zn-finger regions of pp1ab (the POL/Zn site), is processed by the 3CLpro, albeit inefficiently. Mutagenesis of the POL/Zn site, as well as the previously identified HD1/3C site in the 1a region of pp1a and pp1ab, demonstrated that the amino acid residues at the P2 and P1 positions of the cleavage site, occupied by L and Q, respectively, were important determinants of 3CLpro substrate specificity. Finally, a direct comparison of the 3CLpro-mediated cleavages at the HD1/3C and POL/Zn sites was made by determining the rate constants using synthetic peptides. The results show that while a larger polypeptide substrate carrying the HD1/3C site was processed more efficiently than a polypeptide substrate carrying the POL/Zn site, cleavage of the synthetic peptide substrates containing these two cleavage sites occurred at similar efficiencies. This indicates that the overall conformation of a large polyprotein substrate is important in the accessibility of the cleavage site to the proteinase.     

Prevalence of antibodies to Norwalk virus in England: detection by enzyme-linked immunosorbent assay using baculovirus-expressed Norwalk virus capsid antigen.

A total of 3,250 serum specimens collected in England in 1991 and 1992 were tested by an indirect enzyme-linked immunosorbent assay for antibody to Norwalk virus using baculovirus-expressed capsid antigen, and 2,382 (73.3%) were positive. The prevalence of Norwalk virus antibody differed regionally. It was lowest (24.6%) in 6- to 11-month-old infants and increased to 89.7% in persons over 60 years old.     

Prevalence of antibody to the Norwalk virus in various countries.

Serum samples from children and adults from several countries were tested by radioimmunoassay for antibody to the Norwalk virus. Antibody was commonly found in adults from all the countries tested. Antibody appears to be acquired more rapidly in children from underdeveloped countries than in children from the United States.     

Complete nucleotide sequence of the chiba virus genome and functional expression of the 3C-like protease in Escherichia coli

We cloned the genome RNA of the Chiba virus (ChV; Hu/NLV/Chiba 407/1987/JP) and determined its complete nucleotide sequence. The genome is predicted to be a positive-sense, single-stranded RNA of 7697 bases, excluding a poly(A) tract. Comparison of the nucleotide and amino acid sequences with those of other members of the species Norwalk virus (NV) revealed that ChV belongs to genogroup I NV. The ChV genome contains three open reading frames (ORFs). A large 5'-terminal ORF (ORF1) encodes a polyprotein with 1785 amino acids that are likely processed into functional proteins, including RNA helicase, VPg, protease, and RNA-dependent RNA polymerase. ORF2 encodes the capsid protein with 544 amino acids, and a small 3'-terminal ORF (ORF3) encodes a basic protein with 208 amino acids. The amino acid sequences of five cleavage sites in ORF1 are highly conserved compared with those of other members of NV. When expressed in Escherichia coli, the glutathione-S-transferase (GST) fusion protein of the ChV protease connected via a short peptide containing a human rhinovirus 3C protease cleavage site was cleaved into GST and the protease; however, this cleavage did not occur when the Cys mutation was introduced into the putative active site of the protease. Moreover, the ChV protease recognized and cleaved the predicted proteolytic sites between VPg and protease and between protease and RNA polymerase. Therefore, the ChV protease expressed in E. coli retained an enzymatic activity and a substrate specificity similar to that of the human rhinovirus 3C protease.     

Detection of Norwalk virus in stools by enzyme immunoassay

The development of a solid-phase microtiter enzyme immunoassay (EIA) for detection of Norwalk virus antigen in stool samples is described. The EIA was compared with a previously developed radioimmunoassay (RIA) for detection of Norwalk virus antigen in stools obtained from 30 volunteers who received Norwalk virus. The EIA detected viral antigen in stools from 17 of the volunteers and the RIA detected viral antigen in 15. Seroconversion was a more sensitive indicator of infection in some patients. However, two samples from volunteers who were clinically ill but did not show seroconversion to Norwalk virus were positive for Norwalk virus antigen by both immunoassays. This indicates that antigen detection may be important for use in epidemiological studies. Neither of the immunoassays gave positive reactions for stools known to contain enteric adenovirus, rotavirus, or Hawaii virus, or in stools from patients with acute diarrhea of unknown cause. The stability of the EIA reagents and ease of use should provide a means for more extensive testing for Norwalk virus in outbreaks of gastroenteritis.     

Comparison of the reactivities of baculovirus-expressed recombinant Norwalk virus capsid antigen with those of the native Norwalk virus antigen in serologic assays and some epidemiologic observations.

Since the discovery of the Norwalk virus (NV) by immune electron microscopy (IEM) in 1972, serologic studies with this virus have relied on particle-positive fecal material from infected volunteers as the source of antigen because it has not been possible to propagate this virus in cell culture. However, the recent cloning of the NV (strain 8FIIa) genome and expression of the capsid protein in a baculovirus system to form "virus-like particles" has provided a consistent source of antigen (designated rNV). The purpose of the present study was to compare the antigenicities of these rNV particles with those of native NV antigen derived from human fecal material by using well-characterized sera obtained from earlier studies. In IEM studies, the rNV antigen reacted with NV-specific antibodies in a manner similar to that observed previously when particle-positive fecal material was used as antigen. In addition, a direct enzyme-linked immunosorbent assay, in which the rNV antigen was used as antigen, proved efficient and specific for the detection of serologic responses to NV compared with the previously established techniques of IEM and blocking antibody immunoassays in which particle-positive fecal material was used as the antigen. The availability of an unlimited source of antigen will enable serologic studies that will greatly increase our understanding of the epidemiology of NV and its role in human enteric illness.