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PubMed1980

Biochemical and immunological characterization of polyproteins coded for by the McDonough, Gardner-Arnstein, and Snyder-Theilen strains of feline sarcoma virus.

The McDonough (SM), Gardner-Arnstein (GA), and Snyder-Theilen (ST) strains of feline sarcoma virus (FeSV) code for high-molecular-weight polyproteins that contain varying amounts of the amino-terminal region of the FeLV gag gene-coded precursor protein and a polypeptide(s) of an as yet undetermined nature. The SM-FeSV primary translational product is a 180,000-dalton polyprotein which is immediately processed into a highly unstable 60,000-dalton molecule containing the p15-p12-p30 fragment of the FeLV gag gene-coded precursor protein and a 120,000-dalton FeSV-specific polypeptide. The GA- and ST-FeSV genomes code for polyproteins of 95,000 and 85,000 daltons, respectively, which in addition to the amino-terminal moiety (p15-12 and a portion of p30) of the FeLV gag gene-coded precursor protein also contain FeSV-specific polypeptides. However, the GA- and ST-FeSV polyproteins appear to be relatively stable molecules (half-lives of around 16 h) and are not significantly processed into smaller polypeptides. Immunological and biochemical analysis of each of the above FeSV translational products revealed that the sarcoma-specific regions of the GA- and ST-FeSV polyproteins are antigenically cross-reactive and exhibit common methionine-containing peptides. These findings favor the concept that these sarcoma-specific polypeptides are coded for by the similar subsets of cellular sequences incorporated into the GA- and ST-FeSV genomes during the generation of these transforming agents.

Animals↗
PubMed1981

Involvement of a high-molecular-weight polyprotein translational product of Snyder-Theilen Feline sarcoma virus in malignant transformation.

The previously described high-molecular-weight polyprotein major translational product of the Snyder-Theilen strain of feline sarcoma virus (FeSV) was shown to possess protein kinase activity with specificity for tyrosine acceptor sites. Cells transformed by Snyder-Theilen FeSV exhibited constitutively elevated levels of phosphotyrosine and a concomitant reduction in epidermal growth factor (EGF) binding sites. By endpoint cloning in microtiter plates, a number of transformation-defective (tf) mutants of the Snyder-Theilen strain of FeSV were isolated. Mink cells nonproductively infected by such mutants were morphologically nontransformed, failed to grow in soft agar, bound EGF as efficiently as control mink cells, and lacked rescuable transforming virus. Although the level of expression of the major viral polyprotein translational product in td mutant-infected clones was comparable to that of wild-type (wt) transformants, the polyprotein in mutant clones lacked detectable protein kinase activity and total cellular phosphotyrosine levels were not elevated significantly above control values. Of a large number of wt Snyder-Theilen FeSV-transformed mink cell clones isolated, the majority were found to revert to a nontransformed morphology upon continuous passage in cell culture. Such nontransformed variants, as well as a Gardner FeSV-transformed mink cell revertant, lacked detectable polyprotein expression and exhibited levels of phosphotyrosine and EGF binding similar to those of control mink cells. These findings provide strong evidence favoring the involvement of the Snyder-Theilen FeSV-encoded high-molecular-weight polyprotein and its associated tyrosine-specific protein kinase activity in transformation.

Animals↗
PubMed1982

Monoclonal antibodies specific to transforming polyproteins encoded by independent isolates of feline sarcoma virus.

Hybridomas secreting monoclonal antibodies directed against polyprotein gene products of the Gardner, Snyder-Theilen, and McDonough strain of feline sarcoma virus have been isolated. Antibody produced by one hybridoma recognizes immunological determinants localized within a feline leukemia virus gag gene structural component (p15) common to polyproteins encoded by each feline sarcoma virus isolate while antibody produced by a second is specific for p30 determinants unique to P170gag-fms. Additional hybridomas secrete antibody directed against v-fes specific determinants common to the Gardner and Snyder-Theilen feline sarcoma virus-encoded polyproteins and to v-fms determinants unique to P170gas-fms polyprotein. GA P110gas-fes and ST P85gas-fes immunoprecipitated by antibody directed against p15 exhibit readily detectable levels of protein kinase activity but lack such activity when precipitated by antibody specific for their acquired sequence (v-fes) components. P170gas-fms immunoprecipitated by monoclonal antibody to either p15 or p30 lacks detectable levels of autophosphorylation but represents a substrate for the GA P110gag-fes and ST P85gag-fes enzymatic activities. These findings argue that the v-fes-associated protein kinase represents an intrinsic property of the v-fes gene product and recognizes tyrosine acceptor sites within polyprotein gene products of all three strains of feline sarcoma virus.

Animals↗
PubMed1989

Processing the nonstructural polyproteins of sindbis virus: nonstructural proteinase is in the C-terminal half of nsP2 and functions both in cis and in trans.

The processing of the Sindbis virus nonstructural polyprotein translated in vitro has been studied. When Sindbis virus genomic RNA was translated in a reticulocyte lysate, polyprotein P123 was cleaved efficiently to produce nsP1, nsP2, and nsP3. Inhibition of this processing by anti-nsP2 antibodies, but not by antibodies specific for nsP1, nsP3, or nsP4, suggested that the viral proteinase was present in nsP2. To localize the proteolytic activity more precisely, deletions were made in a full-length cDNA clone of Sindbis virus, and RNA was transcribed from these constructs with SP6 RNA polymerase and translated in vitro. Although virtually all of the nsP1, nsP3, and nsP4 sequences could be deleted without affecting processing, deletions in the N-terminal half of nsP2 led to aberrant processing, and deletions in the C-terminal half abolished proteolysis. However, inactive polyproteins containing the nsP2 deletions could be processed by exogenously supplied proteins translated from virion RNA, demonstrating that cleavage was virus specific and not due to a protease present in the reticulocyte lysate and that the deleted polyproteins still served as substrates for the enzyme. From these results and from experiments in which processing was studied at increasingly higher dilution, we have concluded the following: (i) the viral nonstructural proteinase is located in the C-terminal half of nsP2; (ii) in the P123 precursor the cleavage between nsP2 and nsP3 occurs efficiently as a bimolecular reaction (in trans) to remove nsP3, while the bond between nsP1 and nsP2 is cleaved inefficiently, but detectably, in trans, but no autoproteolysis of P123 was detected; (iii) once nsP3 has been removed, the bond between nsP1 and nsP2 in the P12 precursor is cleaved efficiently by autoproteolysis (in cis). This mode of processing leads to a slow rate of cleavage, particularly early in infection, suggesting that the polyproteins might play roles in virus RNA replication distinct from those of the cleaved products. A hypothesis is presented that the proteinase is a thiol protease related to papain.

Amino Acid Sequence↗
PubMed1991

The hepatitis A virus polyprotein expressed by a recombinant vaccinia virus undergoes proteolytic processing and assembly into viruslike particles.

Hepatitis A virus (HAV) contains a single-stranded, plus-sense RNA genome with a single long open reading frame encoding a polyprotein of approximately 250 kDa. Viral structural proteins are generated by posttranslational proteolytic processing of this polyprotein. We constructed recombinant vaccinia viruses which expressed the HAV polyprotein (rV-ORF) and the P1 structural region (rV-P1). rV-ORF-infected cell lysates demonstrated that the polyprotein was cleaved into immunoreactive 29- and 33-kDa proteins which comigrated with HAV capsid proteins VP0 and VP1. The rV-P1 construct produced a 90-kDa protein which showed no evidence of posttranslational processing. Solid-phase radioimmunoassays with human polyclonal anti-HAV sera and with murine or human neutralizing monoclonal anti-HAV antibodies recognized the rV-ORF-infected cell lysates. Sucrose density gradients of rV-ORF-infected cell lysates contained peaks of HAV antigen with sedimentation coefficients of approximately 70S and 15S, similar to those of HAV empty capsids and pentamers. Immune electron microscopy also demonstrated the presence of viruslike particles in rV-ORF-infected cell lysates. Thus, the HAV polyprotein expressed by a recombinant vaccinia virus demonstrated posttranslational processing into mature capsid proteins which assembled into antigenic viruslike particles.

Base Sequence↗
PubMed1992

Cleavage of the dengue virus polyprotein at the NS3/NS4A and NS4B/NS5 junctions is mediated by viral protease NS2B-NS3, whereas NS4A/NS4B may be processed by a cellular protease.

The cleavage mechanism utilized for processing of the NS3-NS4A-NS4B-NS5 domain of the dengue virus polyprotein was studied by using the vaccinia virus expression system. Recombinant vaccinia viruses vNS2B-NS3-NS4A-NS4B-NS5, vNS3-NS4A-NS4B-NS5, vNS4A-NS4B-NS5, and vNS4B-NS5 were constructed. These recombinants were used to infect cells, and the labeled lysates were analyzed by immunoprecipitation. Recombinant vNS2B-NS3-NS4A-NS4B-NS5 expressed the authentic NS3 and NS5 proteins, but the other recombinants produced uncleaved polyproteins. These findings indicate that NS2B is required for processing of the downstream nonstructural proteins, including the NS3/NS4A and NS4B/NS5 junctions, both of which contain a dibasic amino acid sequence preceding the cleavage site. The flavivirus NS4A/NS4B cleavage site follows a long hydrophobic sequence. The polyprotein NS4A-NS4B-NS5 was cleaved at the NS4A/NS4B junction in the absence of other dengue virus functions. One interpretation for this finding is that NS4A/NS4B cleavage is mediated by a host protease, presumably a signal peptidase. Although vNS3-NS4A-NS4B-NS5 expressed only the polyprotein, earlier results demonstrated that cleavage at the NS4A/NS4B junction occurred when an analogous recombinant, vNS3-NS4A-84%NS4B, was expressed. Thus, it appears that uncleaved NS3 plus NS5 inhibit NS4A/NS4B cleavage presumably because the putative signal sequence is not accessible for recognition by the responsible protease. Finally, recombinants that expressed an uncleaved NS4B-NS5 polyprotein, such as vNS4A-NS4B-NS5 or vNS4B-NS5, produced NS5 when complemented with vNS2B-30%NS3 or with vNS2B plus v30%NS3. These results indicate that cleavage at the NS4B/NS5 junction can be mediated by NS2B and NS3 in trans.

Animals↗
PubMed1993

NS3 is a serine protease required for processing of hepatitis C virus polyprotein.

Hepatitis C virus (HCV) possesses a positive-sense RNA genome which encodes a large polyprotein of 3,010 amino acids. Previous data and sequence analysis have indicated that this polyprotein is processed by cellular proteases and possibly by a virally encoded serine protease localized in the N-terminal domain of nonstructural protein NS3. To characterize the molecular aspects of HCV protein biogenesis and to clearly identify the protein products derived from the HCV genome, we have examined HCV polyprotein expression by using the vaccinia virus T7 transient expression system in transfected cells and by cell-free translation studies. HCV proteins were identified by immunoprecipitation with region-specific antisera. Here we show that the amino-terminal region of the HCV polyprotein is processed in vitro by cellular proteases releasing three structural proteins: p21 (core), gp37 (E1), and gp61 (E2). Processing of the nonstructural region of HCV was evident in transfected cells. Two proteins of 24 and 68 kDa were immunoprecipitated with anti-NS2 and NS3 antisera, respectively. Antiserum against NS4 recognized three proteins of 6, 26, and 31 kDa, while antisera specific for NS5 immunoprecipitated two polypeptides of 56 and 65 kDa, indicating that each of these two genes encodes at least two different proteins. When the NS3 protease domain was inactivated by replacing the proposed catalytic Ser-1165 with Ala, processing at several sites was abolished. When Ser-1164 was mutated to Ala, no effect on the processing was observed. Cleavage activities at three of the four sites affected by NS3 were shown to occur in trans, while processing at the carboxy terminus of NS3 could not be mediated in trans. These results provide a detailed description of the protein products obtained from the processing of the HCV polyprotein. Furthermore, the data obtained implicate NS3 as a serine protease and demonstrate that a catalytically active NS3 is necessary for cleavage of the nonstructural region of HCV.

Antigens, Viral↗
PubMed1997

Proteolytic processing in African swine fever virus: evidence for a new structural polyprotein, pp62.

We have identified an open reading frame (ORF), CP530R, within the EcoRI C' fragment of the African swine fever virus (ASFV) genome that encodes a polyprotein of 62 kDa (pp62). Antisera raised against different regions of ORF CP530R recognized a polypeptide of 62 kDa in ASFV-infected cells during the late phase of virus replication, after the onset of viral DNA synthesis. Pulse-chase experiments showed that polyprotein pp62 is posttranslationally processed to give rise to two proteins of 35 kDa (p35) and 15 kDa (p15). This proteolytic processing was found to take place at the consensus sequence Gly-Gly-X through an ordered cascade of proteolytic cleavages like that which also occurs with ASFV polyprotein pp220 (C. Simón-Mateo, G. Andrés, and E. Viñuela, EMBO J. 12:2977-2987, 1993). Immunofluorescence studies showed that polyprotein pp62 is localized in the viral factories. In addition, immunoprecipitation analysis of purified virus particles showed that mature products p35 and p15 are major structural proteins. According to these results, polyprotein processing represents an essential strategy for the maturation of ASFV structural proteins.

African Swine Fever Virus↗
PubMed1998

Mouse hepatitis virus 3C-like protease cleaves a 22-kilodalton protein from the open reading frame 1a polyprotein in virus-infected cells and in vitro.

The 3C-like proteinase (3CLpro) of mouse hepatitis virus (MHV) is predicted to cleave at least 11 sites in the 803-kDa gene 1 polyprotein, resulting in maturation of proteinase, polymerase, and helicase proteins. However, most of these cleavage sites have not been experimentally confirmed and the proteins have not been identified in vitro or in virus-infected cells. We used specific antibodies to identify and characterize a 22-kDa protein (p1a-22) expressed from gene 1 in MHV A59-infected DBT cells. Processing of p1a-22 from the polyprotein began immediately after translation, but some processing continued for several hours. Amino-terminal sequencing of p1a-22 purified from MHV-infected cells showed that it was cleaved at a putative 3CLpro cleavage site, Gln_Ser4014 (where the underscore indicates the site of cleavage), that is located between the 3CLpro domain and the end of open reading frame (ORF) 1a. Subclones of this region of gene 1 were used to express polypeptides in vitro that contained one or more 3CLpro cleavage sites, and cleavage of these substrates by recombinant 3CLpro in vitro confirmed that amino-terminal cleavage of p1a-22 occurred at Gln_Ser4014. We demonstrated that the carboxy-terminal cleavage of the p1a-22 protein occurred at Gln_Asn4208, a sequence that had not been predicted as a site for cleavage by MHV 3CLpro. Our results demonstrate the usefulness of recombinant MHV 3CLpro in identifying and confirming cleavage sites within the gene 1 polyprotein. Based on our results, we predict that at least seven mature proteins are processed from the ORF 1a polyprotein by 3CLpro and suggest that additional noncanonical cleavage sites may be used by 3CLpro during processing of the gene 1 polyprotein.

3C Viral Proteases↗
PubMed2003

Properly folded nonstructural polyprotein directs the semliki forest virus replication complex to the endosomal compartment.

The late RNA synthesis in alphavirus-infected cells, generating plus-strand RNAs, takes place on cytoplasmic vacuoles (CPVs), which are modified endosomes and lysosomes. The cytosolic surface of CPVs consists of regular membrane invaginations or spherules, which are the sites of RNA synthesis (P. Kujala, A. Ikäheimonen, N. Ehsani, H. Vihinen, P. Auvinen, and L. Kääriäinen J. Virol. 75:3873-3884, 2001). To understand how CPVs arise, we have expressed the individual Semliki Forest virus (SFV) nonstructural proteins nsP1 to nsP4 in different combinations, as well as their precursor polyprotein P1234 and its cleavage intermediates. A complex of nsPs was obtained from P123 or P1234, indicating that the precursor stage is essential for the assembly of the polymerase complex. To prevent the processing of the polyprotein and its cleavage intermediates, constructs with the mutation C478A (designated with a superscript CA) in the active site of the protease domain of nsP2 were used. Uncleaved polyproteins containing nsP1 were membrane bound and palmitoylated, and those containing nsP3 were phosphorylated, reflecting properties of authentic nsP1 and nsP3, respectively. Similarly, polyproteins containing nsP1 or nsP2 had enzymatic activities specific for the individual proteins, indicating that they were correctly folded in the precursor state. Uncleaved P12(CA) was localized almost exclusively to the plasma membrane and filopodia, like nsP1 alone, whereas P12(CA)3 and P12(CA)34 were found on cytoplasmic vesicles, some of which contained late endosomal markers. In immunoelectron microscopy these vesicles resembled CPVs in SFV-infected cells. Our results indicate that the nsP1 domain alone is responsible for the membrane association of the nonstructural polyprotein, whereas the nsP1 domain together with the nsP3 domain targets it to the intracellular vesicles.

Endosomes↗
PubMed1993

Cleavage of potato virus Y polyprotein in Escherichia coli depending on the proteolytic activity of viral protease.

We have cloned a 5-kb cDNA corresponding to the 3'-half of genomic RNA of potato virus Y (PVY), the type member of plant potyvirus. Based on its nucleotide sequence, the cDNA was presumed to encode PVY protease responsible for the self-processing of polyprotein precursor expressed from PVY genome. To test its proteolytic activity, the cDNA was modified so as to express the protease-polymerase-coat protein (CP) portion of PVY polyprotein in Escherichia coli. By Western blotting analysis of the cell extract of E. coli expressing the polyprotein, mature CP was detected. A large deletion in the polymerase-coding region did not affect the emergence of mature CP, but a small deletion in the putative protease region resulted in disappearance of mature CP and appearance of the intact polyprotein. These results indicated that the polyprotein expressed in E. coli was cleaved depending on the proteolytic activity of PVY protease.

Amino Acid Sequence↗
PubMed1993

Determination of diabetogenicity attributable to a single amino acid, Ala776, on the polyprotein of encephalomyocarditis virus.

The best experimental evidence indicating that viruses have an etiological role in the pathogenesis of diabetes comes from studies of mice infected with EMC virus. For this study we generated mutant viruses from stocks of diabetogenic EMC-D and nondiabetogenic EMC-B viruses by serial passages of the viruses in L-cell cultures at high MOI. The genomic sequence information and the biological activities of three different plaque-purified diabetogenic variants of EMC virus (EMC-D, EMC-D1/6A, EMC-D2/4) and six different plaque-purified nondiabetogenic variants (EMC-B, EMC-BS, EMC-B1/G, EMC-DV1, EMC-D4/1B, EMC-D3/1) revealed that only one amino acid, Ala (776th amino acid on the polyprotein), is critical for the diabetogenicity of EMC virus. The G base at the nucleotide position 3155 (Ala[GCC]776 in the polyprotein) is unique to all diabetogenic variants, whereas the A base at the same position (Thr[ACC]776 in the polyprotein) is identical to all nondiabetogenic variants. A single-point mutation (G to A; Ala to Thr) results in the conversion of the diabetogenic variant into a nondiabetogenic variant of EMC virus. On the basis of these observations, we conclude that a single amino acid, Ala776, on the polyprotein of EMC virus appears responsible for the inducement of diabetes in susceptible mice. Conversion of Ala776 into Thr776 on the polyprotein by a point mutation, G to A at the nucleotide position 3155, results in the loss of diabetogenicity.

Alanine↗
PubMedDate not supplied

Processing of infectious bursal disease virus (IBDV) polyprotein and self-assembly of IBDV-like particles in Hi-5 cells.

The capsid of infectious bursal disease virus (IBDV), with a size of 60-65 nm, is formed by an initial processing of polyprotein (pVP2-VP4-VP3) by VP4, subsequent assemblage of pVP2 and VP3, and the maturation of VP2. In Sf9 cells, the processing of polyprotein expressed was restrained in the stage of VP2 maturation, leading to a limited production of capsid, i.e., IBDV-like particles (VLPs). In the present study, another insect cell line, High-Five (Hi-5) cells, was demonstrated to efficiently produce VLPs. Meanwhile, in this system, polyprotein was processed to pVP2 and VP3 protein and pVP2 was further processed to the matured form of VP2. Consequently, Hi-5 cells are better in terms of polyprotein processing and formation of VLPs than Sf9. In addition to the processing of pVP2, VP3 was also degraded. With insufficient intact VP3 protein present for the formation of VLPs, the excessive VP2 form subviral particles (SVPs) with a size of about 25 nm. The ratio of VLPs to SVPs is dependent on the multiplicity of infections (MOIs) used, and an optimal MOI is found for the production of both particles. VLPs were separated from SVPs with a combination of ultracentrifugation and gel-filtration chromatography, and a large number of purified particles of both were obtained. In conclusion, the insect cell lines and MOIs were optimized for the production of VLPs, and pure VLPs with morphology similar to that of the wild-type viruses can be effectively prepared. The efficient production and purification of VLPs benefits not only the development of an antiviral vaccine against IBDV but also the understanding of the structure of this avian virus that is economically important.

Animals↗
PubMed2005

Recognition and processing of a nuclear-encoded polyprotein precursor by mitochondrial processing peptidase.

The nuclear-encoded protein RPS14 (ribosomal protein S14) of rice mitochondria is synthesized in the cytosol as a polyprotein consisting of a large N-terminal domain comprising preSDHB (succinate dehydrogenase B precursor) and the C-terminal RPS14. After the preSDHB-RPS14 polyprotein is transported into the mitochondrial matrix, the protein is processed into three peptides: the N-terminal prepeptide, the SDHB domain and the C-terminal mature RPS14. Here we report that the general MPP (mitochondrial processing peptidase) plays an essential role in processing of the polyprotein. Purified yeast MPP cleaved both the N-terminal presequence and the connector region between SDHB and RPS14. Moreover, the connector region was processed more rapidly than the presequence. When the site of cleavage between SDHB and RPS14 was determined, it was located in an MPP processing motif that has also been shown to be present in the N-terminal presequence. Mutational analyses around the cleavage site in the connector region suggested that MPP interacts with multiple sites in the region, possibly in a similar manner to the interaction with the N-terminal presequence. In addition, MPP preferentially recognized the unfolded structure of preSDHB-RPS14. In mitochondria, MPP may recognize the stretched polyprotein during passage of the precursor through the translocational apparatus in the inner membrane, and cleave the connecting region between the SDHB and RPS14 domains even before processing of the presequence.

Cell Nucleus↗
PubMed2002

Antibody responses to HPV6b E polyproteins and production of monoclonal antibodies.

A range of fusion constructs (expressed in Escherichia coli) were produced that contained two or more HPV6b E proteins, producing a single continuous amino acid sequence corresponding to the sequences of the individual E proteins. The constructs also included a C-terminal hexahistidine tag fused in-frame to aid purification. The fusion proteins (polyproteins) were semipurified by Ni(++) metal affinity chromatography under denaturing conditions. Immunization of BALB/c mice with these polyproteins resulted in the production of specific E protein antibodies. The draining lymph nodes from these mice were used to produce monoclonal antibodies (MAbs). The specificity of the polyclonal and MAbs was confirmed by immunoblotting and by screening for reaction with a series of synthetic peptides of E proteins. HPV E polyproteins were found to be immunogenic and immunization with the polyproteins resulted in specific antibody responses to the component E proteins.

Amino Acids↗
PubMed2005

Translation of the F protein of hepatitis C virus is initiated at a non-AUG codon in a +1 reading frame relative to the polyprotein.

The hepatitis C virus (HCV) genome contains an internal ribosome entry site (IRES) followed by a large open reading frame coding for a polyprotein that is cleaved into 10 proteins. An additional HCV protein, the F protein, was recently suggested to result from a +1 frameshift by a minority of ribosomes that initiated translation at the HCV AUG initiator codon of the polyprotein. In the present study, we reassessed the mechanism accounting for the synthesis of the F protein by measuring the expression in cultured cells of a luciferase reporter gene with an insertion encompassing the IRES plus the beginning of the HCV-coding region preceding the luciferase-coding sequence. The insertion was such that luciferase expression was either in the +1 reading frame relative to the HCV AUG initiator codon, mimicking the expression of the F protein, or in-frame with this AUG, mimicking the expression of the polyprotein. Introduction of a stop codon at various positions in-frame with the AUG initiator codon and substitution of this AUG with UAC inhibited luciferase expression in the 0 reading frame but not in the +1 reading frame, ruling out that the synthesis of the F protein results from a +1 frameshift. Introduction of a stop codon at various positions in the +1 reading frame identified the codon overlapping codon 26 of the polyprotein in the +1 reading frame as the translation start site for the F protein. This codon 26(+1) is either GUG or GCG in the viral variants. Expression of the F protein strongly increased when codon 26(+1) was replaced with AUG, or when its context was mutated into an optimal Kozak context, but was severely decreased in the presence of low concentrations of edeine. These observations are consistent with a Met-tRNA(i)-dependent initiation of translation at a non-AUG codon for the synthesis of the F protein.

Base Sequence↗
PubMed2006

Identification of the cleavage sites of sapovirus open reading frame 1 polyprotein.

Sapovirus (SaV), a member of the family Caliciviridae, is a causative agent of acute gastroenteritis in humans and swine and is currently divided into five genogroups, GI-GV. The proteolytic processing of the SaV open reading frame 1 (ORF1) polyprotein with a human GII SaV Mc10 strain has recently been determined and the products are arranged in the following order: NH(2)-p11-p28-p35 (NTPase)-p32-p14 (VPg)-p70 (Pro-Pol)-p60 (VP1)-COOH. The cleavage site between p14 (VPg) and p70 (Pro-Pol) was identified as E(1055)/A(1056) by N-terminal amino acid sequencing. To identify other cleavage sites, a series of GII SaV Mc10 full-length clones containing disrupted potential cleavage sites in the ORF1 polyprotein were constructed and used to generate linear DNA templates for in vitro coupled transcription-translation. The translation products were analysed by SDS-PAGE or by immunoprecipitation with region-specific antibodies. N-terminal amino acid sequencing with Escherichia coli-expressed recombinant proteins was also used to identify the cleavage site between p32 and p14. These approaches enabled identification of the six cleavage sites of the Mc10 ORF1 polyprotein as E(69)/G(70), Q(325)/G(326), Q(666)/G(667), E(940)/A(941), E(1055)/A(1056) and E(1722)/G(1723). The alignment of the SaV full-length ORF1 amino acid sequences indicated that the dipeptides used for the cleavage sites were either E or Q at the P1 position and A, G or S at the P1' position, which were conserved in the GI, GII, GIII, GIV and GV SaV ORF1 polyprotein.

Binding Sites↗
PubMed2006

Cleavage map and proteolytic processing of the murine norovirus nonstructural polyprotein in infected cells.

Murine norovirus (MNV) is presently the only member of the genus Norovirus in the Caliciviridae that can be propagated in cell culture. The goal of this study was to elucidate the proteolytic processing strategy of MNV during an authentic replication cycle in cells. A proteolytic cleavage map of the ORF1 polyprotein was generated, and the virus-encoded 3C-like (3CL) proteinase (Pro) mediated cleavage at five dipeptide cleavage sites, 341E/G342, Q705/N706, 870E/G871, 994E/A995, and 1177Q/G1178, that defined the borders of six proteins with the gene order p38.3 (Nterm)-p39.6 (NTPase)-p18.6-p14.3 (VPg)-p19.2 (Pro)-p57.5 (Pol). Bacterially expressed MNV 3CL Pro was sufficient to mediate trans cleavage of the ORF1 polyprotein containing the mutagenized Pro sequence into products identical to those observed during cotranslational processing of the authentic ORF1 polyprotein in vitro and to those observed in MNV-infected cells. Immunoprecipitation and Western blot analysis of proteins produced in virus-infected cells demonstrated efficient cleavage of the proteinase-polymerase precursor. Evidence for additional processing of the Nterm protein in MNV-infected cells by caspase 3 was obtained, and Nterm sequences 118DRPD121 and 128DAMD131 were mapped as caspase 3 cleavage sites by site-directed mutagenesis. The availability of the MNV nonstructural polyprotein cleavage map in concert with a permissive cell culture system should facilitate studies of norovirus replication.

Amino Acid Sequence↗