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At least 1,153 records · Page 64Linked to original sources
PubMed1996

Processing of the E1 glycoprotein of hepatitis C virus expressed in mammalian cells.

The structural part of the hepatitis C virus (HCV) genome encodes a capsid protein, C and two envelope glycoproteins, E1 and E2, released from the virus polyprotein precursor by signalase(s) cleavage(s). The processing of E1 was investigated by infecting simian cells with recombinant vaccinia viruses expressing parts of the HCV structural proteins. When the predicted E1 sequence was expressed alone (amino acid residues 174-370 of the polyprotein) or with the capsid protein gene (residues 1-370). it showed an apparent molecular mass of 35 kDa as measured by SDS-PAGE analysis. However, when E1 was expressed as part of a truncated C-E1-truncated E2 polypeptide (residues 132-383), the processed E1 product had the expected apparent molecular mass of 31 kDa, suggesting that flanking sequences are necessary for the generation of the mature 31 kDa El form. The N-terminal sequence of the two E1 forms was found to be the same. Analysis of the glycosylation pattern showed that, in both species, only four of the five potential N-linked glycosylation sites were recognized, indicating that glycosylation was not involved in the molecular mass difference. We showed that expression of E1 with or without the hydrophobic stretch of amino acids residues 371-383, defined as the E2 signal sequence, may be responsible for the difference in electrophoretic mobility of the two E1 species. In vitro translation assays and site-directed mutagenesis experiments suggest that this sequence remains part of the 31 kDa E1 mature protein.

Amino Acid Sequence↗
PubMed1996

European brown hare syndrome virus: molecular cloning and sequencing of the genome.

The genome of the European brown hare syndrome virus (EBHSV), a calicivirus related to rabbit haemorrhagic disease virus (RHDV), was fully sequenced. It was 7442 bases long and contained two ORFs. In RHDV, the 5' large ORF (ORF1) is predicted to encode a polyprotein precursor to the non-structural and capsid proteins. The small ORF (ORF2) encodes a predicted protein of 12 kDa. Alignment of sequences of EBHSV and RHDV showed 71% nucleotide identity; the changes were uniformly scattered over the whole genome. Minor differences could be detected when comparing two EBHSV sequences, indicating that EBHSV could vary to the same extent as RHDV. Four cleavage sites previously identified on the RHDV polyprotein were conserved in EBHSV. These sequencing data clearly show that EBHSV and RHDV share a similar genomic organization and confirm that EBHSV and RHDV are two distinct members within the family Caliciviridae.

Amino Acid Sequence↗
PubMed1997

Complete nucleotide sequence of a type 4 hepatitis C virus variant, the predominant genotype in the Middle East.

Hepatitis C virus (HCV) type 4 is the predominant genotype found throughout the Middle East and parts of Africa, often in association with high population prevalence as in Egypt. To investigate more fully its evolutionary relationship with other genotypes of HCV, and to study its overall genome organization, we have determined the entire sequence encompassing the coding region of the genotype 4a isolate ED43, obtained from an HCV-infected individual from Egypt. The sequence of ED43 contained a single open reading frame encoding a polyprotein of 3008 amino acids (aa), smaller than that reported for other HCV genotypes which vary from 3010 aa to 3037 aa. The nucleotide and amino acid sequences were compared with the full-length sequences already reported for genotypes 1a, 1b, 1c, 2a, 2b, 2c, 3a, 3b and those of isolates JKO49 and JKO46 described as types 10a and 11a. The differences in length of the polyprotein originated in variable regions in the E2 and NS5A genes. The complete sequence of ED43 confirmed the classification of type 4 as a separate major genotype.

Amino Acid Sequence↗
PubMed1998

Nucleotide sequence, genetic organization and expression strategy of the double-stranded RNA associated with the '447' cytoplasmic male sterility trait in Vicia faba.

The entire nucleotide sequence of the double-stranded (ds) RNA associated with the unconventional '447' cytoplasmic male sterility (CMS) trait in Vicia faba was determined from overlapping cDNA clones and by RT-PCR. Confirming previous observations, it was found that the negative-strand was continuous and 17,635 nt long, while the positive-strand featured an interruption, probably a nick, that could potentially define two subgenomic RNAs of 2735 nt and 14,900 nt, with the smaller RNA being located on the 5' side. The entire positive-strand could encode a single in-frame ORF starting at the first AUG at position 42-44 and ending with a TGA at 17,517-17,519. This long potential polypeptide with a predicted molecular mass of 654,109 is the largest described to date in the plant kingdom and contains conserved amino acid sequence motifs typical of viral helicases and RNA-dependent RNA polymerases (RDRP). Only limited sequence homology was detected with the ORF B encoded by the hypovirulence-associated dsRNA of chestnut blight fungus, a dsRNA replicon similarly contained in host-derived membranous vesicles and considered to share a common ancestry with potyviruses. By contrast, the helicase and RDRP domains were in the same respective arrangement and shared extensive sequence homologies with those identified in the polyprotein encoded by the dsRNA isolated from Japonica rice, another dsRNA replicon featuring a specific nick in the positive-strand. Although no proteolytic self-cleavage activity has yet been demonstrated, it appears likely that this long ORF is a polyprotein that undergoes proteolytic maturation, with one of the polypeptides derived by self-cleavage being the determinant of the CMS trait.

Amino Acid Sequence↗
PubMed1998

Nucleotide sequence and genome organization of grapevine leafroll-associated virus-2 are similar to beet yellows virus, the closterovirus type member.

The entire genome of grapevine leafroll-associated closterovirus-2 (GLRaV-2), except the exact 5' terminus, was cloned and sequenced. The sequence encompasses nine open reading frames (ORFs) which include, in the 5' to 3' direction, an incomplete ORF1a encoding a putative viral polyprotein and eight ORFs that encode proteins of 52 kDa (ORF1b), 6 kDa (ORF2), 65 kDa (ORF3), 63 kDa (ORF4), 25 kDa (ORF5), 22 kDa (ORF6), 19 kDa (ORF7) and 24 kDa (ORF8) respectively, and 216 nucleotides of the 3' untranslated region. An incomplete ORF1a potentially encoded a large polyprotein containing the conserved domains characteristic of a papain-like protease, methyltransferase and helicase. ORF1b potentially encoded a putative RNA-dependent RNA polymerase. The expression of ORF1b may be via a +1 ribosomal frameshift mechanism, similar to other closteroviruses. A unique gene array, which is conserved in other closteroviruses, was also identified in GLRaV-2; it includes genes encoding a 6 kDa small hydrophobic protein, 65 kDa heat shock protein 70, 63 kDa protein of function unknown, 25 kDa coat protein duplicate and 22 kDa coat protein. Identification of ORF6 (22 kDa) as the coat protein gene was further confirmed by in vivo expression in E. coli and immunoblotting. Phylogenetic analysis comparing different genes of GLRaV-2 with those of other closteroviruses demonstrated a close relationship with beet yellows virus (BYV), beet yellow stunt virus and citrus tristeza virus. GLRaV-2 is the only closterovirus, so far, that matches the genome organization of the type member of the group, BYV, and thus can be unambiguously classified as a definitive member of the genus Closterovirus.

Amino Acid Sequence↗
PubMed1999

Avian encephalomyelitis virus is a picornavirus and is most closely related to hepatitis A virus.

The complete RNA genome of avian encephalomyelitis virus (AEV) has been molecularly cloned and sequenced. This revealed AEV to be a member of the Picornaviridae and consequently it is the first avian picornavirus for which the genome has been sequenced. Excluding the poly(A) tail the genome comprises 7032 nucleotides, which is shorter than that of any mammalian picornavirus sequenced to date. An open reading frame commencing at nucleotide 495 and terminating at position 6896 (6402 nucleotides) potentially encodes a polyprotein of 2134 amino acids. The polyprotein sequence has 39% overall amino acid identity with hepatitis A virus (HAV; genus Hepatovirus), compared to 19 to 21% for viruses from the other five picornavirus genera. Eleven cleavage products were predicted. The highest identity (49%) with HAV was in the P1 region, encoding the capsid proteins. The 5' and 3' untranslated regions (UTRs) comprise 494 and 136 nucleotides, respectively. The 5' UTR is the shortest of any picornavirus sequenced to date and, unlike HAV, it does not contain a long polypyrimidine tract.

Amino Acid Sequence↗
PubMed1999

Analysis of the glycosylation sites of hepatitis C virus (HCV) glycoprotein E1 and the influence of E1 glycans on the formation of the HCV glycoprotein complex.

The hepatitis C virus (HCV) genome encodes two membrane-associated envelope glycoproteins (E1 and E2), which are released from the viral polyprotein precursor by host signal peptidase cleavages. These glycoproteins interact to form a noncovalent heterodimeric complex, which is retained in the endoplasmic reticulum. HCV glycoproteins, E1 and E2, are heavily modified by N-linked glycosylation. A recent study has revealed that upon partial deglycosylation with endoglycosidase H only four of the five potential glycosylation sites of HCV glycoprotein E1 are utilized. In this work, the unused glycosylation site on the E1 glycoprotein was identified and the influence of N-linked glycosylation on the formation of the HCV glycoprotein complex was studied by expressing a panel of E1 glycosylation mutants in HepG2 cells. Each of the five potential N-linked glycosylation sites, located at amino acid positions 196, 209, 234, 305 and 325, respectively, on the HCV polyprotein, was mutated separately as well as in combination with the other sites. Expression of the mutated E1 proteins in HepG2 cells indicated that the fifth glycosylation site is not used for the addition of N-linked oligosaccharides and the Pro immediately following the sequon (Asn-Trp-Ser) precludes core glycosylation. The effect of each mutation on the formation of noncovalent E1E2 complexes was also analysed. As determined with the use of a conformation-sensitive monoclonal antibody, mutations at positions N2 and N3 had no, or only minor, effects on the assembly of the E1E2 complex, whereas a mutation at position N1 and predominantly at position N4 dramatically reduced the efficiency of the formation of noncovalent E1E2 complexes.

Animals↗
PubMed1999

The nucleotide sequence of sacbrood virus of the honey bee: an insect picorna-like virus.

We have determined the nucleotide sequence of sacbrood virus (SBV), which causes a fatal infection of honey bee larvae. The genomic RNA of SBV is longer than that of typical mammalian picornaviruses (8832 nucleotides) and contains a single, large open reading frame (179-8752) encoding a polyprotein of 2858 amino acids. Sequence comparison with other virus polyproteins revealed regions of similarity to characterized helicase, protease and RNA-dependent RNA polymerase domains; structural genes were located at the 5' terminus with non-structural genes at the 3' end. Picornavirus-like agents of insects have two distinct genomic organizations; some resemble mammalian picornaviruses with structural genes at the 5' end and non-structural genes at the 3' end, and others resemble caliciviruses in which this order is reversed; SBV thus belongs to the former type. Sequence comparison suggested that SBV is distantly related to infectious flacherie virus (IFV) of the silk worm, which possesses an RNA of similar size and gene order.

Amino Acid Sequence↗
PubMed2000

Identification of a new cleavage site of the 3C-like protease of rabbit haemorrhagic disease virus.

The calicivirus rabbit haemorrhagic disease virus (RHDV) possesses a 3C-like protease which processes the RHDV polyprotein. In order to monitor the proteolytic activity of the RHDV 3C-like protease on its putative target sequences, i.e. the 10 EG dipeptide bonds distributed along the large polyprotein, a new approach was carried out. Preliminary experiments showed that the luciferase gene when fused in-frame with a long gene yielded a fusion protein almost devoid of luciferase activity. This reporter system was used to test which EG dipeptide bonds were cleaved by the RHDV protease when the coding sequences of the dipeptides and their flanking sequences were inserted at the junction between the protease and luciferase genes. The coding sequences of the fusion proteins were cloned downstream of the T7 promoter and the proteolytic activity was evaluated by measuring the luciferase activity in both in vitro and 'in vivo' systems. The EG dipeptide bonds at positions 718-719, 1108-1109 and 1767-1768 were confirmed as cleavage sites and a new cleavage site EG (143-144) was identified.

Amino Acid Sequence↗
PubMed2000

Role of Ser-652 and Lys-692 in the protease activity of infectious bursal disease virus VP4 and identification of its substrate cleavage sites.

The polyprotein of infectious bursal disease virus (IBDV), an avian birnavirus, is processed by the viral protease, VP4. Previous data obtained on the VP4 of infectious pancreatic necrosis virus (IPNV), a fish birnavirus, and comparative sequence analysis between IBDV and IPNV suggest that VP4 is an unusual eukaryotic serine protease that shares properties with prokaryotic leader peptidases and other bacterial peptidases. IBDV VP4 is predicted to utilize a serine-lysine catalytic dyad. Replacement of the members of the predicted catalytic dyad (Ser-652 and Lys-692) confirmed their indispensability. The two cleavage sites at the pVP2-VP4 and VP4-VP3 junctions were identified by N-terminal sequencing and probed by site-directed mutagenesis. Several additional candidate cleavage sites were identified in the C-terminal domain of pVP2 and tested by cumulative site-directed mutagenesis and expression of the mutant polyproteins. The results suggest that VP4 cleaves multiple (Thr/Ala)-X-Ala downward arrowAla motifs. A trans activity of the VP4 protease of IBDV, and also IPNV VP4 protease, was demonstrated by co-expression of VP4 and a polypeptide substrate in Escherichia coli. For both proteases, cleavage specificity was identical in the cis- and trans-activity assays. An attempt was made to determine whether VP4 proteases of IBDV and IPNV were able to cleave heterologous substrates. In each case, no cleavage was observed with heterologous combinations. These results on the IBDV VP4 confirm and extend our previous characterization of the IPNV VP4, delineating the birnavirus protease as a new type of viral serine protease.

Amino Acid Sequence↗
PubMed2000

Gill-associated virus of Penaeus monodon prawns: an invertebrate virus with ORF1a and ORF1b genes related to arteri- and coronaviruses.

A 20089 nucleotide (nt) sequence was determined for the 5' end of the (+)-ssRNA genome of gill-associated virus (GAV), a yellow head-like virus infecting Penaeus monodon prawns. Clones were generated from a approximately 22 kb dsRNA purified from lymphoid organ total RNA of GAV-infected prawns. The region contains a single gene comprising two long overlapping open reading frames, ORF1a and ORF1b, of 4060 and 2646 amino acids, respectively. The ORFs are structurally related to the ORF1a and ORF1ab polyproteins of coronaviruses and arteriviruses. The 99 nt overlap between ORF1a and ORF1b contains a putative AAAUUUU 'slippery' sequence associated with -1 ribosomal frameshifting. A 131 nt stem-loop with the potential to form a complex pseudoknot resides 3 nt downstream of this sequence. Although different to the G/UUUAAAC frameshift sites and 'H-type' pseudoknots of nidoviruses, in vitro transcription/translation analysis demonstrated that the GAV element also facilitates read-through of the ORF1a/1b junction. As in coronaviruses, GAV ORF1a encodes a 3C-like cysteine protease domain located between two hydrophobic regions. However, its sequence suggests some structural relationship to the chymotrypsin-like serine proteases of arteriviruses. ORF1b encodes homologues of the 'SDD' polymerase, which among (+)-RNA viruses is unique to nidoviruses, as well as metal-ion-binding and helicase domains. The presence of a dsRNA replicative intermediate and ORF1a and ORF1ab polyproteins translated by a-1 frameshift suggests that GAV represents the first invertebrate member of the Order NIDOVIRALES:

Amino Acid Sequence↗
PubMed2001

Determination of the substrate specificity of turnip mosaic virus NIa protease using a genetic method.

The RNA genome of turnip mosaic potyvirus (TuMV) encodes a large polyprotein that is processed to mature proteins by virus-encoded proteases. The TuMV NIa protease is responsible for the cleavage of the polyprotein at seven different locations. These cleavage sites are defined by a conserved sequence motif Val-Xaa-His-Gln decreased, with the scissile bond located after Gln. To determine the substrate specificity of the NIa protease, amino acid sequences cleaved by the NIa protease were obtained from randomized sequence libraries using a screening method referred to as GASP (genetic assay for site-specific proteolysis). Based on statistical analysis of the obtained sequences, a consensus substrate sequence was deduced: Yaa-Val-Arg-His-Gln decreased Ser, with Yaa being an aliphatic amino acid and the scissile bond being located between Gln and Ser. This result is consistent with the conserved cleavage sequence motif, and should provide insight into the molecular activity of the NIa protease.

Binding Sites↗
PubMed2001

Molecular identification of new picornaviruses and characterization of a proposed enterovirus 73 serotype.

Enteroviruses (EV) have traditionally been identified by using serotype-specific antisera in a virus-neutralization test. Three EV strains isolated in California, USA, in 1955, 1964 and 1978, and a 1995 Oman isolate, were found to be antigenically related to one another; however, the strains were not neutralized by standard EV typing antisera, suggesting that they may represent a new EV serotype. The isolates were characterized genetically by RT-PCR coupled with amplicon sequencing and comparison to a database of enterovirus nucleotide sequences. The strains were 75.3 to 87.2% identical to one another in complete VP1 nucleotide sequence, but no more than 68% identical in sequence to the prototype strain of any EV serotype. Their complete capsid sequences were closely related to one another, but only distantly related to those of any EV prototype strain. The California and Oman isolates were most closely related to members of EV cluster B, suggesting that they are unclassified members (i.e. a new serotype) of cluster B. The complete genome sequence was determined for one isolate, CA55-1988, and the predicted polyprotein sequence was 86.5 to 89.2% identical to those of other cluster B EV and 56.7 to 61.9% identical to the polyprotein sequences of EV belonging to other clusters. Isolation of this new EV serotype from samples obtained on two continents and over a period of 40 years suggests continued circulation over a wide geographical area. In keeping with standard picornavirus nomenclature, we propose that this new serotype be named 'enterovirus 73' (EV73).

Antigens, Viral↗
PubMed2001

Extended nucleocapsid protein is cleaved from the Gag-Pol precursor of human immunodeficiency virus type 1.

Human immunodeficiency virus type 1 Gag and Gag-Pol precursors are translated from an mRNA which is indistinguishable from the full-length genomic RNA. The ratio of Gag to Gag-Pol polyproteins is approximately 20:1 and is controlled by a frameshift of the reading frame, which takes place downstream of the p7 nucleocapsid (NC) in the N terminus of the p1 peptide. The viral precursors Gag and Gag-Pol are cleaved by the virus-encoded protease (PR) into the structural proteins, and into p6(Pol), PR, reverse transcriptase and integrase. Due to the frameshift event, the cleavage site at the C terminus of NC coded in the Gag frame (ERQAN-FLGKI) changes either to ERQANFLRED or ERQANFFRED. The results presented in this report demonstrate that the NC released from the Gag-Pol precursor is 8 amino acid residues longer than the NC cleaved from the Gag polyprotein. Our results also show that truncated Gag-Pol precursors bearing cleavage site mutation at the NC/p6(Pol), and/or p6(Pol)/PR junctions, undergo autoprocessing in bacterial and eukaryotic cells, indicating that PR is active when part of the precursor.

Animals↗
PubMed2002

Conservation of substrate specificities among coronavirus main proteases.

The key enzyme in coronavirus replicase polyprotein processing is the coronavirus main protease, 3CL(pro). The substrate specificities of five coronavirus main proteases, including the prototypic enzymes from the coronavirus groups I, II and III, were characterized. Recombinant main proteases of human coronavirus (HCoV), transmissible gastroenteritis virus (TGEV), feline infectious peritonitis virus, avian infectious bronchitis virus and mouse hepatitis virus (MHV) were tested in peptide-based trans-cleavage assays. The determination of relative rate constants for a set of corresponding HCoV, TGEV and MHV 3CL(pro) cleavage sites revealed a conserved ranking of these sites. Furthermore, a synthetic peptide representing the N-terminal HCoV 3CL(pro) cleavage site was shown to be effectively hydrolysed by noncognate main proteases. The data show that the differential cleavage kinetics of sites within pp1a/pp1ab are a conserved feature of coronavirus main proteases and lead us to predict similar processing kinetics for the replicase polyproteins of all coronaviruses.

Amino Acid Sequence↗
PubMed2003

Analysis of hepatitis C virus/classical swine fever virus chimeric 5'NTRs: sequences within the hepatitis C virus IRES are required for viral RNA replication.

Hepatitis C virus (HCV) is classified in the genus Hepacivirus of the family Flaviviridae, whose members have a single-stranded RNA genome of positive polarity, which encodes a single polyprotein. Within this family, HCV is closely related to viruses of the genus Pestivirus, which includes classical swine fever virus (CSFV). Translation of the hepaci- and pestiviral polyprotein is initiated by internal entry of ribosomes, promoted by the 5'NTR. The secondary and tertiary RNA structures of the HCV and pestivirus 5'NTRs are well conserved, despite the fact that their sequences differ significantly from one another. By analogy with other positive-stranded RNA viruses, the 5'NTR of HCV is likely to contain cis-acting determinants for replication as well as the determinants for translation. Studies on both signals could be complicated, as these signals might overlap. In this study, this problem was addressed by constructing chimeric HCV/CSFV 5'NTRs. A two-step analysis of these 5'NTRs was performed: (a) in a translation assay, which provided the possibility to study translation independently of the possible effects on replication; and (b) in a replication assay, in which were studied only the chimeric 5'NTRs for which IRES-dependent translation was demonstrated. An overlap was observed between HCV RNA elements involved in these processes. Exchange of domain II had a minor effect on the translation efficiency of the chimeric 5'NTRs, while replication of subgenomic replicons with these chimeric 5'NTRs was abolished. Exchange of domain III subdomains severely decreased translation activity, while replication was maintained.

5' Untranslated Regions↗
PubMed2004

Topogenesis in membranes of the NTB-VPg protein of Tomato ringspot nepovirus: definition of the C-terminal transmembrane domain.

The putative NTP-binding protein (NTB) of Tomato ringspot nepovirus (ToRSV) contains a hydrophobic region at its C terminus consisting of two adjacent stretches of hydrophobic amino acids separated by a few amino acids. In infected plants, the NTB-VPg polyprotein (containing the domain for the genome-linked protein) is associated with endoplasmic reticulum-derived membranes that are active in ToRSV replication. Recent results from proteinase K protection assays suggested a luminal location for the VPg domain in infected plants, providing support for the presence of a transmembrane domain at the C terminus of NTB. In this study, we have shown that NTB-VPg associates with canine microsomal membranes in the absence of other viral proteins in vitro and adopts a topology similar to that observed in vivo in that the VPg is present in the lumen. Truncated proteins containing 60 amino acids at the C terminus of NTB and the entire VPg exhibited a similar topology, confirming that this region of the protein contains a functional transmembrane domain. Deletion of portions of the C-terminal hydrophobic region of NTB by mutagenesis and introduction of glycosylation sites to map the luminal regions of the protein revealed that only the first stretch of hydrophobic amino acids traverses the membrane, while the second stretch of hydrophobic amino acids is located in the lumen. Our results provide additional evidence supporting the hypothesis that the NTB-VPg polyprotein acts as a membrane-anchor for the replication complex.

Amino Acid Sequence↗
PubMed2004

Sequence analysis and genomic organization of a new insect picorna-like virus, Ectropis obliqua picorna-like virus, isolated from Ectropis obliqua.

The complete nucleotide sequence of a new insect picorna-like virus, Ectropis obliqua picorna-like virus (EoPV), which causes a fatal infection of Ectropis obliqua larvae, has been determined. The genomic RNA of EoPV is 9394 nt in length and contains a single, large open reading frame (nt 391-9351) encoding a polyprotein of 2987 aa. Sequence comparisons with other viral polyproteins revealed that the consensus sequences for picornavirus RNA helicase, protease and RNA-dependent RNA polymerase proteins are found on the genome in order in the 5'-->3' direction. All structural genes were located at the 5' terminus. In terms of sequence similarity, identity and genome organization, EoPV resembles mammalian picornaviruses and three other insect picorna-like viruses: Infectious flacherie virus of silkworm, Sacbrood virus of honeybee and Perina nuda picorna-like virus (PnPV). Phylogenetic analysis showed that EoPV is most closely related to PnPV and suggests that these four insect picorna-like viruses might constitute a new group of insect-infectious RNA viruses.

Amino Acid Sequence↗