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At least 919 records · Page 51Linked to original sources
PubMed1997

Petunia vein-clearing virus: a plant pararetrovirus with the core sequences for an integrase function.

Petunia vein-clearing virus (PVCV) is a plant pararetrovirus that has some features of retrotransposons. It encapsidates dsDNA and has isometric particles and inclusion bodies similar to those of caulimoviruses. The PVCV genome of 7205 bp has two large ORFs in the transcribed strand and a methionine tRNA primer-binding site in its 663-bp intergenic region. The N-terminal position of the large protein (126 kDa) encoded by ORF I has similarity to the movement protein of caulimoviruses. Toward the C-terminus of this same polyprotein are the two distinctive sequence elements [HHCC and DD(35)E] of the integrase function of retroviruses and retrotransposons. ORF II of PVCV encodes a protein of 125 kDa with domains for an RNA-binding element, common to the gag gene of retroelements, followed by consensus sequences for an acid protease, reverse transcriptase, and ribonuclease H. Hence, the gag equivalent (capsid protein) and pol gene of PVCV are part of the same polyprotein. Phylogenetic comparison of the reverse transcriptase of PVCV with that of various other retroelements grouped PVCV between caulimoviruses and the Ty3/gypsy retrotransposons, suggesting that PVCV is a divergent member of the caulimoviruses.

Amino Acid Sequence↗
PubMed1998

Regulation of intracellular human immunodeficiency virus type-1 protease activity.

The maturation of HIV-1 virions is accomplished through the proteolytic cleavage of Gag and GagPol precursor polyproteins by the viral-encoded protease (PR). Since virions are assembled from unprocessed polyproteins, the intracellular activation of PR must be limited. An experimental system was established that allows the investigation of the intracellular regulation of PR activity. By expressing Gag in trans with the GagPol precursor, downregulation of the intracellular PR activity associated with GagPol was demonstrated. Inhibition of PR activity was dependent upon the context of PR expression. Sequences capable of mediating this inhibition were localized to capsid. A mechanism through which Gag regulates PR activity is proposed whereby the disproportionate synthesis of Gag inhibits the activation of PR in the cytoplasm. Further elucidation of the mechanism of intracellular inhibition of PR activity may facilitate the development of novel PR inhibitors capable of inhibiting viral replication in vivo.

Amino Acid Sequence↗
PubMed1998

Characterization of the two overlapping papain-like proteinase domains encoded in gene 1 of the coronavirus infectious bronchitis virus and determination of the C-terminal cleavage site of an 87-kDa protein.

In a previous report, we showed that proteolytic processing of an 87-kDa mature viral protein from the coronavirus infectious bronchitis virus (IBV) 1a and 1a/1b polyproteins was mediated by two putative overlapping papain-like proteinase domains (PLPDs) encoded within the region from nucleotides 4243 to 5553 of ORF 1a (Liu et al., 1995). In this study, we demonstrate that only the first domain, PLPD-1, is responsible for this cleavage, as deletion of the second domain did not affect the formation of the 87-kDa protein. Site-directed mutagenesis studies further showed that a previously predicted nucleophilic cysteine residue (Cys1274) and a histidine residue (His1437) were essential for the proteinase activity, indicating that they may be important components of the catalytic center of the proteinase. Meanwhile, expression of a series of deletion mutants revealed that the 87-kDa protein was encoded by the 5'-most 2.6 kb of ORF 1a. Deletion and amino acid substitution mutation studies demonstrated that the Gly673-Gly674 dipeptide bond was most likely the cleavage site responsible for releasing the C-terminus of the 87-kDa protein from the 1a and 1a/1b polyproteins.

Coronavirus Papain-Like Proteases↗
PubMed1998

Direct cleavage of elF4G by poliovirus 2A protease is inefficient in vitro.

Previously, the purified recombinant 2A proteases (2Apro) of coxsackievirus B4 (CVB4) and human rhinovirus type 2 (HRV2) were shown to cleave synthetic peptides derived from human or rabbit elF4G as well as elF4G protein purified from rabbit reticulocytes. These results were in contrast to previous evidence which supported the view that elF4G cleavage activity in poliovirus-infected HeLa cells required a cellular factor(s) activated by poliovirus (PV) 2Apro. In the present study, recombinant PV 2Apro was shown to cleave either rabbit or human elF4G or their derived peptides in direct cleavage reactions, but cleaved the 4G-derived peptides with 100-fold lower efficiency than with a peptide derived from the poliovirus polyprotein. In these experiments, up to 25-fold molar excess of 2Apro over elF4G protein was required to cause greater than 50% cleavage. CVB4 2Apro was also tested in peptide cleavage assays under the same conditions as PV 2Apro and was found to cleave all elF4G substrates with efficiencies similar to PV 2Apro. Finally, cleavage reactions utilizing recombinant elF4G containing a G486E substitution at the cleavage site for CVB4 and HRV2 proteases resulted in drastically reduced cleavage by PV 2Apro, similar to the reduction previously seen with HRV2 and CVB4 2Apro, confirming that all three viral 2A proteases recognize the same cleavage site on elF4G. These data show that PV 2Apro can directly cleave elF4G in vitro with efficiencies similar to those of CVB 2Apro, but cleavage efficiency of elF4G is approximately 1000-fold lower than cleavage of a peptide derived from the authentic 2A cleavage site on the poliovirus polyprotein.

Animals↗
PubMed1999

Rice tungro bacilliform virus open reading frame 3 encodes a single 37-kDa coat protein.

Rice tungro bacilliform virus (RTBV) is a plant pararetrovirus and a member of the Caulimoviridae family and closely related to viruses in the Badnavirus genus. The coat protein of RTBV is part of the large polyprotein encoded by open reading frame 3 (ORF3). ORF3 of an RTBV isolate from Malaysia was sequenced (accession no. AF076470) and compared with published sequences for the region that encodes the coat protein or proteins. Molecular mass of virion proteins was determined by mass spectrometry (matrix-assisted laser desorption/ionization-TOF) performed on purified virus particles from three RTBV isolates from Malaysia. The N- and C-terminal amino acid sequences of the coat protein were deduced from the mass spectral analysis, leading to the conclusion that purified virions contain a single coat protein of 37 kDa. The location of the coat protein domain in ORF3 was reinforced as a result of immunodetection reactions using antibodies raised against six different segments of ORF3 using Western immunoblots after SDS-PAGE and isoelectrofocusing of proteins purified from RTBV particles. These studies demonstrate that RTBV coat protein is released from the polyprotein as a single coat protein of 37 kDa.

Animals↗
PubMed1999

Cyclophilin A incorporation is not required for human immunodeficiency virus type 1 particle maturation and does not destabilize the mature capsid.

The cellular protein cyclophilin A (CypA) is packaged into human immunodeficiency virus type 1 (HIV-1) virions through a specific interaction with the capsid (CA) domain of the Gag polyprotein. CypA is important for infectivity, but its role in viral replication is currently unknown. Previous reports suggested that CypA promotes uncoating or enhances maturation. We analyzed the morphology and capsid stability of HIV-1 variants defective in CypA binding and of virus grown in the presence of cyclosporin. Both cyclosporin treatment and alteration of Gly89 or Pro90 in the CypA-binding site of CA caused a 5- to 20-fold decrease in CypA incorporation. Virus produced from cyclosporin-treated cells and variants G89V and G89A were 10- to 100-fold less infectious but exhibited normal virion morphologies with regular cone-shaped capsids. Irregular capsid morphologies and lower infectivities were observed for some other variants in the CypA-binding region. Decreased CypA incorporation did not reduce the kinetics of intracellular polyprotein processing or of virus release. No increase in immature particles was observed. These results suggest that CypA does not promote virion maturation. Furthermore, detergent stripping of virus particles with various CypA contents revealed no difference in capsid stability. Based on these results and those reported in the accompanying paper, it appears likely that CypA also is not an uncoating factor. Alternative models for CypA function are discussed.

Capsid↗
PubMed1999

Hepatitis C virus: an infectious molecular clone of a second major genotype (2a) and lack of viability of intertypic 1a and 2a chimeras.

Of the six major genotypes of hepatitis C virus (HCV), infectious cDNA clones of only genotype 1 have been reported. Here, we report the construction of an infectious cDNA clone representing a second major HCV genotype, genotype 2. This infectious clone (pJ6CF) encodes the consensus polyprotein of strain HC-J6(CH), genotype 2a. Its encoded polyprotein differs from those of the infectious clones of genotypes 1a and 1b by approximately 30%. Intertypic chimeric cDNA clones constructed from infectious clones of genotypes 1a and 2a of HCV were not infectious. RNA transcripts of four chimeras containing the 2a structural genes (C, E1, and E2) in the backbone of an infectious genotype 1a clone (pCV-H77C) were not viable in a chimpanzee regardless of whether p7 was from the 1a or 2a clone. However, the chimpanzee was subsequently infected with RNA transcripts of each of the two infectious parent clones, indicating that the inability of the chimeras to replicate was intrinsic to the clones and not the result of preexisting protective immune responses.

3' Untranslated Regions↗
PubMed1999

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.

Amino Acid Sequence↗
PubMed2000

Sequence requirements for translation initiation of Rhopalosiphum padi virus ORF2.

Rhopalosiphum padi virus (RhPV) is an aphid-infecting virus with a 10-kb ssRNA genome that contains two large open reading frames (ORFs). ORF1 and ORF2 encode the nonstructural and structural polyproteins, respectively. Both ORFs are preceded by noncoding regions of 500 nt that could function as internal ribosome entry segments (IRESes). The sequence for ORF2 lacks an obvious initiation codon, but an out-of-frame AUG codon is present that could translate ORF2 through a +1 frameshift. To investigate the mechanisms of translation initiation of ORF2, a series of point and deletion mutations were constructed and transcribed and translated in vitro. A bicistronic plasmid containing two copies of the RhPV intergenic region translated both ORFs efficiently, indicating that the region functioned as an IRES in vitro. Deletion analysis showed that the region required for activity of the IRES extended from 178 nt upstream and 6 nt downstream of the 5' border of ORF2. Changes in the out-of-frame AUG codon had little effect on translation initiation, but mutations that included the first and second codons of ORF2 or that disrupted a putative pseudoknot structure near the 3' end of the IRES failed to initiate protein synthesis. Sequence analysis of the in vitro synthesized proteins showed that the first amino acid of the polyprotein corresponded to the second codon of ORF2. These results show that in vitro the noncoding region upstream of RhPV ORF2 functions as an IRES that contains a pseudoknot that directs translation initiation at a non-AUG codon. If the in vitro synthesized proteins have not been processed by an aminopeptidase, translation is initiated at the second (GCA) codon of ORF2.

5' Untranslated Regions↗
PubMed2000

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.

Amino Acid Sequence↗
PubMed2000

Yellow fever virus NS2B-NS3 protease: charged-to-alanine mutagenesis and deletion analysis define regions important for protease complex formation and function.

Charged-to-alanine substitutions and deletions within the yellow fever virus NS2B-NS3(181) protease were analyzed for effects on protease function. During cell-free translation of NS2B-3(181) polyproteins, mutations at three charge clusters markedly impaired cis cleavage activity: a single N-terminal cluster in the conserved domain of NS2B (residues ELKK(52-55)) and two in NS3 (ED(21-22), and residue H(47)). These mutations inhibited other protease-dependent cleavages of a transiently expressed nonstructural polyprotein, although differential effects occurred. NS2B and NS3(181) proteins harboring these mutations were impaired in their ability to associate for trans cleavage activity. N-terminal deletions in NS3 also implicated residues ED(21-22) in the association with NS2B. Deletions within NS2B revealed that the conserved domain alone provided minimal cofactor activity, with optimal function requiring both flanking hydrophobic regions. NS2B-3(181)- and NS3(181)-green fluorescent protein fusion proteins were used to determine the intracellular distribution of the protease complex. The former localized in membrane-based vesicular structures, whereas the latter localized poorly. The data suggest that NS2B-NS3 complex formation requires charge interactions involving the N-terminus of the conserved domain of NS2B and 22 N-terminal residues of NS3. A role for the putative transmembrane regions of NS2B in targeting of NS3 to intracellular membranes is also suggested.

Amino Acid Sequence↗
PubMed2000

Different architectures in the assembly of infectious bursal disease virus capsid proteins expressed in insect cells.

Infectious bursal disease virus (IBDV) capsid is formed by the processing of a large polyprotein and subsequent assembly of VPX/VP2 and VP3. To learn more about the processing of the polyprotein and factors affecting the correct assembly of the viral capsid in vitro, different constructs were made using two baculovirus transfer vectors, pFastBac and pAcYM1. Surprisingly, the expression of the capsid proteins gave rise to different types of particles in each system, as observed by electron microscopy and immunofluorescence. FastBac expression led to the production of only rigid tubular structures, similar to those described as type I in viral infection. Western blot analysis revealed that these rigid tubules are formed exclusively by VPX. These tubules revealed a hexagonal arrangement of units that are trimer clustered, similar to those observed in IBDV virions. In contrast, pAcYM1 expression led to the assembly of virus-like particles (VLPs), flexible tubules, and intermediate assembly products formed by icosahedral caps elongated in tubes, suggesting an aberrant morphogenesis. Processing of VPX to VP2 seems to be a crucial requirement for the proper morphogenesis and assembly of IBDV particles. After immunoelectron microscopy, VPX/VP2 was detected on the surface of tubules and VLPs. We also demonstrated that VP3 is found only on the inner surfaces of VLPs and caps of the tubular structures. In summary, assembly of VLPs requires the internal scaffolding of VP3, which seems to induce the closing of the tubular architecture into VLPs and, thereafter, the subsequent processing of VPX to VP2.

Animals↗
PubMed2002

Site-directed mutagenesis of Avibirnavirus VP4 gene.

Virus protein VP4 of infectious bursal disease virus (IBDV) is a protease which separates VPX and VP3 from the polyprotein. We studied the importance of serine and aspartic acid on cleavage at the VPX/VP4 junction and analysed the role of the proposed H547, D590, and S653 catalytic site using five different mutations on VP4. Our results suggest that the replacement of serine by lysine in AXAAS motifs in serotype II IBDV influences polyprotein (PP) processing by VP4 and also indicate the presence of an alternative cleavage site. Mutation on D ((510)TLAADK(515)) prevented the cleavage at the VPX/VP4 junction, but we have found that independently of the importance of those alanines in LAA, D has an important role as part of the cleavage site. Replacement of histidine by proline H547P completely abolished PP processing. Mutation on D590 induced a partial PP processing when it was replaced by proline and the replacement of serine by proline at S653P induced a prominent change in PP processing. These results permit us to conclude that IBDV VP4 has the ability to act according to structural and topographical changes during translational and posttranslational processes and allow multiple hit sites, which serve to increase effectiveness.

Amino Acid Sequence↗
PubMed1991

Mutational analysis of a native substrate of the HIV-1 proteinase.

The purpose of this study was to define further the determinants of substrate specificity of HIV-1 PR. Rather than using small peptides, we used an in vitro system which permitted us to evaluate the effect of a mutated site within the context of its natural precursor. We made single-amino-acid substitutions around two sites which are processed by the HIV-1 PR. The Tyr/Pro site within gag appears to encode highly specific determinants which direct proteinase processing between MA and CA. The Phe/Pro site in pol, however, appears to be far more tolerant to amino acid substitutions, as none of our single-amino-acid substitutions blocked cleavage at or around this site. The increased tolerance of the Phe/Pro site may indicate that at this site, structural features are more important determinants of cleavage than primary amino acid sequence. We have shown that sequences outside of those encoding mature PR can inhibit proteolytic processing in this system. By preventing PR from cleaving itself from the polyprotein prematurely, p6* sequences would regulate morphogenesis and infectious particle formation. Late in infection, when the protein concentration of gag and gag/pol polyproteins at the cell surface becomes very high, cooperative protein-protein interactions may cause alterations of a p6*-PR interaction, relieving repression and permitting autocatalysis.

Amino Acid Sequence↗
PubMed1991

Bovine viral diarrhea virus proteins and their antigenic analyses.

Bovine Viral Diarrhea Virus (BVDV) polypeptides present in infected cells are the result of the processing of the polyprotein translated from the large single open reading frame of the BVDV genomic RNA. The presence of these proteins in infected cells was studied by radiolabeling under hypertonic conditions and with the aid of radioimmunoprecipitation. The genomic mapping of these polypeptides suggests a complex pattern of processing which involves cellular and viral proteases. The consistent absence of 80k in noncytopathic isolates of BVDV suggests that the processing of the viral polyprotein is different in cytopathic and noncytopathic biotypes of BVDV. The antigenic structure of BVDV was studied with a panel of monoclonal antibodies (MABs) prepared against the Singer isolate of BVDV. Neutralizing MABs were found to bind the 56-58k polypeptide, providing evidence that this glycoprotein is present on the surface of the virion and carries neutralization epitopes. Antigenic analyses with the panel of MABs reveals extensive antigenic heterogeneity among BVDV field isolates. MABs were used to determine the frequency of neutralization escape mutants in stocks of BVDV. Plaque-purified BVDV stocks contain neutralization escape mutants with a frequency of 10(-2.47).

Animals↗
PubMed1994

Processing of dengue type 4 and other flavivirus nonstructural proteins.

Dengue type 4 (DEN4) and other flaviviruses employ host and viral proteases for polyprotein processing. Most proteolytic cleavages in the DEN4 nonstructural protein (NS) region are mediated by the viral NS2B-NS3 protease. The N-terminal third of NS3, containing sequences homologous to serine protease active sites, is the protease domain. To determine required sequences in NS2B, deletions were introduced into DEN4 NS2B-30% NS3 cDNA and the expressed polyproteins assayed for self-cleavage. A 40 amino acid segment within NS2B was essential. Sequence analysis of NS2B predicts that this segment constitutes a hydrophilic domain surrounded by hydrophobic regions. Hydrophobicity profiles of other flavivirus NS2Bs show similar patterns. Cleavage of DEN4 NS1-NS2A requires an octapeptide sequence at the NS1 C terminus and downstream NS2A. Comparison of the analogous octapeptide sequences among flaviviruses indicates a consensus cleavage sequence of (P8)/Met/Leu-Val-Xaa-Ser-Xaa-Val-Ala(P1), where Xaa are non-conserved amino acids. The effects on cleavage of amino acid substitutions in this consensus sequence were analyzed. Most substitutions of the conserved residues interfered with cleavage, whereas substitutions of non-conserved residues had little or no effect. These findings indicate that the responsible enzyme recognizes well-defined sequences at the cleavage site.

Amino Acid Sequence↗
PubMed1994

In vitro characterization of a cassette to accumulate multiple proteins through synthesis of a self-processing polypeptide.

The strategy for processing the polyprotein encoded by plant potyviruses has been mimicked by constructing an expression cassette based on the nuclear inclusion (Nla) proteinase from tobacco etch virus (TEV). This cassette (pPR01), includes the TEV Nla coding region flanked on each side by its heptapeptide cleavage sequence and cloning sites for the in frame insertion of two different open reading frames. pPR01 allows the synthesis, under the control of a single transcriptional promoter, of two proteins in equimolar amounts as part of a polyprotein which is cleaved into individual mature products by the TEV protease. In in vitro reactions the cassette functioned as expected when several different protein-coding sequences were used. The potential uses of pPR01 are discussed.

Amino Acid Sequence↗
PubMed1993

Human T-cell leukemia virus type 1 protease protein expressed in Escherichia coli possesses aspartic proteinase activity.

We amplified the human T-cell leukemia virus type 1 (HTLV-1) protease gene fragment by polymerase chain reaction (PCR) and cloned it into a pUC plasmid vector. DNA sequencing data of the protease gene fragment indicated that it contained an open reading frame capable of encoding the active HTLV-1 protease. To express a fusion protein of beta-galactosidase linked with the HTLV-1 protease in Escherichia coli, a plasmid DNA was constructed by inserting the HTLV-1 protease gene DNA into a procaryotic expression vector, pUEX2, consisting of a lacZ gene directed by a lambda phage Pr promoter and designated pUEX-pro. By Western blot analysis using anti-beta-galactosidase antibody, a bigger molecular size band than that of the control beta-galactosidase molecule was observed in E. coli cells transformed with pUEX-pro but not with control pUEX2, suggesting that the particular fusion protein was successfully expressed. This recombinant protease protein in the E. coli cell lysate was demonstrated to be able to cleave the decapeptide substrates composed of amino acid sequences containing proteolytic cleavage sites in the HTLV-1 gag precursor polyprotein. The gag precursor polyprotein expressed in the mammalian cells by the recombinant vaccinia virus system was also expectedly cleaved by this enzyme. Significant inhibition of this protease activity by pepstatin A, an aspartic proteinase-specific inhibitor, confirms that HTLV-1 protease is a member of the aspartic proteinase group as suggested previously. Since the crude lysate without purification is utilized sufficiently as a native HTLV-1 protease reagent, this protease preparation is easily applicable to the large scale screening of HTLV-1 protease inhibitors for the treatment of diseases caused by HTLV-1.

Amino Acid Sequence↗