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At least 1,261 records · Page 70Linked to original sources
PubMed1998

Complete nucleotide sequence and genetic organization of Aichi virus, a distinct member of the Picornaviridae associated with acute gastroenteritis in humans.

The complete nucleotide sequence of a novel enteric virus, Aichi virus, associated with nonbacterial acute gastroenteritis in humans was determined. The Aichi virus genome proved to be a single-stranded positive-sense RNA molecule with 8,251 bases excluding a poly(A) tail; it contains a large open reading frame with 7,302 nucleotides that encodes a potential polyprotein precursor of 2,433 amino acids. The genome contains a 5' nontranslated region (NTR) with 712 bases and a 3' NTR with 240 bases followed by a poly(A) tail. The structure of the genome, VPg-5' NTR-leader protein-structural proteins-nonstructural proteins-3' NTR-poly(A), was found to be typical of a picornavirus. The VP0-VP3 and VP3-VP1 cleavage sites were determined to be Q-H and Q-T, respectively, by N-terminal amino acid sequence analyses using purified virion proteins. Possible cleavage sites, Q-G, Q-A, and Q-S, which cleave P2 and P3 polyproteins were found to be similar to those of picornaviruses. A dendrogram based on 3Dpol proteins indicated that Aichi virus is genetically distinct from the known six genera of picornaviruses including entero-, rhino-, cardio-, aphtho-, and hepatovirus and echovirus 22. Considering this together with other properties of the virus (T. Yamashita, S. Kobayashi, K. Sakae, S. Nakata, S. Chiba, Y. Ishihara, and S. Isomura, J. Infect. Dis. 164:954-957, 1991), we propose that Aichi virus be regarded as a new genus of the family Picornaviridae.

Acute Disease↗
PubMed1998

A protein critical for a Theiler's virus-induced immune system-mediated demyelinating disease has a cell type-specific antiapoptotic effect and a key role in virus persistence.

TO subgroup strains of Theiler's murine encephalomyelitis virus (TMEV) induce a persistent central nervous system infection and demyelinating disease in mice. This disease serves as an experimental model of multiple sclerosis (MS) because the two diseases have similar inflammatory white matter pathologies and because the immune system appears to mediate demyelination in both processes. We previously reported (H. H. Chen, W. P. Wong, L. Zhang, P. L. Ward, and R. P. Roos, Nat. Med. 1:927-931, 1995) that TO subgroup strains use an alternative initiation codon (in addition to the AUG used to synthesize the picornavirus polyprotein from one long open reading frame) to translate L*, a novel protein that is out of frame with the polyprotein and which plays a key role in the demyelinating disease. We now demonstrate that L* has antiapoptotic activity in macrophage cells and is critical for virus persistence. The antiapoptotic action of L* as well as the differential translation of L* and virion capsid proteins may foster virus persistence in macrophages and interfere with virus clearance. The regulation of apoptotic activity in inflammatory cells may be important in the pathogenesis of TMEV-induced demyelinating disease as well as MS.

Animals↗
PubMed1998

Sequence-specific binding of human immunodeficiency virus type 1 nucleocapsid protein to short oligonucleotides.

We have analyzed the binding of recombinant human immunodeficiency virus type 1 nucleocapsid protein (NC) to very short oligonucleotides by using surface plasmon resonance (SPR) technology. Our experiments, which were conducted at a moderate salt concentration (0.15 M NaCl), showed that NC binds more stably to runs of d(G) than to other DNA homopolymers. However, it exhibits far more stable binding with the alternating base sequence d(TG)n than with any homopolymeric oligodeoxyribonucleotide; thus, it shows a strong sequence preference under our experimental conditions. We found that the minimum length of an alternating d(TG) sequence required for stable binding was five nucleotides. Stable binding to the tetranucleotide d(TG)2 was observed only under conditions where two tetranucleotide molecules were held in close spatial proximity. The stable, sequence-specific binding to d(TG)n required that both zinc fingers be present, each in its proper position in the NC protein, and was quite salt resistant, indicating a large hydrophobic contribution to the binding. Limited tests with RNA oligonucleotides indicated that the preferential sequence-specific binding observed with DNA also occurs with RNA. Evidence was also obtained that NC can bind to nucleic acid molecules in at least two distinct modes. The biological significance of the specific binding we have detected is not known; it may reflect the specificity with which the parent Gag polyprotein packages genomic RNA or may relate to the functions of NC after cleavage of the polyprotein, including its role as a nucleic acid chaperone.

Capsid↗
PubMed1998

Signal peptidase cleavage at the flavivirus C-prM junction: dependence on the viral NS2B-3 protease for efficient processing requires determinants in C, the signal peptide, and prM.

Signal peptidase cleavage at the C-prM junction in the flavivirus structural polyprotein is inefficient in the absence of the cytoplasmic viral protease, which catalyzes cleavage at the COOH terminus of the C protein. The signal peptidase cleavage occurs efficiently in circumstances where the C protein is deleted or if the viral protease complex is present. In this study, we used cDNA of Murray Valley encephalitis virus (MVE) to examine features of the structural polyprotein which allow this regulation of a luminal cleavage by a cytoplasmic protease. We found that the inefficiency of signal peptidase cleavage in the absence of the viral protease is not attributable solely to features of the C protein. Inhibition of cleavage still occurred when charged residues in C were mutated to uncharged residues or when an unrelated protein sequence (that of ubiquitin) was substituted for C. Also, fusion of the C protein did not inhibit processing of an alternative adjacent signal sequence. The cleavage region of the flavivirus prM translocation signal is unusually hydrophobic, and we established that altering this characteristic by making three point mutations near the signal peptidase cleavage site in MVE prM dramatically increased the extent of cleavage without requiring removal of the C protein. In addition, we demonstrated that luminal sequences downstream from the signal peptidase cleavage site contributed to the inefficiency of cleavage.

Amino Acid Sequence↗
PubMed1998

N-terminal protease of pestiviruses: identification of putative catalytic residues by site-directed mutagenesis.

Pestiviruses are the only members of the Flaviviridae that encode a nonstructural protease at the N terminus of their polyproteins. This N-terminal protease (Npro) cleaves itself off of the nascent polyprotein autocatalytically and thereby generates the N terminus of the adjacent viral capsid protein C. In previous reports, sequence similarities between Npro and the catalytic residues of papain-like cysteine proteases were put forward. To test this hypothesis, substitutions of cysteine and histidine residues within Npro were carried out by site-directed mutagenesis. Translation of the mutagenized Npro-C proteins in cell-free lysates confirmed that only the predicted Cys69 was an essential amino acid for proteolysis, not His130. Further essential residues were identified with His49 and Glu22. While it remains speculative whether Glu22-His49-Cys69 actually build a catalytic triad, these results invalidate the assumption that Npro is a papain-like cysteine protease.

Amino Acid Sequence↗
PubMed1998

Type D retrovirus capsid assembly and release are active events requiring ATP.

Mason-Pfizer monkey virus (M-PMV), the prototype type D retrovirus, differs from most other retroviruses by assembling its Gag polyproteins into procapsids in the cytoplasm of infected cells. Once assembled, the procapsids migrate to the plasma membrane, where they acquire their envelope during budding. Because the processes of M-PMV protein transport, procapsid assembly, and budding are temporally and spatially unlinked, we have been able to determine whether cellular proteins play an active role during the different stages of procapsid morphogenesis. We report here that at least two stages of morphogenesis require ATP. Both procapsid assembly and procapsid transport to the plasma membrane were reversibly blocked by treating infected cells with sodium azide and 2-deoxy-D-glucose, which we show rapidly and reversibly depletes cellular ATP pools. Assembly of procapsids in vitro in a cell-free translation/assembly system was inhibited by the addition of nonhydrolyzable ATP analogs, suggesting that ATP hydrolysis and not just ATP binding is required. Since retrovirus Gag polyproteins do not bind or hydrolyze ATP, these results demonstrate that cellular components must play an active role during retrovirus morphogenesis.

Adenosine Triphosphate↗
PubMed1998

Mutations of the human immunodeficiency virus type 1 p6Gag domain result in reduced retention of Pol proteins during virus assembly.

One of the crucial steps in the assembly of the human immunodeficiency virus type 1 (HIV-1) and other retroviruses is the incorporation and retention of all the key viral enzymes in released virions. The viral enzymes protease, reverse transcriptase, and integrase of HIV-1 are initially synthesized as Gag-Pol fusion polyproteins. It has been shown that the incorporation of Gag-Pol polyproteins during virus assembly requires the Gag domains that are shared by the Gag and Gag-Pol precursors. We now report that truncation of the C-terminal p6 domain of HIV-1 Gag, which is present in the Gag precursor but not in the Gag-Pol precursor, drastically reduced the amount of Pol proteins in the mutant virions. Mutations in the lentivirus conserved motif P(T/S)APP in p6 also drastically reduced the amount of Pol proteins in mutant virions. The steady-state levels of Gag-Pol precursors and cleaved Pol proteins in the transfected cells were not affected by mutations in p6. The incorporation of unprocessed Gag-Pol precursors into p6 mutant virions was detected when the viral protease was mutated, suggesting that the interactions among mutant Gag molecules and Gag-Pol precursors were not significantly affected. These results suggest that the p6 domain of HIV-1 Gag may play an important role in recruiting or retaining cleaved Pol proteins during virus assembly.

Amino Acid Sequence↗
PubMed1998

Insertion of a sequence encoding light chain 3 of microtubule-associated proteins 1A and 1B in a pestivirus genome: connection with virus cytopathogenicity and induction of lethal disease in cattle.

Pestiviruses represent the first RNA viruses for which recombination with cellular protein-coding sequences has been reported. As a result of such recombinations cytopathogenic (cp) pestiviruses can develop from noncytopathogenic (noncp) viruses. In the case of bovine viral diarrhea virus (BVDV), the generation of cp mutants is linked to the induction of the lethal syndrome mucosal disease (MD) in cattle. The cp BVDV JaCP was isolated from an animal which had come down with MD. The genome of JaCP contains a novel kind of cellular insertion (LC3*) which is flanked by duplicated pestivirus sequences. Neither insertion nor duplication is present in the genome of the accompanying noncp virus JaNCP. As part of the viral polyprotein, the insertion in the JaCP genome is translated into a polypeptide almost identical to a fragment of light chain 3, a subunit of the microtubule-associated proteins 1A and 1B from the rat. Transient-expression studies revealed that the LC3* sequence is able to induce an additional cleavage of the viral polyprotein. The respective cleavage occurs directly downstream of the LC3*-encoded sequence and is not dependent on the NS3 serine protease. Insertion of LC3* into an infectious noncp pestivirus cDNA clone without duplicated viral sequences resulted in recovery of a defective cp virus able to replicate only in the presence of a noncp helper virus. In contrast, introduction of both insertion and duplication led to an autonomously replicating cp virus.

Amino Acid Sequence↗
PubMed1998

ORF1a-encoded replicase subunits are involved in the membrane association of the arterivirus replication complex.

Among the functions of the replicase of equine arteritis virus (EAV; family Arteriviridae, order Nidovirales) are important viral enzyme activities such as proteases and the putative RNA polymerase and RNA helicase functions. The replicase is expressed in the form of two polyproteins (open reading frame 1a [ORF1a] and ORF1ab), which are processed into 12 nonstructural proteins by three viral proteases. In immunofluorescence assays, the majority of these cleavage products localized to the perinuclear region of the cell. A dense granular and vesicular staining was observed, which strongly suggested membrane association. By using confocal microscopy and double-label immunofluorescence, the distribution of the EAV replicase was shown to overlap with that of PDI, a resident protein of the endoplasmic reticulum and intermediate compartment. An in situ labeling of nascent viral RNA with bromo-UTP demonstrated that the membrane-bound complex in which the replicase subunits accumulate is indeed the site of viral RNA synthesis. A number of ORF1a-encoded hydrophobic domains were postulated to be involved in the membrane association of the arterivirus replication complex. By using various biochemical methods (Triton X-114 extraction, membrane purification, and sodium carbonate treatment), replicase subunits containing these domains were shown to behave as integral membrane proteins and to be membrane associated in infected cells. Thus, contribution to the formation of a membrane-bound scaffold for the viral replication-transcription complex appears to be an important novel function for the arterivirus ORF1a replicase polyprotein.

Animals↗
PubMed1999

Molecular characterization of a bovine enteric calicivirus: relationship to the Norwalk-like viruses.

Jena virus (JV) is a noncultivatable bovine enteric calicivirus associated with diarrhea in calves and was first described in Jena, Germany. The virus was serially passaged 11 times in colostrum-deprived newborn calves and caused diarrheal disease symptoms at each passage. The complete JV genome sequence was determined by using cDNA made from partially purified virus obtained from a single stool sample. JV has a positive-sense single-stranded RNA genome which is 7,338 nucleotides in length, excluding the poly(A) tail. JV genome organization is similar to that of the human Norwalk-like viruses (NLVs), with three separate open reading frames (ORFs) and a 24-nucleotide sequence motif located at the 5' terminus of the genome and at the start of ORF 2. The polyprotein (ORF 1) consists of 1,680 amino acids and has the characteristic 2C helicase, 3C protease, and 3D RNA polymerase motifs also found in the NLVs. However, comparison of the N-terminal 100 amino acids of the JV polyprotein with those of the group 1 and group 2 NLVs showed a considerable divergence in sequence. The capsid protein (ORF 2) at 519 amino acids is smaller than that of all other caliciviruses. JV ORF 2 was translated in vitro to produce a 55-kDa protein that reacted with postinfection serum but not preinfection serum. Phylogenetic studies based on partial RNA polymerase sequences indicate that within the Caliciviridae JV is most closely related to the group 1 NLVs.

Amino Acid Sequence↗
PubMed1999

Mutagenesis of the NS2B-NS3-mediated cleavage site in the flavivirus capsid protein demonstrates a requirement for coordinated processing.

Analysis of flavivirus polyprotein processing has revealed the presence of a substrate for the virus-encoded NS2B-NS3 protease at the carboxy-terminal end of the C (capsid or core) protein. Cleavage at this site has been implicated in the efficient generation of the amino terminus of prM via signal peptidase cleavage. Yellow fever virus has four basic residues (Arg-Lys-Arg-Arg) in the P1 through P4 positions of this cleavage site. Multiple alanine substitutions were made for these residues in order to investigate the substrate specificity and biological significance of this cleavage. Mutants were analyzed by several methods: (i) a cell-free trans processing assay for direct analysis of NS2B-NS3-mediated cleavage; (ii) a trans processing assay in BHK-21 cells, using a C-prM polyprotein, for analysis of prM production; (iii) an infectivity assay of full-length transcripts to determine plaque-forming ability; and (iv) analysis of proteins expressed from full-length transcripts to assess processing in the context of the complete genome. Mutants that exhibited severe defects in processing in vitro and in vivo were incapable of forming plaques. Mutants that contained two adjacent basic residues within the P1 through P4 region were processed more efficiently in vitro and in vivo, and transcripts bearing these mutations were fully infectious. Furthermore, two naturally occurring plaque-forming revertants were analyzed and shown to have restored protein processing phenotypes in vivo. Finally, the efficient production of prM was shown to be dependent on the proteolytic activity of NS3. These data support a model of two coordinated cleavages, one that generates the carboxy terminus of C and another that generates the amino terminus of prM. A block in the viral protease-mediated cleavage inhibits the production of prM by the signal peptidase, inhibits particle release, and eliminates plaque formation.

Capsid↗
PubMed1999

Distinct attenuation phenotypes caused by mutations in the translational starting window of Theiler's murine encephalomyelitis virus.

Upon initiation of translation of picornavirus RNA, the ribosome is believed to bind the internal ribosome entry site of the template and then to form a productive complex with a downstream RNA segment, the starting window. The presence or absence of an AUG triplet within the starting window of the RNA of Theiler's murine encephalomyelitis virus (a picornavirus) is known to modulate its neurovirulence. In this study, mutants of this virus in which the starting windows, lying upstream of the viral polyprotein reading frame, had AUGs with different nonoptimal contexts were engineered. Upon intracerebral inoculation of mice, the mutants proved to be partially attenuated, as judged by a significant increase in the dose causing paralysis in 50% of the animals (PD50). Mutants with similar PD50s might differ from one another by eliciting either a severe, fatal tetraplegy or only mild, recoverable neurologic lesions. Some of the mutants triggered a chronic inflammatory reaction in the white matter of the spinal cord in the absence of detectable viral RNA or antigen. Thus, point mutations changing the context of an AUG within the starting window outside the polyprotein reading frame may differently affect the morbidity and mortality caused by a viral infection and may result in distinct attenuation phenotypes.

Animals↗
PubMed1999

Colocalization and membrane association of murine hepatitis virus gene 1 products and De novo-synthesized viral RNA in infected cells.

Murine hepatitis virus (MHV) gene 1, the 22-kb polymerase (pol) gene, is first translated into a polyprotein and subsequently processed into multiple proteins by viral autoproteases. Genetic complementation analyses suggest that the majority of the gene 1 products are required for viral RNA synthesis. However, there is no physical evidence supporting the association of any of these products with viral RNA synthesis. We have now performed immunofluorescent-staining studies with four polyclonal antisera to localize various MHV-A59 gene 1 products in virus-infected cells. Immunoprecipitation experiments showed that these antisera detected proteins representing the two papain-like proteases and the 3C-like protease encoded by open reading frame (ORF) 1a, the putative polymerase (p100) and a p35 encoded by ORF 1b, and their precursors. De novo-synthesized viral RNA was labeled with bromouridine triphosphate in lysolecithin-permeabilized MHV-infected cells. Confocal microscopy revealed that all of the viral proteins detected by these antisera colocalized with newly synthesized viral RNA in the cytoplasm, particularly in the perinuclear region of infected cells. Several cysteine and serine protease inhibitors, i.e., E64d, leupeptin, and zinc chloride, inhibited viral RNA synthesis without affecting the localization of viral proteins, suggesting that the processing of the MHV gene 1 polyprotein is tightly associated with viral RNA synthesis. Dual labeling with antibodies specific for cytoplasmic membrane structures showed that MHV gene 1 products and RNA colocalized with the Golgi apparatus in HeLa cells. However, in murine 17CL-1 cells, the viral proteins and viral RNA did not colocalize with the Golgi apparatus but, instead, partially colocalized with the endoplasmic reticulum. Our results provide clear physical evidence that several MHV gene 1 products, including the proteases and the polymerase, are associated with the viral RNA replication-transcription machinery, which may localize to different membrane structures in different cell lines.

Animals↗
PubMed1999

Localization of mouse hepatitis virus nonstructural proteins and RNA synthesis indicates a role for late endosomes in viral replication.

The aim of the present study was to define the site of replication of the coronavirus mouse hepatitis virus (MHV). Antibodies directed against several proteins derived from the gene 1 polyprotein, including the 3C-like protease (3CLpro), the putative polymerase (POL), helicase, and a recently described protein (p22) derived from the C terminus of the open reading frame 1a protein (CT1a), were used to probe MHV-infected cells by indirect immunofluorescence (IF) and electron microscopy (EM). At early times of infection, all of these proteins showed a distinct punctate labeling by IF. Antibodies to the nucleocapsid protein also displayed a punctate labeling that largely colocalized with the replicase proteins. When infected cells were metabolically labeled with 5-bromouridine 5'-triphosphate (BrUTP), the site of viral RNA synthesis was shown by IF to colocalize with CT1a and the 3CLpro. As shown by EM, CT1a localized to LAMP-1 positive late endosomes/lysosomes while POL accumulated predominantly in multilayered structures with the appearance of endocytic carrier vesicles. These latter structures were also labeled to some extent with both anti-CT1a and LAMP-1 antibodies and could be filled with fluid phase endocytic tracers. When EM was used to determine sites of BrUTP incorporation into viral RNA at early times of infection, the viral RNA localized to late endosomal membranes as well. These results demonstrate that MHV replication occurs on late endosomal membranes and that several nonstructural proteins derived from the gene 1 polyprotein may participate in the formation and function of the viral replication complexes.

Animals↗
PubMed2000

Isolation and characterization of monoclonal antibodies that inhibit hepatitis C virus NS3 protease.

A series of mouse monoclonal antibodies (MAbs) to the nonstructural protein 3 (NS3) of hepatitis C virus was prepared. One of these MAbs, designated 8D4, was found to inhibit NS3 protease activity. This inhibition was competitive with respect to the substrate peptide (K(i) = 39 nM) but was significantly decreased by the addition of the NS4A peptide, a coactivator of the NS3 protease. 8D4 also showed marked inhibition of the NS3-dependent cis processing of the NS3/4A polyprotein but had virtually no effect on the succeeding NS3/4A-dependent trans processing of the NS5A/5B polyprotein in vitro. Epitope mapping of 8D4 with a random peptide library revealed a consensus sequence, DxDLV, that matched residues 79 to 83 (DQDLV) of NS3, a region containing the catalytic residue Asp-81. Furthermore, synthetic peptides including this sequence were shown to block the ability of 8D4 to bind to NS3, indicating that 8D4 interacts with the catalytic region of NS3. The data showing decreased inhibition potency of 8D4 against the NS3/4A complex suggest that 8D4 recognizes the conformational state of the protease active site caused by the association of NS4A with the protease.

Amino Acid Sequence↗
PubMed2000

Human immunodeficiency virus type 1 virion density is not determined by nucleocapsid basic residues.

The human immunodeficiency virus type 1 (HIV-1) Gag polyprotein is sufficient for assembly and release of virion-like particles from the plasma membrane. To promote assembly, the Gag polyprotein must polymerize to form a shell that lines the inner membrane of nascent virions. Several techniques have been used to functionally map the domain required for Gag polymerization (the I domain). Among these methods, isopycnic centrifugation has been used under the assumption that changes in virion density reflect impairment in Gag-Gag interaction. If virion density is determined by efficient Gag-Gag interaction, then mutation of basic residues in the nucleocapsid (NC) domain should disrupt virion density, since these residues constitute the I domain. However, we have previously shown that simultaneous disruption of up to 10 HIV-1 NC basic residues has no obvious effect on virion density. To rule out the possibility that HIV-1 NC basic residues other than those previously mutated might be important for virion density, mutations were introduced at the remaining sites and the ability of these mutations to affect Gag-Gag interaction and virion density was analyzed. Included in our analysis is a mutant in which all NC basic residues are replaced with alanine. Our results show that disruption of HIV-1 NC basic residues has an enormous effect on Gag-Gag interaction but only a minimal effect on the density of those virions that are still produced. Therefore, the determinants of the I domain and of virion density are genetically distinguishable.

Amino Acid Sequence↗
PubMed2000

Characterization of an internal ribosomal entry segment in the 5' leader of murine leukemia virus env RNA.

The 5' untranslated region, also called the leader, of oncoretroviruses and lentiviruses is long and is formed of several structured domains critically important in virus replication. The 5' leader of murine leukemia virus (MLV) RNA contains an internal ribosomal entry segment (IRES) which promotes synthesis of Gag and glyco-Gag polyprotein precursors. In the present study we investigated the translational features of the 5' leader of MLV subgenomic RNA (env RNA) encoding the Env polyprotein precursor. When the env leader was inserted between two genes, such as lacZ and the neomycin resistance cassette, in a dicistronic vector, it allowed IRES-dependent translation of the 3' cistron in the rabbit reticulocyte lysate system and in murine cells. The drug rapamycin and the foot-and-mouth disease virus L protease, known to inhibit cap-dependent translation, caused an enhancement of the translation driven by the env leader sequence, consistent with an IRES activity promoting Env expression. Analysis of several deletion mutants led us to localize the minimal env IRES between the splice junction and the env AUG start codon.

3T3 Cells↗
PubMed2000

Basic residues in human immunodeficiency virus type 1 nucleocapsid promote virion assembly via interaction with RNA.

Retroviral Gag polyproteins drive virion assembly by polymerizing to form a spherical shell that lines the inner membrane of nascent virions. Deletion of the nucleocapsid (NC) domain of the Gag polyprotein disrupts assembly, presumably because NC is required for polymerization. Human immunodeficiency virus type 1 NC possesses two zinc finger motifs that are required for specific recognition and packaging of viral genomic RNA. Though essential, zinc fingers and genomic RNA are not required for virion assembly. NC promiscuously associates with cellular RNAs, many of which are incorporated into virions. It has been hypothesized that Gag polymerization and virion assembly are promoted by nonspecific interaction of NC with RNA. Consistent with this model, we found an inverse relationship between the number of NC basic residues replaced with alanine and NC's nonspecific RNA-binding activity, Gag's ability to polymerize in vitro and in vivo, and Gag's capacity to assemble virions. In contrast, mutation of NC's zinc fingers had only minor effects on these properties.

Amino Acid Sequence↗