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Results for “viral complexity”

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PubMed1994

An outbreak of viral hepatitis in a housing complex of north Calcutta.

In a housing complex of North Calcutta, a sudden outbreak of hepatitis occurred between September 1988 and January 1989. Of 620 residents, 8.5 per cent were affected. Majority of the cases occurred among the adult population and the case fatality rate was 3.8 per cent. The epidemic was common source with peak incidence in the month of November. The source of infection was most likely drinking water supplied by the Municipal Corporation which was found to be contaminated by faecal coli. Epidemic was suspected to be caused by enterically transmitted Non A Non B hepatitis virus as the serological testing of all 18 blood samples were negative for anti-HAVIgM titre and only one sample was positive for HBsAg.

Adolescent↗

PubMed2005

Specific interactions between HIV-1 nucleocapsid protein and the TAR element.

During retroviral reverse transcription, the minus-strand strong-stop DNA (ss-cDNA) is transferred to the 3' end of the genomic RNA and this requires the repeat (R) sequences present at both ends of the genome. In vitro, the human immunodeficiency virus type 1 (HIV-1) R sequence can promote DNA strand transfer when present in ectopic internal positions. Using HIV-1 model systems, the R sequences and nucleocapsid protein (NC) were found to be key determinants of ss-cDNA transfer. To gain insights into specific interactions between HIV-1 NC and RNA and the influence of NC on R folding, we investigated the secondary structures of R in two natural contexts, namely at the 5' or 3' end of RNAs representing the terminal regions of the genome, and in two ectopic internal positions that also support efficient minus-strand transfer. To investigate the roles of NC zinc fingers and flanking basic domains in the NC/RNA interactions, we used NC mutants. Analyses of the viral RNA/NC complexes by chemical and enzymatic probings, and gel retardation assays were performed under conditions allowing ss-cDNA transfer by reverse transcriptase. We report that NC binds the TAR apical loop specifically in the four genetic contexts without changing the folding of the TAR hairpin and R region significantly, and this requires the NC zinc fingers. In addition, we show that efficient annealing of cTAR DNA to the 3' R relies on sequence complementarities between TAR and cTAR terminal loops. These findings suggest that the TAR apical loop in the acceptor RNA is the initiation site for the annealing reaction that is chaperoned by NC during the minus-strand transfer.

Amino Acid Sequence↗

PubMed2002

Rotavirus protein involved in genome replication and packaging exhibits a HIT-like fold.

Rotavirus, the major cause of life-threatening infantile gastroenteritis, is a member of the Reoviridae. Although the structures of rotavirus and other members of the Reoviridae have been extensively studied, little is known about the structures of virus-encoded non-structural proteins that are essential for genome replication and packaging. The non-structural protein NSP2 of rotavirus, which exhibits nucleoside triphosphatase, single-stranded RNA binding, and nucleic-acid helix-destabilizing activities, is a major component of viral replicase complexes. We present here the X-ray structure of the functional octamer of NSP2 determined to a resolution of 2.6 A. The NSP2 monomer has two distinct domains. The amino-terminal domain has a new fold. The carboxy-terminal domain resembles the ubiquitous cellular histidine triad (HIT) group of nucleotidyl hydrolases. This structural similarity suggests that the nucleotide-binding site is located inside the cleft between the two domains. Prominent grooves that run diagonally across the doughnut-shaped octamer are probable locations for RNA binding. Several RNA binding sites, resulting from the quaternary organization of NSP2 monomers, may be required for the helix destabilizing activity of NSP2 and its function during genome replication and packaging.

Amino Acid Sequence↗

PubMed1993

In vitro translation of fractionated virus-specific RNA isolated from plasma of chicken infected by avian myeloblastosis virus. Unprocessed and processed myeloblastosis-associated virus env-polypeptide precursors.

The 60 S viral RNA complex isolated from leukaemic plasma of chicken infected by avian myeloblastosis virus (AMV) was denatured, the poly(A)-RNA selected and centrifuged in a linear sucrose density gradient. RNA from each fraction was translated in vitro and the products were analyzed by slab polyacrylamide gel electrophoresis (PAGE). Unprocessed primary translation product (p64env) of MAV env gene from 21 S RNA fraction was immunoprecipitated by anti-gp85 serum. If, however, this RNA was translated in the presence of dog pancreas microsomal membranes (DPM), the processed 92 K MAV glycoprotein precursor (p92env) was immunoprecipitated by anti-gp85 serum. This precursor, unlike p64env was resistant to exogenous protease.

Animals↗

PubMed2000

Cell-to-cell movement of TMV RNA is temperature-dependent and corresponds to the association of movement protein with microtubules.

The movement protein (MP) of tobacco mosaic virus (TMV) is essential for spread of the viral RNA genome from cell to cell. During infection, the MP associates with microtubules, and it has been proposed that the cytoskeleton transports the viral ribonucleoprotein complex from ER sites of synthesis to plasmodesmata through which infection spreads into adjacent cells. However, microtubule association of MP was observed in cells undergoing late infection rather than in cells undergoing early infection at the leading edge of expanding infection sites where virus RNA cell-to-cell spread occurs. Therefore, alternative roles for microtubules in virus infection have been proposed, including a role in MP degradation. To further investigate the role of microtubules in virus pathogenesis, we tested the efficiency of cell-to-cell spread of infection and microtubule association of the MP in response to changes in temperature. We show that the subcellular distribution of MP is temperature-dependent and that a higher efficiency of intercellular transport of virus RNA at elevated temperatures corresponds to an increased association of MP with microtubules early in infection.

Green Fluorescent Proteins↗

PubMed1999

hnRNP C and polypyrimidine tract-binding protein specifically interact with the pyrimidine-rich region within the 3'NTR of the HCV RNA genome.

Like other members of the Flaviviridae family, the 3' non-translated region (NTR) of the hepatitis C virus (HCV) is believed to function in the initiation and regulation of viral RNA replication by interacting with components of the viral replicase complex. To inves-tigate the possibility that host components may also participate in this process, we used UV cross-linking assays to determine if any cellular proteins could bind specifically to the 3'NTR RNA. We demonstrate the specific interaction of two host proteins with the extensive pyrimidine-rich region within the HCV 3'NTR. One host protein migrates as a doublet with a molecular weight of 57 kDa and is immunoreactive with antisera specific for polypyrimidine tract-binding protein (PTB), and the other protein (35 kDa) is recognized by a monoclonal antibody specific for heterogeneous nuclear ribonucleoprotein C (hnRNP C). These results suggest that recognition of the large pyrimidine-rich region by PTB and hnRNP C may play a role in the initiation and/or regulation of HCV RNA replication.

3' Untranslated Regions↗

PubMed2006

Subcellular localization and membrane topology of the Dengue virus type 2 Non-structural protein 4B.

Dengue virus (DV) is a member of the family Flaviviridae. These positive strand RNA viruses encode a polyprotein that is processed in case of DV into 10 proteins. Although for most of these proteins distinct functions have been defined, this is less clear for the highly hydrophobic non-structural protein (NS) 4B. Despite its possible role as an antagonist of the interferon-induced antiviral response, this protein may play an additional more direct role for viral replication. In this study we determined the subcellular localization, membrane association, and membrane topology of DV NS4B. We found that NS4B resides primarily in cytoplasmic foci originating from the endoplasmic reticulum. NS4B colocalizes with NS3 and double-stranded RNA, an intermediate of viral replication, arguing that NS4B is part of the membrane-bound viral replication complex. Biochemical analysis revealed that NS4B is an integral membrane protein, and that its preceding 2K signal sequence is not required for this integration. We identified three membrane-spanning segments in the COOH-terminal part of NS4B that are sufficient to target a cytosolic marker protein to intracellular membranes. Furthermore, we established a membrane topology model of NS4B in which the NH2-terminal part of the protein is localized in the endoplasmic reticulum lumen, whereas the COOH-terminal part is composed of three trans-membrane domains with the COOH-terminal tail localized in the cytoplasm. This topology model provides a good starting point for a detailed investigation of the function of NS4B in the DV life cycle.

Amino Acid Sequence↗

PubMed2001

Identification of a central DNA flap in feline immunodeficiency virus.

A duplication of the polypurine tract (PPT) at the center of the human immunodeficiency virus type 1 (HIV-1) genome (the cPPT) has been shown to prime a separate plus-strand initiation and to result in a plus-strand displacement (DNA flap) that plays a role in nuclear import of the viral preintegration complex. Feline immunodeficiency virus (FIV) is a lentivirus that infects nondividing cells, causes progressive CD4(+) T-cell depletion, and has been used as a substrate for lentiviral vectors. However, the PPT sequence is not duplicated elsewhere in the FIV genome and a central plus-strand initiation or strand displacement has not been identified. Using Southern blotting of S1 nuclease-digested FIV preintegration complexes isolated from infected cells, we detected a single-strand discontinuity at the approximate center of the reverse-transcribed genome. Primer extension analyses assigned the gap to the plus strand, and mapped the 5' terminus of the downstream (D+) segment to a guanine residue in a purine-rich tract in pol (AAAAGAAGAGGTAGGA). RACE experiments then mapped the 3' terminus of the upstream plus (U+)-strand segment to a T nucleotide located 88 nucleotides downstream of the D+ strand 5' terminus, thereby identifying the extent of D+ strand displacement and the central termination sequence of this virus. Unlike HIV, the FIV cPPT is significantly divergent in sequence from its 3' counterpart (AAAAAAGAAAAAAGGGTGG) and contains one and in some cases two pyrimidines. An invariant thymidine located -2 to the D+ strand origin is neither required nor optimal for codon usage at this position. Although the mapped cPPTs of FIV and HIV-1 act in cis, they encode homologous amino acids in integrase.

Animals↗

PubMed1992

Nucleolin forms a specific complex with a fragment of the viral (minus) strand of minute virus of mice DNA.

Nucleolin, a major nucleolar protein, forms a specific complex with the genome (a single-stranded DNA molecule of minus polarity) of parvovirus MVMp in vitro. By means of South-western blotting experiments, we mapped the binding site to a 222-nucleotide motif within the non-structural transcription unit, referred to as NUBE (nucleolin-binding element). The specificity of the interaction was confirmed by competitive gel retardation assays. DNaseI and nuclease S1 probing showed that NUBE folds into a secondary structure, in agreement with a computer-assisted conformational prediction. The whole NUBE may be necessary for the interaction with nucleolin, as suggested by the failure of NUBE subfragments to bind the protein and by the nuclease footprinting experiments. The present work extends the previously reported ability of nucleolin to form a specific complex with ribosomal RNA, to a defined DNA substrate. Considering the tropism of MVMp DNA replication for host cell nucleoli, these data raise the possibility that nucleolin may contribute to the regulation of the parvoviral life-cycle.

Base Sequence↗

PubMed1978

Transcription of a defective polyoma virus genome.

The circular genome of the cloned defective polyoma virus D-50 consists of tandemly repeated copies of the DNA sequence between 67 and 84 units on the wild-type polyoma virus DNA map. Each repeated copy thus contains the origin of viral DNA replication, which is located at about 71 map units. Viral RNA was synthesized in vitro using viral transcription complexes extracted late (30 hr) after infection from mouse cells co-infected with D-50 and helper wild-type virus. Both wild-type and D-50 DNA molecules were active as templates for in vitro transcription. Approximately 84% of the RNA transcribed in vitro from wild-type DNA was complementary to the L DNA strand. This is normal for wild-type transcription late after infection. By contrast, at least 90% of the RNA transcribed from D-50 DNA molecules was complementary to the E DNA strand. After normalization of the data to account for the observed molar ratio of D-50 DNA repeated sequences to unit length wild-type DNA, we estimate that transcription of the E DNA strand of each D-50 repeated unit is about 1.4 times as efficient as transcription of the wild-type E DNA strand. Transcription of the D-50 L DNA strand, however, is only 0.03 times as efficient as transcription of the wild-type L DNA strand. The implications of these results concerning the nature and location of promoter sequences in polyoma DNA are discussed.

Binding Sites↗

PubMed1984

SV40 deletion mutant (d1861) with agnoprotein shortened by four amino acids.

d1861 is an SV40 deletion mutant which was thought to lack the agnoprotein coding region and was used to verify the role of agnoprotein in the life cycle of SV40. In the present study the region flanking the deletion was sequenced and, in contrast to the available information, it was found that d1861 lacks 12 nt in phase, downstream from the AUG start codon of agnoprotein (residues 347-358). Using the runoff protocol with viral transcriptional complexes (VTC), that in vitro elongate the in vivo preinitiated nascent RNA, it was found that in vivo the major initiation site for late transcription is at residue 325, the same as in wild type (WT). In comparison with WT, d1861 encodes information for agnoprotein shortened by four amino acids and it has been identified in d1861 infected cells. However, pulse-chase experiments indicated that the rate of synthesis of d1861 agnoprotein is slower than that of WT agnoprotein and that it has a turnover rate of 1 hr as compared to 3 hr of WT agnoprotein. The reduced rate of synthesis of d1861 agnoprotein can be explained by nuclease S1 analyses in which the major leader of d1861 16 S RNA, that encodes the agnoprotein, appeared in significantly lower amounts as compared to the major leader of WT 16 S RNA. Furthermore, analysis of the potential secondary structures at the 5' end of the leader of d1861 16 S RNA has revealed stable structures in which the start codon of agnoprotein is sequestered in a stem. The involvement of RNA secondary structures in regulating the synthesis of agnoprotein is discussed.

Amino Acid Sequence↗

PubMed2003

Proteasome activator PA28gamma-dependent nuclear retention and degradation of hepatitis C virus core protein.

Hepatitis C virus (HCV) core protein plays an important role in the formation of the viral nucleocapsid and a regulatory protein involved in hepatocarcinogenesis. In this study, we have identified proteasome activator PA28gamma (11S regulator gamma) as an HCV core binding protein by using yeast two-hybrid system. This interaction was demonstrated not only in cell culture but also in the livers of HCV core transgenic mice. These findings are extended to human HCV infection by the observation of this interaction in liver specimens from a patient with chronic HCV infection. Neither the interaction of HCV core protein with other PA28 subtypes nor that of PA28gamma with other Flavivirus core proteins was detected. Deletion of the PA28gamma-binding region from the HCV core protein or knockout of the PA28gamma gene led to the export of the HCV core protein from the nucleus to the cytoplasm. Overexpression of PA28gamma enhanced the proteolysis of the HCV core protein. Thus, the nuclear retention and stability of the HCV core protein is regulated via a PA28gamma-dependent pathway through which HCV pathogenesis may be exerted.

Active Transport, Cell Nucleus↗

PubMed1980

Comparison of the nuclear ribonucleoproteins containing the transcripts of adenovirus-2 and HeLa cell dna.

A method as devised allowing the preparation of hnRNA-containing ribonucleproteins (hnRNP) frm HeLa cells infected with adenovirus type 2 under conditions where the extraction of viral replication complexes as minimal. Approximately 60% of the RNA from such hnRNP hybridized with adenovirus DNA. The hnRNP from infected cells had the same general characteristics as those from uninfected cells. Their size was heterogeneous (30-260 S) and depended upon that of their RNA. Their CsCl densities were identical (1.39-1.40 g/ml), indicating the same protein:RNA ratio. Their proteins were found in the same molecular weight range, between 25 000 and 200 000. The major proteins of hnRNP from HeLa cells were present in hnRNP from adenovirus-infected cells. As 60% of the cellular RNA was replaced by adenovirus RNA in hnRNP, this in dicated that there was not stringent specificity in the RNA-protein interactions. The relative proportions of the proteins were identifical in both cases, suggesting that the hnRNP assembly was independent of nucleotide sequences at least for the major proteins. The hnRNP from infected and uninfected cells differed by the size of their RNA, which was larger after infection, and by the presence of six additional minor polypeptides after infection. However, it cannot be excluded that the presence of these polypeptides in hnRNP resulted from non-specific adsorption.

Adenoviruses, Human↗

PubMed1983

Pausing of RNA polymerase molecules during in vivo transcription of the SV40 leader region.

Viral transcription complexes were isolated from SV40-infected cells and incubated in vitro in the presence of [alpha-32P]UTP to allow elongation of the promoter-proximal RNA up to the attenuation sites. The 94 nucleotide attenuated RNA (spanning nucleotides 243-336) was purified, digested with RNase T1 and fingerprinted. The labeled oligonucleotides were then isolated, digested with RNase T2 and their base composition was determined. Based on these analyses 10 consecutive oligonucleotides, spanning residues 259-336, were identified. As the in vivo synthesized oligonucleotides are unlabeled the junctions between labeled and unlabeled oligonucleotides define the in vivo pause sites of RNA polymerase molecules. The characterization of the 10 radioactive spots and their relative intensities allowed the localization of two in vivo pause sites: one at 13-16 nucleotides downstream from the major initiation site presumably at the initial opening of the DNA helix and the second at approximately 40 nucleotides downstream from the major initiation site, just past a GC-rich region of dyad symmetry. It is postulated that pausing of RNA polymerase molecules in the leader region is an essential process in the control of SV40 late transcription.

5' Untranslated Regions↗

PubMed1984

S1 mapping of purified nascent transcripts of simian virus 40.

We purified nascent simian virus 40 late transcripts by incubating viral transcriptional complexes, isolated from infected BSC-1 cells, in a reaction mixture that contained mercurated CTP; RNA molecules that had incorporated mercurated residues in vitro were isolated by sulfhydrylcellulose affinity chromatography. The nascent RNA was hybridized to an end-labeled HindIII C probe fragment (0.646 to 0.86 map unit), and the hybrids were analyzed by S1 mapping. Most of the products of digestion corresponded to unspliced transcripts with 5' ends mapping at nucleotides 325, 260, and 195, which are positions of the 5' ends of mature, cytoplasmic late mRNA species. In addition, two minor products diagnostic of splicing at the acceptor junctions mapping at nucleotides 556 and 443 were detected. Because the abundance of these products was not diminished by repurifying the nascent RNA through a second round of sulfhydrylcellulose chromatography, these products did not originate from contaminating non-nascent RNA. Moreover, the generation of these products was not affected when a higher salt concentration and lower temperature were used for S1 digestion, conditions that should decrease artifactual cleavage by S1 in A + U-rich regions of colinear hybrids. Therefore, it is likely that some simian virus 40 RNA chains are spliced before release from the template.

Base Sequence↗

PubMed1994

Protection of mice against an influenza virus infection by oral vaccination with viral nucleoprotein incorporated into immunostimulating complexes.

Influenza A virus nucleoprotein (NP) was integrated into immunostimulating complexes (ISCOMs) after attachment of bacterial lipopolysaccharide to the antigen. Oral immunization with these NP-ISCOMs protected mice fully against an otherwise lethal challenge infection with an unrelated influenza virus subtype without the appearance of severe clinical signs or extensive pathological lesions in the lungs. Mice immunized with analogous bovine serum albumine-incorporated ISCOMs all died. After oral immunization, high titers of NP-specific antibodies, particularly IgA, could be detected in the bronchoalveolar fluid and in the blood serum. No cytotoxic lymphocytes could be demonstrated in the spleens or the lungs of vaccinated mice, and no anti-NP antibody-dependent cytolysis of infected host cells was mediated by complement or in the form of an antibody-dependent cell cytotoxicity. However, a vigorous delayed-type hypersensitivity reaction was produced after probing vaccinated animals with purified NP. No comparable protective immunity or antibody response was induced by a strictly intragastric administration of NP-ISCOMs. It appears, therefore, that the general and local immune response in the lungs was primarily stimulated through contact of NP-ISCOMs with the mucous membrane of the oro-pharyngeal cavity and that cytotoxic effects did not play a major role for the establishment of the protective immunity. Partial protection against a lethal challenge was observed in chickens immunized with NP-ISCOMs in the drinking water.

Administration, Oral↗

PubMed1999

Selective stimulation of hepatitis C virus and pestivirus NS5B RNA polymerase activity by GTP.

NS5B of the hepatitis C virus is an RNA template-dependent RNA polymerase and therefore the key player of the viral replicase complex. Using a highly purified enzyme expressed with recombinant baculoviruses in insect cells, we demonstrate a stimulation of RNA synthesis up to 2 orders of magnitude by high concentrations of GTP but not with ATP, CTP, UTP, GDP, or GMP. Enhancement of RNA synthesis was found with various heteropolymeric RNA templates, with poly(C)-oligo(G)12 but not with poly(A)-oligo(U)12. Several amino acid substitutions in polymerase motifs B, C, and D previously shown to be crucial for RdRp activity were tested for GTP stimulation of RNA synthesis. Most of these mutations, in particular those affecting the GDD motif (motif C) strongly reduced or completely abolished activation by GTP, suggesting that the same NTP-binding site is used for stimulation and RNA synthesis. Since GTP did not affect the overall RNA binding properties or the elongation rate, high concentrations of GTP appear to accelerate a rate-limiting step at the level of initiation of RNA synthesis. Finally, enhancement of RNA synthesis by high GTP concentrations was also found with NS5B of the pestivirus classical swine fever virus, but not with the 3D polymerase of poliovirus. Thus, stimulation of RdRp activity by GTP is evolutionarily conserved between the closely related hepaciviruses and pestiviruses but not between these and the more distantly related picornaviruses.

DNA-Directed RNA Polymerases↗

PubMed2006

Binding site-based classification of coronaviral papain-like proteases.

The coronavirus replicase gene encodes one or two papain-like proteases (termed PL1pro and PL2pro) implicated in the N-terminal processing of the replicase polyprotein and thus contributing to the formation of the viral replicase complex that mediates genome replication. Using consensus fold recognition with the 3D-JURY meta-predictor followed by model building and refinement, we developed a structural model for the single PLpro present in the severe acute respiratory syndrome coronavirus (SCoV) genome, based on significant structural relationships to the catalytic core domain of HAUSP, a ubiquitin-specific protease (USP). By combining the SCoV PLpro model with comparative sequence analyses we show that all currently known coronaviral PLpros can be classified into two groups according to their binding site architectures. One group includes all PL2pros and some of the PL1pros, which are characterized by a restricted USP-like binding site. This group is designated the R-group. The remaining PL1pros from some of the coronaviruses form the other group, featuring a more open papain-like binding site, and is referred to as the O-group. This two-group, binding site-based classification is consistent with experimental data accumulated to date for the specificity of PLpro-mediated polyprotein processing and PLpro inhibition. It also provides an independent evaluation of the similarity-based annotation of PLpro-mediated cleavage sites, as well as a basis for comparison with previous groupings based on phylogenetic analyses.

Amino Acid Sequence↗