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At least 19 recordsLinked to original sources
PubMed1995

Detection of Cellular Proteins and Viral Core Protein Interacting with the 5' Untranslated Region of Hepatitis C Virus RNA.

Hepatitis C virus (HCV) is a pesti- and flavi-like virus, which contains a highly conserved 5'-untranslated region (UTR). This region is implicated in the regulation of both translation and RNA replication. To examine the possible cellular factors involved in HCV replication, we performed UV cross-linking experiments to detect cellular protein binding to 5'-UTR of HCV RNA. No cytoplasmic proteins were found to cross-link to 5'-UTR. Surprisingly, when nuclear extracts were used for UV cross-linking, a major protein of 110 kD and several other minor proteins were detected. Competition assays confirmed that the binding of the 110-kD protein was specific to the 5'-UTR. The protein-binding site was mapped within the 78-nt region between nucleotides 199 and 277 from the 5' end of the viral RNA. This protein was present in several different cell lines tested. No cellular proteins specifically bound to the complementary strands of the 5'-UTR. We have also shown by an RNA-protein blotting assay that 5'-UTR bound to the HCV core protein, which can be translocated to the nuclei. These findings suggest that HCV RNA may enter nuclei by complexing with the viral core protein and interact with nuclear proteins that are involved in the regulation of RNA replication or translation. It is thus possible that HCV employs a replication strategy distinct from its related pestiviruses or flaviviruses. Copyright 1995 S. Karger AG, Basel

Journal Article↗
PubMed1985

Sequence analysis of the viral core protein and the membrane-associated proteins V1 and NV2 of the flavivirus West Nile virus and of the genome sequence for these proteins.

Cell-associated flaviviruses contain the two membrane proteins V3 and NV2 besides the viral core protein V2 whereas extracellular viruses do contain V2 protein and the two membrane proteins V3 and V1. Since the V1 protein could not be detected in infected cells it has been suggested that V1 is generated from NV2 by proteolytic cleavage during the release of virus from cells (D. Shapiro, W. E. Brandt, and P. K. Russell (1972), Virology 50, 906-911). We have isolated the viral structural proteins V1, V2, and NV2 from the flavivirus West Nile virus and determined their amino-terminal amino acid sequences and amino acid sequences of peptides derived from these proteins. We have also transcribed parts of the viral genome into cDNA and cloned and sequenced this cDNA. The analyses of the protein structure of V1, V2, and NV2 together with the determination of the amino-terminal sequence of V3 (data not shown) have allowed us to identify the nucleotide region coding for the structural proteins V2, NV2, and V1. The primary structure of this nucleotide sequence is presented in this report. The data show that the amino terminus of the viral core protein V2 is followed by the amino termini of the proteins NV2, V1, and V3, respectively. These data for the first time identify the exact order of all structural proteins of a flavivirus identified so far. Our data strongly support the above-mentioned hypothesis that V1 is derived from NV2 by proteolytic cleavage and furthermore indicate that V1 represents the nonglycosylated carboxy-terminal part of NV2 which contains those sequences which anchor NV2 in the viral membrane. A working hypothesis is presented in which two species of cellular enzymes, signalase(s) removing signal sequences and enzymes involved in cleaving polyproteins after a pair of basic amino acids, do generate the proteins V2, NV2, and V1 from the growing peptide chain synthesized during translation of the 42 S genome RNA which functions as mRNA for these proteins.

Amino Acid Sequence↗
PubMed2002

Hepatitis B viral core protein activates the hepatitis B viral enhancer II/pregenomic promoter through the nuclear factor kappaB binding site.

We here demonstrated that the hepatitis B viral (HBV) core protein (HBc) functions as a transcriptional activator on the pregenomic promoter of HBV. Detailed analyses on the HBV pregenomic promoter by serial deletion, mutation, and heterologous promoter system showed that the site responsible for activation was the nuclear factor kappaB (NF-kappaB) binding site (GGGACGTACT, nucleotides 1408-1417) upstream of the enhancer II/pregenomic promoter. The electrophoretic mobility shift assay using the HBc-transfected HepG2 nuclear extracts showed that the HBc enhanced the NF-kappaB DNA-binding ability. These results suggest that the HBc functions as a positive regulator, which may enhance viral replication in hepatocytes.

Binding Sites↗
PubMed1988

Identification of gag precursor of equine infectious anaemia virus with monoclonal antibodies to the major viral core protein, p26.

Monoclonal antibodies (MAbs) against the major core protein p26 of equine infectious anaemia virus (EIAV) were produced and characterized. Sensitive enzyme-linked immunosorbent assay and Western blot immunoassay were employed to confirm the specificity of these MAbs. Western blot analysis also indicated that MAbs to p26 reacted with another EIAV protein of 55,000 apparent Mr (designated here as Pr55gag) present in density gradient-purified virus preparations. Rabbit antiserum prepared against p26 as well as MAbs to p26 detected Pr55gag and several other intermediate clevage products in detergent-soluble lysates of virus-infected cells in Western blot and immunoprecipitation assays. The results suggest that Pr55gag is the gag polyprotein of EIAV.

Antibodies, Monoclonal↗
PubMed1976

Specific binding of the type C viral core protein p12 with purified viral RNA.

The major viral phosphoproteins (p12) of the Rauscher murine leukemia virus (R-MuLV) and the simian sarcoma-associated virus (SSAV) bind in vitro to their homologous 70S and 35S viral RNAs. Using purified 32P-labeled RNA and 125I-labeled p12 protein, complexes that are stabilized by formaldehyde-cross-linking can be readily detected after velocity gradient centrifugation. The in vitro reconstructed ribonucleoprotein complexes are seen only with p12 proteins incubated with viral RNAs isolated from the same type C viruses; no such complexes form with heterologous protein-RNA mixtures. Homologous but not heterologous p12 molecules compete with radiolabeled p12 protein for the specific viral RNA binding sites. The competition assay permits the detection of 10 ng of viral p12 protein. The major internal protein of type C viruses (p30) does not bind to viral RNA using identical assay conditions. From the specific activities of the radiolabeled components and also by equilibrium sedimentation analysis, we estimate that fewer than 15 molecules of p12 protein bind to each molecule of viral RNA. Both the specificity and stoichiometry of the p12-RNA interactions suggest that these RNA tumor virus proteins have a regulatory role in cells.

Binding Sites↗
PubMed1994

Multiple viral core proteins are determinants of reovirus-induced acute myocarditis.

Previously, we showed that the M1 gene (encoding a viral core protein, mu 2, whose function is unknown) was associated with the efficiently myocarditic phenotype of a reovirus variant, 8B. Here, we have extended our genetic analysis of 8B and conducted genetic analyses of two other reovirus strains (T1L [serotype 1 strain Lang] and Abney). Our results demonstrate that multiple viral core proteins are determinants of reovirus-induced myocarditis. In contrast to our previous association of mu 2 with induction of myocarditis, this provides strong evidence that a core function achieved through the interaction of multiple core proteins is responsible for induction of the disease.

Animals↗
PubMed2003

Transcriptional repression of the human p53 gene by hepatitis B viral core protein (HBc) in human liver cells.

Hepatitis B virus (HBV) is a causative agent of chronic and acute hepatitis, and is associated with the development of hepatocellular carcinoma (HCC). We demonstrate here that the Hepatitis B viral core protein (HBc) functions as a repressor on the promoter activity of the human p53 gene. The functional analyses of the promoter of the p53 gene by serial deletion, site-directed mutagenesis, and the heterologous promoter system revealed that the promoter activity was repressed through the E2F1-binding site (nucleotides -28 to -8) by HBc. An electrophoretic mobility shift assay (EMSA) showed that the HBc reduced the DNA-binding ability of E2F1 to the binding site of the p53 promoter. The interaction of HBc with E2F1 was also observed by glutathione S-transferase (GST) fusion protein binding assay. Furthermore, HBc represses the expression of the p53 gene in the human liver cell line HepG2. Finally, HBc and HBx synergistically repress both the promoter activity and the expression of the p53 gene in HepG2 cells. These results, together with our previous study, strongly suggest that HBc, like HBx, represses the expression of the human p53 tumor suppressor gene.

Binding Sites↗
PubMed1984

Identification of a transfer of viral core protein to cellular ribosomes during the early stages of alphavirus infection.

Sindbis virus containing [35S]methionine-labeled structural proteins was allowed to be taken up by primary chick embryo fibroblasts, and the fate of the core protein was studied. The experiments show that core protein of incoming viral particles is transferred to the large subunit of cellular ribosomes during the initial steps of virus infection. A similar transfer occurs in vitro if cores isolated from SIN virus particles are incubated in the postmitochondrial cytoplasmic fraction of cell lysates. In vivo transfer also occurs if the protein synthesis-inhibiting drugs puromycin or cycloheximide are present during virus uptake, whereas in the presence of chloroquine, which inhibits the release of viral cores into the cytoplasm, which is necessary for productive infection, a transfer of core protein to ribosomes cannot be observed. The latter result indicates that the transfer probably is part of the reactions leading to the release of viral genomic RNA into the cellular cytoplasm during the early stages of productive infection, and presumably does not reflect side reactions. It has been shown earlier that newly synthesized alphavirus core protein binds to the large ribosomal subunit prior to the assembly of viral core particles in infected cells [I. Ulmanen, H. Sonderlund, and L. Kääriäinen (1979) Virology 99, 265-276]. These data lead to the suggestion that the disassembly and assembly of alphavirus cores might be regulated by a process which could be called receptor-mediated core disassembly, in which acceptors exist for the protein components of viral nucleoproteins in uninfected cells which early in infection bind these proteins and thereby lead to disassembly of these complexes, and which later on have to be saturated with newly synthesized protein before efficient assembly of nucleoproteins can occur, and that the large ribosomal subunit functions as such a receptor during alphavirus replication.

Animals↗
PubMed1989

The reovirus M1 gene, encoding a viral core protein, is associated with the myocarditic phenotype of a reovirus variant.

Reoviruses contain a genome composed of 10 double-stranded RNA gene segments. A reovirus reassortant, 8B, derived from type 1 Lang (T1L) and type 3 Dearing (T3D), displayed a phenotype unlike that of either of its parents in that it efficiently induced numerous macroscopic external cardiac lesions in neonatal mice (B. Sherry, F. J. Schoen, E. Wenske, and B. N. Fields, J. Virol. 63:4840-4849, 1989). A panel of T1L/T3D reassortants and a panel of reassortants derived from 8B were used to determine whether novel T1L/T3D gene associations in 8B were responsible for its myocarditic phenotype. The results eliminated the possibility that any T1L/T3D gene combination found in 8B, from 2 genes to all 10 genes, was the explanation for its induction of cardiac lesions. This suggested that a mutation(s) in an 8B gene(s) might be responsible for induction of the myocarditis. Statistical analysis of experiments with 31 reassortants derived from 8B revealed a highly significant association (P = 0.002) of the 8B M1 gene with induction of cardiac lesions. The reovirus M1 gene encodes a viral core protein of unknown function, although evidence suggests a potential role in core structure and/or viral RNA synthesis. This represents the first report of the association of a viral gene with induction of myocarditis.

Animals↗
PubMed2006

A vaccinia virus lacking A10L: viral core proteins accumulate on structures derived from the endoplasmic reticulum.

The assembly of the intracellular mature virus (IMV) of vaccinia virus (VV), the prototype member of the poxviridae, is poorly understood and controversial. We have previously proposed that the IMV is composed of a continuous double-membraned cisterna derived from the smooth ER, whereby the genome-containing core is enwrapped by a part of this cisterna. In the present study we characterize a mutant virus in which the synthesis of the major core protein A10L can be conditionally expressed. Without A10L, IMVs are not made; immature viruses (IVs) and regularly stacked membrane structures that contain viral DNA, accumulate instead. By immunolabelling of thawed cryo-sections these stacks contain most of the viral core proteins and low levels of viral membrane proteins. Importantly, the stacked membranes could be labelled with antibodies to an ER marker protein, implying that they are derived from this cellular compartment. By electron tomography (ET) on semi-thin cryo-sections we show that the membranes of the stacks are continuous with the membranes of the IVs. Direct continuities with ER cisternae, to which the stacks are tightly apposed, were, however, not unequivocally seen. Finally, ET revealed how the IV membranes separated to become two-membrane profiles. Taken together, this study shows that VV core proteins and the viral DNA can coassemble onto ER-derived membranes that are continuous with the membranes of the IVs.

Animals↗
PubMed1995

Large scale purification of rubella virus and the isolation of native viral core protein.

A number of structural analyses of viruses are dependent on the availability of purified virus and of pure viral components in milligram amounts. In order to allow such analyses of the Rubella togavirus we have identified a virus-cell-system which produces large amounts of Rubella virus in tissue culture and we have developed a rapid and efficient procedure of Rubella virus purification which involves adsorption and elution of virus to fixed erythrocytes. Furthermore, we describe a procedure which allows the extraction of native core protein from viral cores and its chromatographic purification.

Animals↗
PubMed1991

A possible role of the viral core protein in the regulation of assembly and disassembly of alphavirus cores.

In many viral systems the genome is part of a stable nucleoprotein complex which has to be dissociated early in infection, in order to make the genome accessible for transcription or translation. It is not directly evident how a nucleoprotein generated as a stable complex during virus assembly can be dissociated during the initial steps of viral multiplication. During analyses of the molecular biology of alphavirus replication it has been shown that the core protein of these viruses is a multifunctional protein. In this manuscript I propose that one of the functions of the core protein, namely its ability to bind to the large subunit of the cellular ribosomes both during synthesis and as a mature protein, allows to regulate the assembly and disassembly of alphavirus cores. I propose that in the early stages of virus infection the core protein is transferred from the core of the incoming virus to the cellular ribosomes, whereas in the late stages of infection when a significant part of the ribosomes contain bound, newly synthesized core protein an efficient transfer of newly synthesized core protein into cores can occur.

Alphavirus↗
PubMed1993

Simian immunodeficiency virus (SIVmac251) membrane lipid mixing with human CD4+ and CD4- cell lines in vitro does not necessarily result in internalization of the viral core proteins and productive infection.

The cell binding site of simian immunodeficiency virus (SIV) is believed to be the CD4 molecule. Several CD4+ cell lines are, however, resistant to infection by SIVmac251 in vitro and additional cell membrane molecules have been implicated in SIVmac251 entry. We investigated the binding, envelope fusion and entry of the viral core proteins (p27) of SIVmac251 into two human CD4+ cell lines (H9 and Sup-T1) which are infectible, and one CD4+ (A3.01) and two CD4- cell lines (K562 and Raji) that are resistant to infection. The fusion of the viral and cellular membranes was monitored by a fluorescence assay for lipid mixing. Cell entry of the viral core was evaluated following virus-cell incubation and cell surface trypsinization. We found that SIVmac251 can bind to and fuse (membrane lipid mixing) in a temperature-dependent but pH-independent fashion with CD4+ and CD4- human-derived cell lines. In contrast, lipid mixing with CD4 expressing EL-4 mouse T cells or Mv-1-lu mink lung fibroblasts was absent or limited, suggesting that certain components of human cell membranes in addition to CD4 are involved in SIVmac envelope-cell fusion. Lipid mixing with the human cells was inhibited partially by soluble CD4. Anti-CD4 antibodies inhibited the lipid inter-mixing with H9, but not with Raji cells, whereas neutralizing anti-SIVmac sera inhibited fusion with both CD4+ and CD4- cells. Out of the five human cell lines tested, efficient entry of p27 and productive infection took place only with H9 and Sup-T1 cells. In these two cases, the amounts of p27 internalized during virus-cell fusion correlated with the extent of infection.

Animals↗
PubMed2005

Suppression of hepatitis B virus replication by SRPK1 and SRPK2 via a pathway independent of the phosphorylation of the viral core protein.

The SR-domain protein kinase (SRPK) 1 and 2 are two important kinases involved in cellular RNA splicing. Recently, it was suggested that these two kinases, which could bind to the hepatitis B virus (HBV) core protein, might be the major cellular kinases that phosphorylate the core protein to regulate HBV replication. In this report, we tested the role of SRPK1 and SRPK2 in HBV replication and found that both of them could suppress HBV replication by reducing the packaging efficiency of the pgRNA without affecting the formation of the viral core particles. This suppressive effect of SRPK1 and SRPK2 on HBV replication cannot be explained by their phosphorylation activities on the HBV core protein as the over-expression of these two kinases had no detectable effects on HBV core protein phosphorylation in vivo and their mutants that lacked the kinase activity could still suppress HBV DNA replication. Thus, these findings demonstrate a negative role of SRPK1 and SRPK2 in the regulation of HBV replication through a mechanism not involving the phosphorylation of the core protein.

Cell Line↗
PubMed1986

Precore sequence of hepatitis B virus inducing e antigen and membrane association of the viral core protein.

Hepatitis B virus (HBV) DNA contains a precore (pre-c) sequence of 29 codons with unknown function upstream of its gene for the major core protein. Its significance was studied by expression of core proteins with and without pre-c in Escherichia coli. Core protein without pre-c, P22c, assembled spontaneously to core particles and formed core antigen. It had the same size and antigenicity as core particles from infected liver. Core protein with pre-c, P25e, instead formed membrane-associated e antigen (HBeAg). The data suggest that pre-c functions as a signal peptide for the attachment of core protein P25e to cellular membranes. This hypothesis can explain the not yet understood relation between viremia and HbeAg and the protective role of anti-HBe antibody.

Amino Acid Sequence↗
PubMed2005

Down-regulation of the internal ribosome entry site (IRES)-mediated translation of the hepatitis C virus: critical role of binding of the stem-loop IIId domain of IRES and the viral core protein.

In a previous study, we observed that hepatitis C virus (HCV) core protein specifically inhibits translation initiated by an HCV internal ribosome entry site (IRES). To investigate the mechanism by which down-regulation of HCV translation occurs, a series of mutations were introduced into the IRES element, as well as the core protein, and their effect on IRES activity examined in this study. We found that expression of the core protein inhibits HCV translation possibly by binding to a stem-loop IIId domain, particularly a GGG triplet within the hairpin loop structure of the domain, within the IRES. Basic-residue clusters located at the N-terminus of the core protein have an inhibitory effect on HCV translation, and at least one of three known clusters is required for inhibition. We propose a model in which competitive binding of the core protein for the IRES and 40S ribosomal subunit regulates HCV translation.

5' Untranslated Regions↗
PubMed1998

Reovirus induction of and sensitivity to beta interferon in cardiac myocyte cultures correlate with induction of myocarditis and are determined by viral core proteins.

Reovirus-induced acute myocarditis in mice serves as a model to investigate non-immune-mediated mechanisms of viral myocarditis. We have used primary cardiac myocyte cultures infected with a large panel of myocarditic and nonmyocarditic reassortant reoviruses to identify determinants of viral myocarditic potential. Here, we report that while both myocarditic and nonmyocarditic reoviruses kill cardiac myocytes, viral myocarditic potential correlates with viral spread through cardiac myocyte cultures and with cumulative cell death. To address the role of secreted interferon (IFN), we added anti-IFN-alpha/beta antibody to infected cardiac myocyte cultures. Antibody benefited nonmyocarditic more than myocarditic virus spread (P < 0.001), and this benefit was associated with the reovirus M1 and L2 genes. There was no benefit for a differentiated skeletal muscle cell line culture (C2C12 cells), suggesting cell type specificity. IFN-beta induction in reovirus-infected cardiac myocyte cultures correlated with viral myocarditic potential (P = 0.006) and was associated with the reovirus M1, S2, and L2 genes. Sensitivity to the antiviral effects of IFN-alpha/beta added to cardiac myocyte cultures also correlated with viral myocarditic potential (P = 0.004) and was associated with the same reovirus genes. Several reoviruses induced IFN-beta levels discordant with their myocarditic phenotypes, and for those tested, sensitivity to IFN-alpha/beta compensated for the anomalous induction levels. Thus, the combination of induction of and sensitivity to IFN-alpha/beta is a determinant of reovirus myocarditic potential. Finally, a nonmyocarditic reovirus induced cardiac lesions in mice depleted of IFN-alpha/beta, demonstrating that IFN-alpha/beta is a determinant of reovirus-induced myocarditis. This provides the first identification of reovirus genes associated with IFN induction and sensitivity and provides the first evidence that IFN-beta can be a determinant of viral myocarditis and reovirus disease.

Animals↗
PubMed1994

Trans-suppression of gene expression by hepatitis C viral core protein.

We have demonstrated that the truncated hepatitis C (HCV) core protein with its C-terminal hydrophobic domains deleted is translocated to the nucleus of transfected cells (22). In this study, intact and truncated core proteins of HCV were transiently expressed in a human hepatoblastoma cell line, HepG2, and their effects on the expression of the chloramphenycol acethyl transferase (CAT) gene driven by viral and cellular promoters were examined. The intact core protein of 22 kDa which is localized in the cytoplasm of the transfected cells suppressed the expression in all of the promoters tested. They were promoters of the SV40 early region, the c-fos oncogene, the retinoblastoma susceptibility gene, the beta-interferon gene and the beta-actin gene. In contrast, the truncated HCV core protein located in the nucleus did not show such a suppressive activity. The HCV core protein appears to function not only as a viral structural protein but as a regulator of gene expression and it might act as a suppressive factor for the cellular gene expression.

Antibodies, Viral↗