Phosphorylation of the mouse hepatitis virus nucleocapsid protein

Analysis of the radiolabeled tryptic peptides derived from the nucleocapsid proteins of two serotypes of mouse hepatitis virus showed each to have a small number of unique peptides; however, two biologically distinct variants of the JHM strain appeared identical. Analysis of [32P]-labeled nucleocapsid-derived peptides showed that phosphorylation occurs at only a few sites and that all three viruses differed in the sites of phosphorylation. No differences in the sites of phosphorylation were found between the nucleocapsid proteins derived from purified virions and the membranes or the cytosol of infected cells, suggesting that post-translational phosphorylation plays no role in the regulation of viral assembly. These data show unequivocal evidence that the nucleocapsid proteins of mouse hepatitis virus strains differ in the sites of phosphorylation.     

Mechanisms for inhibition of hepatitis B virus gene expression and replication by hepatitis C virus core protein

We have demonstrated previously that the core protein of hepatitis C virus (HCV) exhibits suppression activity on gene expression and replication of hepatitis B virus (HBV). Here we further elucidated the suppression mechanism of HCV core protein. We demonstrated that HCV core protein retained the inhibitory effect on HBV gene expression and replication when expressed as part of the full length of HCV polyprotein. Based on the substitution mutational analysis, our results suggested that mutation introduced into the bipartite nuclear localization signal of the HCV core protein resulted in the cytoplasmic localization of core protein but did not affect its suppression ability on HBV gene expression. Mutational studies also indicated that almost all dibasic residue mutations within the N-terminal 101-amino acid segment of the HCV core protein (except Arg(39)-Arg(40)) impaired the suppression activity on HBV replication but not HBV gene expression. The integrity of Arg residues at positions 101, 113, 114, and 115 was found to be essential for both suppressive effects, whereas the Arg residue at position 104 was important only in the suppression of HBV gene expression. Moreover, our results indicated that the suppression on HBV gene expression was mediated through the direct interaction of HCV core protein with the trans-activator HBx protein, whereas the suppression of HBV replication involved the complex formation between HBV polymerase (pol) and the HCV core protein, resulting in the structural incompetence for the HBV pol to bind the package signal and consequently abolished the formation of the HBV virion. Altogether, this study suggests that these two suppression effects on HBV elicited by the HCV core protein likely depend on different structural context but not on nuclear localization of the core protein, and the two effects can be decoupled as revealed by its differential targets (HBx or HBV pol) on these two processes of the HBV life cycle.     

Expression of a recombinant DNA gene coding for the vesicular stomatitis virus nucleocapsid protein.

A cDNA clone containing the entire vesicular stomatitis virus nucleocapsid gene was assembled by fusing portions of two partial clones. When the cDNA clone was inserted into a new general-purpose eucaryotic expression vector and introduced into appropriate host cells, abundant N-protein synthesis ensued. The expressed protein was indistinguishable from authentic N protein produced during vesicular stomatitis virus infections. The recombinant N protein was recognized by a polyclonal antibody and two different monoclonal antibodies and could not be resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis from authentic N. Our results suggest that the recombinant N protein produced in transfected cells rapidly aggregates into high-molecular-weight complexes in the absence of vesicular stomatitis virus genomic RNA.     

DNA-based immunization with chimeric vectors for the induction of immune responses against the hepatitis C virus nucleocapsid.

Vectors expressing the first 58 amino acids of the hepatitis C virus (HCV) nucleocapsid alone or as a fusion protein with the middle (pre-S2 and S) or major (S) surface antigens of hepatitis B virus (HBV) were constructed. Intramuscular immunization of BALB/c mice with the chimeric constructs in the form of naked DNA elicited humoral responses to antigens from both viruses within 2 to 6 weeks postinjection. No anti-HCV responses were obtained in mice immunized with the vector expressing the HCV sequence in the nonfusion context. Sera from chimera-injected mice specifically recognized both HCV capsid and HBV surface antigens in enzyme-linked immunosorbent assay and immunoblot testing. Anti-HCV serum titers formed plateaus of approximately 1:3,000; these remained stable until the end of the study (18 weeks postinfection). Anti-HBV immune responses were found to be lower in the chimera-injected animals (< 200 mIU/ml) than in those immunized with the native HBV vector (> 2,000 mIU/ml). This is the first report of the use of DNA-based immunization for the generation of immune responses to an HCV protein. In addition, these findings show that it is possible to elicit responses to viral epitopes from two distinct viruses via DNA immunization with chimeric vectors.     

Definition of a minimal optimal cytotoxic T-cell epitope within the hepatitis B virus nucleocapsid protein.

Residues 11 to 27 of the hepatitis B virus nucleocapsid antigen contain a cytotoxic T-cell epitope that is recognized by cytotoxic T cells from virtually all HLA-A2-positive patients with acute hepatitis B virus infection. Using panels of truncated and overlapping peptides, we now show that the optimal amino acid sequence recognized by cytotoxic T cells is a 10-mer (residues 18 to 27) containing the predicted peptide-binding motif for HLA-A2 and that this peptide can stimulate cytotoxic T cells able to recognize endogenously synthesized hepatitis B core antigen. Since patients with chronic hepatitis B virus infection fail to mount an efficient cytotoxic T-cell response to it, this epitope might serve as the starting point for the design of synthetic peptide-based immunotherapeutic strategies to terminate persistent viral infection.     

Reduction of synonymous substitutions in the core protein gene of hepatitis C virus

Molecular evolutionary analyses were carried out to elucidate the phylogenetic relationships, the evolutionary rate, and the divergence times of hepatitis C viruses. Using the nucleotide sequences of the viruses isolated from various locations in the world, we constructed phylogenetic trees. The trees showed that strains isolated from a single location were not necessarily clustered as a group. This suggests that the viruses may be transferred with blood on a worldwide scale. We estimated the evolutionary rates at synonymous and nonsynonymous sites for all genes in the viral genome. We then found that the rate (1.35 × 10^–3 per site per year) at synonymous sites for the C gene was much smaller than those for the other genes (e.g., 6.29 × 10^–3 per site per year for the E gene). This indicates that a special type of functional constraint on synonymous substitutions may exist in the C gene. Because we found an open reading frame (ORF) with the C gene region, the possibility exists that synonymous substitutions for the C gene are constrained by the overlapping ORF whose reading frame is different from that of the C gene. Applying the evolutionary rates to the trees, we also suggest that major groups of hepatitis C viruses diverged from their common ancestor several hundred years ago. Correspondence to: T. Gojobori     

Conformational maturation of measles virus nucleocapsid protein.

We have obtained a polyclonal antiserum, N-BE, against the denatured, amino-terminal half of the measles virus (MV) nucleocapsid (N) protein and a monoclonal antibody (MAb), N46, which recognizes a conformation-dependent epitope in the same region. Amino acid residues 23 to 239 were required and sufficient for the formation of the conformational epitope. Using these antibodies, we show that the N protein of MV is synthesized as a relatively unfolded protein which first appears in the free-protein pool. This nascent N protein undergoes a conformational change into a more folded mature form. This change does not require the participation of other viral proteins or genomic RNA. The mature N protein does not accumulate in the free-protein pool but is quickly and selectively incorporated into the viral nucleocapsids. The mature N protein is a target for interaction with the phosphoprotein (P protein) of MV. This interaction interferes with the recognition of the N protein by the N46 MAb. This suggests that the association with the P protein may mask the binding site for the N46 MAb or that it induces a conformational change in the N protein.     

The internal open reading frame within the nucleocapsid gene of mouse hepatitis virus encodes a structural protein that is not essential for viral replication.

The coronavirus mouse hepatitis virus (MHV) contains a large open reading frame embedded entirely within the 5' half of its nucleocapsid (N) gene. This internal gene (designated I) is in the +1 reading frame with respect to the N gene, and it encodes a mostly hydrophobic 23-kDa polypeptide. We have found that this protein is expressed in MHV-infected cells and that it is a previously unrecognized structural protein of the virion. To analyze the potential biological importance of the I gene, we disrupted its expression by site-directed mutagenesis using targeted RNA recombination. The start codon for I was replaced by a threonine codon, and a stop codon was introduced at a short interval downstream. Both alterations created silent changes in the N reading frame. In vitro translation studies showed that these mutations completely abolished synthesis of I protein, and immunological analysis of infected cell lysates confirmed this conclusion. The MHV I mutant was viable and grew to high titer. However, the I mutant had a reduced plaque size in comparison with its isogenic wild-type counterpart, suggesting that expression of I confers some minor growth advantage to the virus. The engineered mutations were stable during the course of experimental infection in mice, and the I mutant showed no significant differences from wild type in its ability to replicate in the brains or livers of infected animals. These results demonstrate that I protein is not essential for the replication of MHV either in tissue culture or in its natural host.     

Hybrid hepatitis B virus nucleocapsid bearing an immunodominant region from hepatitis B virus surface antigen.

A hepatitis B core antigen (HBcAg) gene bearing the 39-amino-acid-long domain A of hepatitis B surface antigen (HBsAg) within the HBcAg immunodominant loop has been constructed and expressed in Escherichia coli. Chimeric capsids demonstrated HBs but not HBc antigenicity and elicited in mice B-cell and T-cell responses against native HBcAg and HBsAg.     

A structural protein of hepatitis C virus expressed in E. coli facilitates accurate detection of hepatitis C virus.

A putative core protein derived from hepatitis C virus was expressed in E. coli. More than 5% of the total protein expressed in the bacteria after induction by isopropylthio-beta-D-galactoside was shown to be the expected protein. Western blotting with this E. coli lysate proved to be more efficient than ELISA with a non-structural viral protein, C100, to detect infection of hepatitis C virus in the sera of patients with non-A, non-B chronic hepatitis, hepatocellular carcinoma as well as in sera from healthy persons.     

The conserved N-terminal region of Sendai virus nucleocapsid protein NP is required for nucleocapsid assembly.

Sendai virus nucleocapsid protein NP synthesized in the absence of other viral components assembled into nucleocapsid-like particles. They were identical in density and morphology to authentic nucleocapsids but were smaller in size. The reduction in size was probably due to the fact that they contained RNA only 0.5 to 2 kb in length. Nucleocapsid assembly requires NP-NP and NP-RNA interactions. To identify domains on NP protein involved in nucleocapsid formation, 29 NP protein mutants were tested for the ability to assemble. Any deletion between amino acid residues 1 and 399 abolished formation of nucleocapsid-like particles, but mutants within this region exhibited two different phenotypes. Deletions between positions 83 and 384 completely abolished all interactions. Deletions between residues 1 and 82 and between residues 385 and 399, at the N- and C-terminal ends of the region from 1 to 399, resulted in unstructured aggregates of NP protein, indicating only a partial loss of function. Deletions within the C-terminal 124 amino acids were the only ones that did not affect assembly. The results suggest that NP protein can be divided into at least two separate domains which function independently of each other. Domain I (residues 1 to 399) seems to contain all of the structural information necessary for assembly, while domain II (residues 400 to 524) is not involved in nucleocapsid formation.     

DNA-binding activity of hepatitis B e antigen polypeptide lacking the protaminelike sequence of nucleocapsid protein of human hepatitis B virus.

The characteristics of binding of hepatitis B core antigen (HBcAg) and hepatitis B e antigen (HBeAg) polypeptides to hepatitis B virus (HBV) DNA were analyzed. HBcAg polypeptide from recombinant HBV core particles and HBeAg polypeptide from partially purified serum HBeAg were prepared and verified to have molecular weights of 21,500 (P21.5) and of 17,000 (P17) and 18,000 (P18), respectively, by immunoblot analysis. By reaction of these proteins on a nitrocellulose membrane with cloned 32P-HBV DNA, it was revealed that the HBeAg polypeptide, which lacks the C-terminal 34 amino acids of P21.5, as well as the HBcAg polypeptide, bound to the DNA. The secondary structures of nucleocapsid proteins of HBV, woodchuck hepatitis virus, and ground squirrel hepatitis virus were predicted by the Garnier algorithm. Amino acid sequences which, in addition to those of the C-terminal regions, may contribute to binding were proposed to be the 21-amino-acid residues located at amino acids 100 to 120 of the nucleocapsid proteins of these hepadnaviruses.     

Karyophilic properties of Semliki Forest virus nucleocapsid protein.

Semliki Forest virus capsid (C) protein molecules (Mr, 33,000) can be introduced efficiently into the cytoplasm of various target cells by electroporation, liposome, and erythrocyte ghost-mediated delivery (M. Elgizoli, Y. Dai, C. Kempf, H. Koblet, and M.R. Michel, J. Virol. 63:2921-2928, 1989). Here, we show that the transferred C protein molecules partition rapidly from the cytosolic compartment into the nucleus. Transport of the C protein molecules into the nucleus was reversibly arrested by metabolic inhibitors, indicating that the transfer process is energy dependent. Fractionation of isolated nuclei revealed that the delivered C protein preferentially associates with the nucleoli. This finding was confirmed by morphological studies, showing that in an in vitro system containing ATP isolated nuclei rapidly accumulated rhodamine-labeled C protein in their nucleoli. Furthermore, in this assay system, the lectin wheat germ agglutinin prevented transfer of C protein through nuclear pores. These results are in agreement with our observation that nucleoli contain measurable amounts of newly synthesized C protein as early as 5 h after infection of cells with SFV. Thereafter, nucleolar-associated C protein increased progressively during the course of infection.     

Measles virus nucleocapsid protein protects rats from encephalitis.

Lewis rats immunized with recombinant vaccinia virus expressing the nucleocapsid (N) protein of measles virus were protected from encephalitis when subsequently challenged by intracerebral infection with neurotropic measles virus. Immunized rats revealed polyvalent antibodies to the N protein of measles virus in the absence of any neutralizing antibodies as well as an N protein-specific proliferative lymphocyte response. Depletion of CD8+ T lymphocytes did not abrogate the protective potential of the N protein-specific cell-mediated immune response in rats, while protection could be adoptively transferred with N protein-specific CD4+ T lymphocytes. These results indicate that a CD4+ cell-mediated immune response specific for the N protein of measles virus is sufficient to control measles virus infections of the central nervous system.     

Synthesis and antigenic activity of peptides of the nucleocapsid protein of the hepatitis delta virus]

Hepatitis delta virus (HDV), a recently discovered infectious agent, participates in severe, often lethal forms of acute and chronic hepatitis and liver cirrhosis. Based on theoretical analysis of secondary structure, hydrophilicity and acrophilicity data, several regions of HDV antigen, presumably containing B-epitopes, have been revealed and the corresponding peptides have been synthesized by the solid phase method. All the peptides obtained reacted with the respective antipeptide rabbit sera. The peptides and their conjugates with BSA or KLH were used for ELISA with individual and pooled anti-HD-positive sera from patients with chronic delta hepatitis. The high antigenicity of the peptide 65-80 shows that one of the antigenically active regions of HDAg is situated between these amino acid residues and that the peptide may be used for detection of anti-HD antibodies in patients blood sera.     

Interaction between hepatitis C virus core protein and E1 envelope protein.

Hepatitis C virus has three structural genes named C, E1, and E2. The C gene encodes the core (capsid) protein and the E1 and E2 genes encode the envelope proteins. In an immunoprecipitation experiment, the E1 protein was found to be precipitated by an anti-core antibody in the presence but not in the absence of the core protein, indicating that the E1 protein can interact with the core protein. This interaction is independent of whether the E1 and the C genes are linked in cis or separated in different DNA constructs for expression. The interaction between the core and the E1 proteins is confirmed by the observation that a hybrid protein derived from the core protein and the tissue plasminogen activator is localized in the nucleus in the absence of the E1 protein and in the perinuclear region in the presence of the E1 protein. Deletion-mapping studies indicate that the carboxy-terminal sequences of both the core and the E1 proteins are important for their interaction. Since little E1 sequence is exposed on the cytosolic side of the membrane of the endoplasmic reticulum, the interaction between the core and the E1 proteins most likely takes place in the endoplasmic reticulum membrane. The E2 protein could not be coprecipitated with the core protein by the anti-core antibody in a similar assay and likely does not interact with the core protein. The implications of these findings on the morphogenesis of the hepatitis C virus virion are discussed.