Molecular interactions of Epstein-Barr virus capsid proteins

The capsids of herpesviruses, which comprise major and minor capsid proteins, have a common icosahedral structure with 162 capsomers. An electron microscopic study shows that Epstein-Barr virus (EBV) capsids in the nucleus are immunolabeled by anti-BDLF1 and anti-BORF1 antibodies, indicating that BDLF1 and BORF1 are the minor capsid proteins of EBV. Cross-linking and electrophoresis studies of purified BDLF1 and BORF1 revealed that these two proteins form a triplex that is similar to that formed by the minor capsid proteins, VP19C and VP23, of herpes simplex virus type 1 (HSV-1). Although the interaction between VP23, a homolog of BDLF1, and the major capsid protein VP5 could not be verified biochemically in earlier studies, the interaction between BDLF1 and the EBV major capsid protein, viral capsid antigen (VCA), can be confirmed by glutathione S-transferase (GST) pulldown assay and coimmunoprecipitation. Additionally, in HSV-1, VP5 interacts with only the middle region of VP19C; in EBV, VCA interacts with both the N-terminal and middle regions of BORF1, a homolog of VP19C, revealing that the proteins in the EBV triplex interact with the major capsid protein differently from those in HSV-1. A GST pulldown study also identifies the oligomerization domains in VCA and the dimerization domain in BDLF1. The results presented herein reveal how the EBV capsid proteins interact and thereby improve our understanding of the capsid structure of the virus.     

Effects of S-adenosylhomocysteine and analogs on Epstein-Barr virus-induced transformation, expression of the Epstein-Barr virus capsid antigen, and methylation of Epstein-Barr virus DNA.

S-Adenosylhomocysteine was found to have no effect on Epstein-Barr virus-induced transformation of B-lymphocytes and to stimulate viral capsid antigen expression only slightly in the FF41-1 cell line. In contrast, the S-adenosylhomocysteine analogs sinefungin and S-isobutyladenosine inhibited Epstein-Barr virus transformation and induced a significant increase in the numbers of cells expressing the viral capsid antigen. An inverse relationship between levels of viral DNA methylation and gene expression was demonstrated.     

Adeno-associated virus proteins: origin of the capsid components.

The three primary capsid proteins (A, B, and C) of adeno-associated viruses have been shown previously to contain overlapping amino acid sequences (R. McPherson and J. Rose, J. Virol. 46:523-529, 1983). In the present study we demonstrate definitively that these proteins are encoded in the right half of the adeno-associated virus 2 genome, and one or both of the smallest adeno-associated RNA species (2.3- or 2.6-kilobase RNA) account for their synthesis. Protein A (90 kilodaltons) apparently initiates from a site within the intervening sequence, which is intact in the larger (unspliced) 2.6-kilobase mRNA, and may read through one or more termination codons, including a strong stop signal (UAA) that lies 31 bases downstream from the end of the intervening sequence. Proteins B (72 kilodaltons) and C (60 kilodaltons) are not derived from protein A but apparently originate from independent, in-frame initiations that lie downstream from the splice junction. It thus seems likely that production of the three adeno-associated virus capsid proteins involves at least two mRNA species. The B and C proteins presumably arise from the spliced 2.3-kilobase RNA, whereas protein A should be generated by the 2.6-kilobase RNA or a hitherto unidentified spliced RNA species.     

A bicistronic Epstein-Barr virus mRNA encodes two nuclear proteins in latently infected, growth-transformed lymphocytes.

EBNA2 is a nuclear protein expressed in all cells latently infected with and growth transformed by Epstein-Barr virus (EBV) infection (K. Hennessy and E. Kieff, Science 227:1230-1240, 1985). The nucleotide sequence of the EBNA2 mRNA (J. Sample, M. Hummel, D. Braun, M. Birkenbach, and E. Kieff, Proc. Natl. Acad. Sci. USA 83:5096-5100, 1986) revealed that it begins with a 924-base open reading frame that has an unusual potential translational initiation site (CAAATGG). This open reading frame is followed by 138 nucleotides with only one highly unlikely translational initiation site (TACATGC), which would translate a pentapeptide before the next stop codon. The last part of the mRNA is the open reading frame which encodes EBNA2. In this paper, we demonstrate that the 924-base open reading frame translates a 40-kilodalton protein in vitro or in murine cells transfected with the EBNA2 cDNA under control of the murine leukemia virus long terminal repeat. A protein of identical size was detected in EBV-transformed, latently infected human lymphocyte nuclei by using antibody specific for the leader open reading frame expressed in bacteria. Therefore, this is a rare example of a mRNA which translates two proteins from nonoverlapping open reading frames. Since the protein encoded by the leader of the EBNA mRNA is expressed in all nuclei of a latently infected cell line, it was designated EBNA-LP. EBNA-LP localizes to small intranuclear particles and differs in this respect from EBNA1, EBNA2, or EBNA3. EBNA-LP is not expressed in an EBV-transformed marmoset lymphocyte cell (B95-8) or in one EBV-infected Burkitt tumor cell line (Raji) but is expressed in three other Burkitt tumor cell lines (Namalwa, P3HR-1, and Daudi).     

Identification and characterization of three immunodominant structural proteins of fowlpox virus

Genes encoding fowlpox virus (FWPV) structural proteins have been identified mainly by sequence homology with those from vaccinia virus (VACV), but little is known about the encoded proteins. Production of monoclonal antibodies (MAbs) against Poxine and HP1-440 (Munich) clone FP9 allowed the identification of three immunodominant FWPV proteins: the 39-kDa core protein (encoded by FPV168, homologous to VACV A4L), a 30- and 35-kDa protein doublet, and an abundant 63-kDa protein. The 30- and 35-kDa proteins are nonglycosylated, antigenically related proteins present in the intracellular mature virus membrane and localizing closely with the viral factories. N-terminal sequencing identified the 35-kDa protein as encoded by FPV140 (the FWPV homolog of VACV H3L). The 63-kDa protein forms covalently linked dimers and oligomers. It remained mainly insoluble upon detergent treatment of purified virus but did not localize closely with the viral factory. N-terminal sequencing was unsuccessful, suggesting N-terminal blocking. CNBr digestion generated a peptide encoded by FPV191, predicted to encode one of two FWPV A-type inclusion (ATI) proteins. The characteristics of the 63-kDa protein were inconsistent with published observations on cowpox or VACV ATI proteins (it appears to be essential). The 63-kDa protein, however, shares characteristics with both VACV p4c virus occlusion and 14-kDa fusion proteins. Gene assignment at the poxvirus ATI locus (between VACV A24R and A28L) is complicated by sequence redundancies and variations, often due to deletions and multiple frameshift mutations. The identity of FPV191 in relation to genes at this locus is discussed.     

Identification of proteins encoded by Epstein-Barr virus trans-activator genes.

Specific antisera were generated to characterize Epstein-Barr virus proteins reported to have trans-activating properties. Open reading frame BRLF1 was found to be expressed in two modifications in vivo, with molecular sizes ranging from 94 to 98 kilodaltons (kDa) depending on the cell line, whereas only one protein (Raji cells, 96 kDa) was detected by in vitro translation. Open reading frame BZLF1 encoded polypeptides of 38 and 35 kDa and additional smaller forms. A BZLF1-encoded 30-kDa protein could be detected under conditions in which expression was restricted to immediate early genes. Nuclear localization could be detected under conditions in which expression was restricted to immediate early genes. Nuclear localization could be shown for the proteins derived from reading frames BZLF1 and BMLF1. BMLF1 expression gave a heterogeneous protein pattern, with molecular sizes between 45 and 70 kDa, including a predominant 60-kDa protein detected in different B-cell lines.     

Cell-free synthesis of polyoma virus capsid proteins VP1 and VP2.

Polyadenylated RNA isolated from the cytoplasm of mouse 3T6 cells 28 h after infection with polyoma virus has been isolated and translated in vitro. Polyoma capsid proteins VP1 and VP2 have been identified in the cell-free product by polyacrylamide gel electrophoresis, specific immunoprecipitation, and tryptic peptide fingerprinting. Polyoma mRNA species have been isolated by preparative hybridization to purified viral DNA immobilized on cellulose nitrate filters and shown to code for both VP1 and VP2. These experiments establish conditions for the isolation of late polyoma mRNA and the cell-free synthesis of polyoma capsid proteins and indicate that the active mRNA species are at least partially virus coded.     

Hybrid Sindbis/Epstein-Barr virus episomal expression vector for inducible production of proteins

Alphavirus vectors are attractive as recombinant protein expression systems due to the high level of gene expression achieved. The combination of two mutations in the viral replicase, which render the replicase noncytopathic and temperature-sensitive, allowed the generation of a DNA-based vector (CytTs) that shows temperature inducible expression. This vector is of significant value for the production of toxic protein. However, like for other stable expression systems, tedious screening of individual cell clones are required in order to get a high producer cell clone. To circumvent this, we generated an episomally replicating vector by introducing an Epstein-Barr virus mini-replicon unit into CytTs. This novel vector allowed rapid generation of cell populations that showed tight regulation of expression and comparable expression levels to the ones achieved with high producer cell clones with CytTs. Moreover, protein production with selected cell populations could easily be scaled-up to a fermentation process.     

Fine mapping of an immunodominant domain in the transmembrane glycoprotein of human immunodeficiency virus.

Sera from virtually all individuals infected with human immunodeficiency virus contain antibodies against the viral envelope glycoproteins. By using a series of synthetic peptide antigens, we identified an immunodominant domain at amino acid position 598-609 of gp41. The minimal essential epitope is a 7-amino-acid sequence (amino acids 603-609) containing two cysteine residues. Both cysteine residues are required for the antigenic conformation of the sequence, possibly due to creation of a cyclic structure via disulfide bond formation.     

Chaperone functions common to nonhomologous Epstein-Barr virus gL and Varicella-Zoster virus gL proteins.

Herpesviruses encode the complex-forming, essential glycoproteins gH and gL. Maturation and transport of gH are dependent on coexpression of its chaperone, gL. The gL proteins of alpha herpesviruses and gamma herpesviruses do not have a significant percentage of amino acid sequence homology. Yet, as we report herein, the diverse gL glycoproteins of Epstein-Barr virus (EBV) and varicella-zoster virus (VZV) were functionally interchangeable, although membrane expression and maturation of gH were separate functions for these viruses. In VZV both functions were performed by a single protein. EBV required two separate glycoproteins, one of which can be replaced by its homologous protein from VZV, a distant relative of EBV. Collectively, these results suggested that VZV gL is a simpler form of the gL chaperone protein than EBV gL.     

Modulation of signaling pathways by RNA virus capsid proteins

Capsid proteins are structural components of virus particles. They are nucleic acid-binding proteins whose main recognized function is to package viral genomes into protective structures called nucleocapsids. Research over the last 10 years indicates that in addition to their role as genome guardians, viral capsid proteins modulate host cell signaling networks. Disruption or alteration of intracellular signaling pathways by viral capsids may benefit replication of the virus by affecting innate immunity and in some cases, may underlie disease progression. In this review, we describe how the capsid proteins from medically relevant RNA viruses interact with host cell signaling pathways.     

In vitro immunization of human lymphocytes with Epstein-Barr virus capsid antigen]

Conditions for in vitro immunization of human lymphocytes from adult peripheral blood, tonsils and cord blood with Epstein-Barr Virus (EBV) capsid antigens have been studied. Pokeweed mitogen and B cell growth factor from Namalva cell line were shown to induce a significant production of specific antibodies by human lymphocytes stimulated with EBV. This effect made it possible to generate primary immune response in vitro using lymphocytes from EBV seronegative donors.     

Simian virus 40 chromatin interaction with the capsid proteins

It has been established that both in virions and in infected cells, the cellular core histones fold the SV40 DNA into nucleosomes to form the SV40 chromosome or chromatin. We and others have begun to examine how the capsid proteins assemble the SV40 chromatin into virions and to investigate whether these proteins interact with the encapsidated chromatin. To follow the pathway of virus assembly, we have analyzed the nucleoproteins which accumulate in cells infected with the SV40 mutants temperature-sensitive in assembly: tsC, tsBC, and tsB. (The temperature-sensitivity of these mutants result from alterations in the amino acid sequence of the major capsid protein VP1). We have found that mutants belonging to the same class accumulate similar types of nucleoproteins at the nonpermissive temperature (40 degrees C) and thus, share characteristics in common. For example, the tsC mutants accumulate only the 75 S chromatin. Both tsBC and tsB mutants produce in addition to chromatin, nucleoprotein complexes which sediment broadly from 100-160 S and contain all the three capsid proteins VP1, VP2, and VP3. These nucleoproteins can be distinguished morphologically, however. Under the electron microscope, the tsBC 100-160 S nucleoproteins appear as chromatin to which a small cluster of the capsid proteins is attached; the tsB nucleoproteins appear as partially assembled virions. In addition, we find that the 220 S virions are assembled in cells coinfected with tsB and tsC mutants at 40 degrees C, in agreement with genetic analysis. Our observations favor the hypothesis that the VP1 protein contains three discrete domains. We speculate that each domain may play a specific function in SV40 assembly. To gain more insight into VP1-VP1 interactions, we have examined the nucleoproteins which result from treatment of the mature wild-type virions with increasing concentrations of the reducing agent DTT. In the presence of as low a concentration of DTT as 0.1 mM, the virion shell can be penetrated by micrococcal nuclease, which then cleaves the viral DNA. This result indicates that some of the disulfide bonds bridging the VP1 proteins are on the virion surface.     

Colocalization of adeno-associated virus Rep and capsid proteins in the nuclei of infected cells.

The mechanism of adeno-associated virus (AAV) DNA replication was characterized both genetically and biochemically. In this study, we used monoclonal and polyclonal antibodies to examine the AAV p5 (Rep78 and Rep68) and p19 (Rep52 and Rep40) proteins in infected cells. By overexpressing a truncated Rep78 protein in Escherichia coli, we obtained monoclonal antibody anti-78/68, which is specific for the p5 Rep proteins, and monoclonal antibody anti-52/40, which recognized both the p5 and p19 Rep proteins. In single-fluorochrome indirect immunofluorescence labeling experiments, the viral Rep proteins were localized in distinct intranuclear foci. Analysis of AAV proteins by double-fluorochrome indirect immunofluorescence experiments demonstrated that (i) all four AAV Rep proteins occupied the same intranuclear compartments and (ii) the Rep and capsid proteins colocalized in the nuclei of infected cells. These results suggest that replication centers similar to those established by other viruses exist for AAV. These reagents should provide a useful tool for further delineation of the mechanism of AAV replication in vitro.     

Identification of immunodominant and conformational epitopes in the capsid protein of hepatitis E virus by using monoclonal antibodies

Antibody to the capsid (PORF2) protein of hepatitis E virus (HEV) is sufficient to confer immunity, but knowledge of B-cell epitopes in the intact capsid is limited. A panel of murine monoclonal antibodies (MAbs) was generated following immunization with recombinant ORF2.1 protein, representing the C-terminal 267 amino acids (aa) of the 660-aa capsid protein. Two MAbs reacted exclusively with the conformational ORF2.1 epitope (F. Li, J. Torresi, S. A. Locarnini, H. Zhuang, W. Zhu, X. Guo, and D. A. Anderson, J. Med. Virol. 52:289-300, 1997), while the remaining five demonstrated reactivity with epitopes in the regions aa 394 to 414, 414 to 434, and 434 to 457. The antigenic structures of both the ORF2.1 protein expressed in Escherichia coli and the virus-like particles (VLPs) expressed using the baculovirus system were examined by competitive enzyme-linked immunosorbent assays (ELISAs) using five of these MAbs and HEV patient sera. Despite the wide separation of epitopes within the primary sequence, all the MAbs demonstrated some degree of cross-inhibition with each other in ORF2. 1 and/or VLP ELISAs, suggesting a complex antigenic structure. MAbs specific for the conformational ORF2.1 epitope and a linear epitope within aa 434 to 457 blocked convalescent patient antibody reactivity against VLPs by approximately 60 and 35%, respectively, while MAbs against epitopes within aa 394 to 414 and 414 to 434 were unable to block patient serum reactivity. These results suggest that sequences spanning aa 394 to 457 of the capsid protein participate in the formation of strongly immunodominant epitopes on the surface of HEV particles which may be important in immunity to HEV infection.