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PubMed2000

Interactions in vivo between the proteins of infectious bursal disease virus: capsid protein VP3 interacts with the RNA-dependent RNA polymerase, VP1.

Little is known about the intermolecular interactions between the viral proteins of infectious bursal disease virus (IBDV). By using the yeast two-hybrid system, which allows the detection of protein-protein interactions in vivo, all possible interactions were tested by fusing the viral proteins to the LexA DNA-binding domain and the B42 transactivation domain. A heterologous interaction between VP1 and VP3, and homologous interactions of pVP2, VP3, VP5 and possibly VP1, were found by co-expression of the fusion proteins in Saccharomyces cerevisiae. The presence of the VP1-VP3 complex in IBDV-infected cells was confirmed by co-immunoprecipitation studies. Kinetic analyses showed that the complex of VP1 and VP3 is formed in the cytoplasm and eventually is released into the cell-culture medium, indicating that VP1-VP3 complexes are present in mature virions. In IBDV-infected cells, VP1 was present in two forms of 90 and 95 kDa. Whereas VP3 initially interacted with both the 90 and 95 kDa proteins, later it interacted exclusively with the 95 kDa protein both in infected cells and in the culture supernatant. These results suggest that the VP1-VP3 complex is involved in replication and packaging of the IBDV genome.

Animals↗

PubMed2002

Addition of six-His-tagged peptide to the C terminus of adeno-associated virus VP3 does not affect viral tropism or production.

Production of large quantities of recombinant adeno-associated virus (AAV) is difficult and not cost-effective. To overcome this problem, we have explored the feasibility of creating a recombinant AAV encoding a 6xHis tag on the VP3 capsid protein. We generated a plasmid vector containing a six-His (6xHis)-tagged AAV VP3. A second plasmid vector was generated that contained the full-length AAV capsid capable of producing VP1 and VP2, but not VP3 due to a mutation at position 2809 that encodes the start codon for VP3. These plasmids, necessary for production of AAV, were transfected into 293 cells to generate a 6xHis-tagged VP3mutant recombinant AAV. The 6xHis-tagged VP3 did not affect the formation of AAV virus, and the physical properties of the 6xHis-modified AAV were equivalent to those of wild-type particles. The 6xHis-tagged AAV did not affect the production titer of recombinant AAV and could be used to purify the recombinant AAV using an Ni-nitrilotriacetic acid column. Addition of the 6xHis tag did not alter the viral tropism compared to wild-type AAV. These observations demonstrate the feasibility of producing high-titer AAV containing a 6xHis-tagged AAV VP3 capsid protein and to utilize the 6xHis-tagged VP3 capsid to achieve high-affinity purification of this recombinant AAV.

Base Sequence↗

PubMed2003

The oligomerization domain of VP3, the scaffolding protein of infectious bursal disease virus, plays a critical role in capsid assembly.

Infectious bursal disease virus (IBDV) capsids are formed by a single protein layer containing three polypeptides, pVP2, VP2, and VP3. Here, we show that the VP3 protein synthesized in insect cells, either after expression of the complete polyprotein or from a VP3 gene construct, is proteolytically degraded, leading to the accumulation of product lacking the 13 C-terminal residues. This finding led to identification of the VP3 oligomerization domain within a 24-amino-acid stretch near the C-terminal end of the polypeptide, partially overlapping the VP1 binding domain. Inactivation of the VP3 oligomerization domain, by either proteolysis or deletion of the polyprotein gene, abolishes viruslike particle formation. Formation of VP3-VP1 complexes in cells infected with a dual recombinant baculovirus simultaneously expressing the polyprotein and VP1 prevented VP3 proteolysis and led to efficient virus-like particle formation in insect cells.

Amino Acid Sequence↗

PubMed2005

[Sequencing and analysis of Vibrio cholerae typing phage VP3 genome].

DNA sequence and the genome of phage VP3 (a typing phage of V. cholera) were analyzed. A random library of VP3 DNA was constructed by shot-gun library method. The VP3 genome sequence was assembled with contigs sequences, the gaps between different contigs were filled with sequencing data from primer walking. ORFs were predicted; Phylogeny of DNA polymerase sequences was analyzed to determine the class of VP3; The activity of putative promoter genes were analyzed using lacZ report system. VP3 genome is a 39504bp of circular double-stranded DNA. Twenty-seven out of forty-nine putative ORFs were annotated; twenty gene products were homologous with T7-like phages, including DNAP, DNA replicative protein, capsid, tail tubular, tail fiber protein, and DNA packaged protein. The activity of the putative promoter regions was confirmed through cloning those regions to LacZ-fuse plasmid pRS1274 and analysis of the expression of beta galactosidase. The complete genomic sequence of VP3 and phylogenetic tree analysis suggests VP3 is a member of T7 phage family.

Bacteriophage T7↗

PubMed1998

The SV40 capsid protein VP3 cooperates with the cellular transcription factor Sp1 in DNA-binding and in regulating viral promoter activity.

Chromatin structure and protein-protein interactions play an important role in eukaryotic gene function. Nucleosomal rearrangement at the simian virus 40 (SV40) regulatory region occurs at the late stages of the viral life cycle preceding viral assembly. The SV40 capsid proteins are required for this nucleosomal rearrangement suggesting that they participate in turning-off the viral promoters. In aiming to elucidate the role of the capsid proteins in gene regulation, we studied the interaction between VP3, an internal capsid protein, and the cellular transcription factor Sp1, a major regulator of both the early and late viral promoters. Our results showed that VP3 repressed transcription from the viral early promoter in vitro. We found significant cooperativity between Sp1 and VP3 in specific DNA-binding to the Sp1 binding site. In addition, protein-protein interactions between VP3 and Sp1 in the absence of DNA were observed. These findings have led us to conclude that the novel host-viral Sp1-VP3 complex down regulates viral transcription and further suggest that Sp1 participates in recruiting VP3 to the SV40 minichromosome in SV40 assembly.

Base Sequence↗

PubMed1994

Mutations in the poliovirus P1 capsid precursor at arginine residues VP4-ARG34, VP3-ARG223, and VP1-ARG129 affect virus assembly and encapsidation of genomic RNA.

To begin to identify poliovirus capsid protein determinants required for assembly and RNA encapsidation, we have addressed the functional significance of three arginine residues of the poliovirus capsid in virus assembly and encapsidation of genomic RNA. These studies were conducted by using a recently described system in which recombinant vaccinia viruses are used to supply poliovirus capsid proteins in trans to a poliovirus subgenomic replicon [D. C. Ansardi, D. C. Porter, and C. D. Morrow (1993) J. Virol. 67, 3684-3690]. Two of the arginine residues, located at position 34 of VP4 (VP4-R034) and position 129 of VP1 (VP1-R129), are located within a cavity on the poliovirus capsid interior, whereas the third arginine, residue 223 of VP3 (VP3-R223), is located at a promoter-protomer interface. Five mutants were constructed by site-directed mutagenesis of poliovirus P1 capsid precursor cDNA to separately encode lysine or glutamine substitutions at VP4-R034 (VP4-R034K, VP4-R034Q), lysine or glutamine substitutions at residue 129 of VP1 (VP1-R129K, VP1-R129Q), or a lysine substitution at residue 223 of VP3 (VP3-R223K). Processed capsid proteins derived from the VP3-R223K, VP1-R129K, and VP1-R129Q mutant precursors were unstable and failed to assemble subviral particles or virions at 37 degrees. The assembly defect for cleavage products of the VP3-R223K precursor was partially overcome at 33 degrees, as empty capsids, but not mature virions, assembled from the mutant capsid subunits at the lower temperature. With regard to the third arginine residue analyzed, VP4-R034, processed capsid proteins derived from both the VP4-R034K and the VP4-R034Q mutant precursors assembled 155S virions at 37 degrees; however, capsid proteins derived from the VP4-R034Q precursor were temperature-sensitive for virion formation at 39.5 degrees. The reduced virion formation at 39.5 degrees was apparently a reflection of a defect in forming assembly competent subunits which also prevented accumulation of surplus VP4-R034Q subunits as empty capsids. By using graphics to display the poliovirus three-dimensional structure, the locations of these residues on the poliovirus capsid interior and their interactions with adjacent amino acids were visualized to provide structural explanations for the observed assembly defects which highlight the important role these residues play in capsid assembly and RNA encapsidation.

Arginine↗

PubMed2006

Genetic characterization of VP3 gene of group A rotaviruses.

We sequenced VP3 gene of four rotavirus strains and examined the diversity of VP3 and its other genetic characteristics in a total of 22 Group A rotaviruses. The 22 rotavirus strains were derived from six different host species (human, avian, bovine, equine, simian and porcine). Comparison of the partial VP3 peptide sequences (aa 141-294) showed identities ranging from 49 to 99% among different species. The phylogenetic analysis of VP3 sequences revealed segregation according to the species origin. This was further confirmed by identification of three host specific domains within the VP3 peptide. Thus, the host range restriction or attenuation previously shown to associate with VP3 may be attributed to the species-specific nature of the gene.

Amino Acid Sequence↗

PubMed1988

Nucleotide sequence of UK bovine rotavirus segment 4: possible host restriction of VP3 genes.

The bovine UK and simian SA11 rotaviruses are commonly used VP7-type reference strains. Since the surface protein VP3 is a significant neutralization antigen, it is important to fully characterize the VP3 types associated with current reference strains. Here we present the complete nucleotide and predicted amino acid sequence of VP3 from UK rotavirus (VP7 type 6) and compare it to the published sequences of SA114fm and RV-5. We also compare the deduced amino acid sequence covering the trypsin cleavage region of UK VP3 to 25 other available sequences. The UK protein is clearly different from that of bovine NCDV (another commonly used VP7 type 6 strain) and represents a second VP3 type associated with bovine rotaviruses. Our SA11 sequence differs from that determined by Lopez et al. [1985, Virology 144, 11-19; later referred to as SA114fM by Lopez et al. (1986, Virology 154, 224-227], their sequence being very similar to the published sequence of NCDV VP3. The significance of these results with regard to virus serotypes is discussed. Finally, in analyzing the nucleotide sequence surrounding the initiation codon, a potential hairpin-loop structure was identified which may be involved in translational regulation.

Amino Acid Sequence↗

PubMed1992

Rotavirus VP3 expressed in insect cells possesses guanylyltransferase activity.

We have examined the possible function(s) of the protein VP3 encoded by the rotavirus SA11 genomic segment 3. Viral-associated VP3 in double-shelled and single-shelled particles was shown to bind GTP covalently and reversibly. These properties are similar to the unique characteristics of eukaryotic and viral guanylyltransferases, suggesting that VP3 is associated with a capping enzyme activity. Previous studies have shown that intact viral particles are required for transcription, making it difficult to unequivocally identify the functions of individual proteins within such particles. Characterization of VP3 produced in the baculovirus expression system showed that the expressed VP3 covalently bound GTP. These studies suggest that VP3 alone is the guanylyltransferase. GTP binding also was seen in core virus-like particles and single-shelled virus-like particles that lacked viral nucleic acid and were assembled in insect cells.

Animals↗

PubMed2003

The simian virus 40 minor structural protein Vp3, but not Vp2, is essential for infectious virion formation.

The SV40 capsid is composed of pentameric capsomeres of the major structural protein Vp1. The two minor structural proteins, Vp2 and Vp3, interact with the capsid. Here, the roles of Vp2 and Vp3 were explored during the course of SV40 infection. Start codons of Vp2, Vp3, or both Vp2 and Vp3, were destroyed by site-directed mutagenesis, and mutant genomes were transfected into CV-1 cells. SV40DeltaVp2 produced plaques and infectious virion particles with titres indistinguishable from wild-type. SV40DeltaVp3 and SV40 DeltaVp2/Vp3 were defective in plaque formation and rendered no infectious particles. All three mutants showed normal nuclear localization of T-Ag and Vp1; they also showed packaging of SV40 DNA by nuclease digestion assays. Thus, Vp3 is essential for formation of infectious SV40 particles, whereas Vp2 is not. One critical role of full-length Vp3 appears to be in virus-cell interactions at post-packaging steps of a permissive infection.

Animals↗

PubMed1988

Identification of cross-reactive and serotype 2-specific neutralization epitopes on VP3 of human rotavirus.

The group A rotaviruses are composed of at least seven serotypes. Serotype specificity is defined mainly by an outer capsid protein, VP7. In contrast, the other surface protein, VP3 (775 amino acids), appears to be associated with both serotype-specific and heterotypic immunity. To identify the cross-reactive and serotype-specific neutralization epitopes on VP3 of human rotavirus, we sequenced the VP3 gene of antigenic mutants resistant to each of seven anti-VP3 neutralizing monoclonal antibodies (N-MAbs) which exhibited heterotypic or serotype 2-specific reactivity, and we defined three distinct neutralization epitopes on VP3. The mutants sustained single amino acid substitutions at position 305, 392, 433, or 439. Amino acid position 305 was critical to epitope I, whereas amino acid position 433 was critical to epitope III. In contrast, epitope II appeared to be more dependent upon conformation and protein folding because both amino acid positions 392 and 439 appeared to be critical. These four positions clustered in a relatively limited area of VP5, the larger of the two cleavage products of VP3. At the positions where amino acid substitutions occurred, there was a correlation between amino acid sequence homology among different serotypes and the reactivity patterns of various viruses with the N-MAbs used for selection of mutants. A synthetic peptide (amino acids 296 to 313) which included the sequence of epitope I reacted with its corresponding N-MAb, suggesting that the region contains a sequential antigenic determinant. These data may prove useful in current efforts to develop vaccines against human rotavirus infection.

Amino Acid Sequence↗

PubMed1998

The N terminus of rotavirus VP2 is necessary for encapsidation of VP1 and VP3.

The innermost core of rotavirus is composed of VP2, which forms a protein layer that surrounds the two minor proteins VP1 and VP3, and the genome of 11 segments of double-stranded RNA. This inner core layer surrounded by VP6, the major capsid protein, constitutes double-layered particles that are transcriptionally active. Each gene encoding a structural protein of double-layered particles has been cloned into baculovirus recombinants and expressed in insect cells. Previously, we showed that coexpression of different combinations of the structural proteins of rotavirus double-layered particles results in the formation of virus-like particles (VLPs), and each VLP containing VP1, the presumed RNA-dependent RNA polymerase, possesses replicase activity as assayed in an in vitro template-dependent assay system (C. Q.-Y. Zeng, M. J. Wentz, J. Cohen, M. E. Estes, and R. F. Ramig, J. Virol. 70:2736-2742, 1996). This work reports construction and characterization of VLPs containing a truncated VP2 (VPdelta2, containing amino acids [aa] Met-93 to 880). Expression of VPdelta2 alone resulted in the formation of single-layered delta2-VLPs. Coexpression of VPdelta2 with VP6 produced double-layered delta2/6-VLPs. VLPs formed by coexpression of VPdelta2 and VP1 or VP3, or both VP1 and VP3, resulted in the formation of VLPs lacking both VP1 and VP3. The presence of VP6 with VPdelta2 did not result in encapsidation of VP1 and VP3. To determine the domain of VP2 required for binding VP1, far-Western blot analyses using a series of truncated VP2 constructs were performed to test their ability to bind VP1. These analyses showed that (i) full-length VP2 (aa 1 to 880) binds to VP1, (ii) any N-terminal truncation lacking aa 1 to 25 fails to bind VP1, and (iii) a C-terminal 296-aa truncated VP2 construct (aa 1 to 583) maintains the ability to bind VP1. These analyses indicate that the N terminus of rotavirus VP2 is necessary for the encapsidation of VP1 and VP3.

Animals↗

PubMed1994

Signal- and energy-dependent nuclear transport of SV40 Vp3 by isolated nuclei. Establishment of a filtration assay for nuclear protein import.

Nuclear transport signal (NTS)-containing proteins are transported into the nucleus through the nuclear pore complex by a mechanism that is not well understood. To better characterize the mechanisms of transport, we have established an homologous in vitro system using an NTS-containing structural protein of simian virus 40 (SV40) and isolated nuclei from cultured cells of its natural host. Isolated nuclei accumulated either fluorescently labeled SV40 Vp3-NTS peptide-BSA conjugates (NTSwt-BSA), as assayed cytochemically, or 125I-NTSwt-BSA, as assayed by filtration, in a signal- and ATP-dependent manner. Nuclear accumulation required nuclear membrane integrity and was inhibited by the lectin wheat germ agglutinin but not concanavalin A. Unlike several other systems, this system is not dependent on cytoplasmic extracts for the transport of SV40 proteins. NTSwt-BSA was transported with an apparent Km of 0.8 microM and Vmax of 0.8 nmol/min/10(*) nuclei. Thin section autoradiography confirmed the transport. This system faithfully reproduced what occurs in vivo: nuclear import of the SV40 capsid protein Vp3 was dependent on the presence of its functional NTS. Full-length Vp3, expressed as a glutathione S-transferase fusion protein, and a deletion mutant which retains its NTS, Vp3 delta C13, were transported by the nuclei but Vp3 delta C35, which lacks the NTS, and an NTS mutant, Vp3(202E/204T), were not transported.

Amino Acid Sequence↗

PubMed1991

Phylogenetic analyses of the complete nucleotide sequence of the capsid protein (VP3) of Australian epizootic haemorrhagic disease of deer virus (serotype 2) and cognate genes from other orbiviruses.

The complete nucleotide sequence of the minor capsid protein (VP3) of epizootic haemorrhagic disease of deer virus (EHDV; Australian serotype 2) was determined using a combination of cloning and sequencing methods. Gene segment 3 that coded for the EHDV VP3 capsid protein was 2768 nucleotides in length with a coding region of 2697 nucleotides flanked by 5' and 3' non-coding regions of 17 and 53 nucleotides, respectively. A protein of 899 amino acids (Mr 103,160) having no overall charge at neutral pH was deduced from the nucleotide sequence. Comparisons with equivalent regions from the other Australian EHDV serotypes showed the VP3 genes and the segments that coded for them were similar, varying by a maximum of 5%. Comparisons with known cognate genes from bluetongue viruses showed that their VP3 genes and the proteins translated from them were remarkably similar to those of EHDV, having approximately 70% to 80% homology at either level, respectively. In an attempt to delineate the evolution of orbiviruses, we have obtained sequence data from the VP3 genes from representative members of all Australian orbiviruses now known. Computer analyses of this data enabled a phylogenetic tree for the orbiviruses to be proposed that incorporated the concept of topotypes.

Amino Acid Sequence↗

PubMed2006

SV40 VP2 and VP3 insertion into ER membranes is controlled by the capsid protein VP1: implications for DNA translocation out of the ER.

Nonenveloped viruses such as Simian Virus 40 (SV40) exploit established cellular pathways for internalization and transport to their site of penetration. By analyzing mutant SV40 genomes that do not express VP2 or VP3, we found that these structural proteins perform essential functions that are regulated by VP1. VP2 significantly enhanced SV40 particle association with the host cell, while VP3 functioned downstream. VP2 and VP3 both integrated posttranslationally into the endoplasmic reticulum (ER) membrane. Association with VP1 pentamers prevented their ER membrane integration, indicating that VP1 controls the function of VP2 and VP3 by directing their localization between the particle and the ER membrane. These findings suggest a model in which VP2 aids in cell binding. After capsid disassembly within the ER lumen, VP3, and perhaps VP2, oligomerizes and integrates into the ER membrane, potentially creating a viroporin that aids in viral DNA transport out of the ER.

Capsid Proteins↗

PubMed1998

Enhancement of the immunogenicity of a synthetic peptide bearing a VP3 epitope of hepatitis A virus.

The immune responses elicited in mice by different forms of the VP3(110-121) B-epitope of the hepatitis A virus (HAV) were studied. Different forms of incorporation in liposomes were tested, encapsulation, rather than surface exposure, being the best antigenic preparation. Three larger peptides of the VP3 epitope, two of them containing a hepatitis B virus T-epitope, and a third containing a putative T-epitope of HAV (VP3(102-121)) were assayed. While this latter T-epitope induced an enhancement of the response against the VP3 B-epitope, the artificially coupled T-epitopes failed to induce a significant increase. The administration of two multiple antigenic peptide (MAP) constructs, the first containing the VP3(110-121) and VP1(11-25) HAV sequences and the second only the VP1(11-25) sequence, also suggested the presence of a T-epitope, since the response against the VP1 peptide was increased in the first construct.

Animals↗

PubMed1998

pH-induced destabilization of lipid bilayers by a peptide from the VP3 protein of the capsid of hepatitis A virus.

The membrane destabilizing and fusogenic properties of the synthetic peptide VP3(110-121), corresponding to an immunogenic sequence of the hepatitis A virus (HAV) VP3 capsid protein, were studied. By tryptophan fluorescence and acryalmide quenching it was demonstrated that the peptide binds liposomes of POPC-SM-DPPE (47 + 39 + 14) and POPC-SM-DPPE-DOTAP (40 + 33 + 12 + 15) and penetrates the membrane, at both neutral and acidic pH (POPC = 1-palmitoyl-2-oleoylglycero-sn-3-phosphocholine; SM = sphingomyelin; DPPE = 1,2-dipalmitoylphosphatidylethanolamine; DOTAP = 1,2-dioleoyl-3-trimethylammoniumpropane). VP3(110-121) did not have membrane-destabilizing properties at neutral pH. Acid-induced destabilization of the vesicles was demonstrated by fluorescence techniques and dynamic light scattering. VP3(110-121) induced aggregation of POPC-SM-DPPE-DOTAP (40 + 33 + 12 + 15) vesicles, lipid mixing and leakage of vesicle contents, all consistent with fusion of vesicles. In POPC-SM-DPPE (47 + 39 + 14) vesicles, at acidic pH, VP3(110-121) induced membrane destabilization with leakage of contents but without aggregation of vesicles or lipid mixing. The peptide only showed fusogenic properties when bound to the vesicles at neutral pH before acidification to pH below 6.0, and no effect was seen if the peptide was added to vesicles already set at acidic pH. These results may have physiological significance in the mechanism of infection of host hepatic cells by HAV.

Capsid↗

PubMed1979

Vp1 affects intracellular localization of Vp3 polypeptide during simian virus 40 infection.

In order to understand the functions of simian virus 40 genes, permissive cells (TC7) were infected with mutants temperature sensitive in the complementation groups A, B, C, BC, and D at permissive and nonpermissive temperatures. Cells were examined for the localization of viral polypeptide antigens by immunofluorescent staining with monospecific antibodies. The results are as follows: (i) The appearance of Vp1 antigen in cells infected by tsB, C, or BC mutants was not affected appreciably by the mutations. (ii) The appearance of Vp3 antigen was affected by the mutations in B, C, or BC. Vp3 antigen is confined to the nuclei in cells infected by wild-type virus. With mutant virus infection, Vp3 antigen is found in the cytoplasm, perinuclear region, and nucleoli. (iii) The tsD mutants and the tsA mutants did not express either Vp1 or Vp3 antigens at the nonpermissive temperature. (iv) Nucleoli seem to play an essential role in the biosynthesis and assembly of viral polypeptides. Thus, mutations in any one of complementation groups B, C, or BC, which are within the structural gene for Vp1, cause an alteration of intracellular distribution of another late gene product, Vp3. These results suggest that the amino acid sequences of Vp1 polypeptide play a role(s) in the transport of viral antigens across internal membranes or in virus assembly processes or in both.

Antigens, Viral↗