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At least 19 recordsLinked to original sources
PubMed2026

MARK1 suppresses infectious bursal disease virus replication via phosphorylating VP3.

Infectious bursal disease virus (IBDV) of the Birnaviridae family is a non-envelope, double-stranded RNA virus that encodes a VP3 protein with multiple functions, which controls viral genome replication, IFN-β production, and virus traffic in infected cells. Posttranslational modifications (PTMs), such as ubiquitination, of VP3 have been demonstrated for affecting its function and stability. To clarify the mechanism by which VP3 is regulated in IBDV infected cells, we focused on the phosphorylation of VP3. Mass spectrometry analysis identified that microtubule-affinity regulating kinases 1 (MARK1) was a kinase interacting protein of VP3. Inhibitory function of MARK1 in affecting viral replication was validated. We describe the phosphorylation event at the serine 130 (S130) and serine 163 (S163) residues of VP3 mediated by MARK1 via mass spectrometry analysis. Alanine replacement of the phosphorylation sites in VP3 significantly enhanced its RNA-binding activity. Additionally, the mutation of two serine residues led to remarkably improved in its polymerase-enhancing function. We then incorporated the two mutations to rescue recombinant IBDV. Viral growth curve analysis revealed that replication of mutant IBDV was significantly enhanced relative to wild type (WT) virus. In conclusion, we found that VP3 functions are specifically regulated by MARK1 mediated phosphorylation at S130 and S163 and that this regulation suppresses IBDV replication ultimately.

Infectious bursal disease virus↗
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↗
PubMed1978

Characterization of the mRNA's for the polyoma virus capsid proteins VP1, VP2, and VP3.

Polyadenylated cytoplasmic RNA from polyoma virus-infected cells can be translated in the wheat germ system to yield all there polyoma virus capsid proteins, VP1, VP2, and VP3. The translation products of RNA selected from total cytoplasmic RNA of infected cells by hybridization to polyoma virus DNA showed a high degree of enrichment for VP1, VP2, and VP3. The identity of the in vitro products with authentic virion proteins was established in two ways. First, tryptic peptide maps of the in vitro products were found to be essentially identical to those of their in vivo counterparts. Second, the mobilities of the in vitro products on two-dimensional gels were the same as those of viral proteins labeled in vivo. VP1, VP2, and vp3 were all labeled with [35S] formylmethionine when they were synthesized in the presence of [35S] formylmethionyl-tRNAfmet. We determined the sizes of the polyadenylated mRNA's for VP1, VP2, and VP3 by fractionation on gels. The sizes of the major mRNA species for the capsid proteins are as follows: VP2, 8.5 X 10(5) daltons; VP3, 7.4 X 10(5) daltons; and VP1, 4.6 X 10(5) daltons. We conclude that all three viral capsid proteins are synthesized independently in vitro, that all three viral capsid proteins are virally coded, and that each of the capsid proteins has a discrete mRNA.

Capsid↗
PubMed1977

Induction of neutralizing antibodies and immunity in vaccinated guinea pigs by cyanogen bromide-peptides of VP3 of foot-and-mouth disease virus.

The specificity of guinea pig antisera against large cyanogen bromide-cleaved peptides of the virus capsid protein VP3 of foot-and-mouth disease virus type O1, strain Kaufbeuren has been characterized by double immunodiffusion, virus neutralization and protection tests. Antibodies to purified 146S particles and the cleavage peptides of VP3 showed an incomplete cross-section against VP3 peptide antigen when reacted in immunodiffusion tests, indicating that new antigenic determinants are exhibited by the peptides which are not recognized by the antiserum against the native virus proteins. The immune response against the reduced, unfolded chain constituents of VP3 was lower in comparison to that of native virus particles but still some immunological determinants remained actively capable of inducing virus-neutralizing antibodies in immunized guinea pigs.

Animals↗
PubMed1977

Overlapping of the VP2-VP3 gene and the VP1 gene in the SV40 genome.

The nucleotide sequence of the SV40 Hind E fragment has been determined mainly by the partial chemical degradation procedure of Maxam and Gilbert (1977). The sequence of the strand with the same polarity as the late messenger RNA shows only one open reading frame for translation. Considering that VP3 corresponds to the carbosyl terminal part of VP2, and considering various evidence which indicates that the SV40 Hind E segment is part of the amino acid sequence of VP2-VP3. It continues clockwise in Hind K, where it terminates with a UAA signal. The latter is located 110 nucleotides beyond the initiation signal for the major structural protein VP1 (Fiers et al., 1975; Van de Voorde et al., 1976). Hence this small overlapping region of the genome codes for the synthesis of three different proteins in two different reading frames. The deduced amino acid sequence covers a major part of the vp3 poly peptide, and the amino acid composition is in good agreement with published values (Greenaway and Levine, 1973).

Base Sequence↗
PubMed1978

Nucleotides sequence of the genes for the simian virus 40 proteins VP2 and VP3.

We have determined the nucleotide sequence of the DNA of simian virus 40. The proceeding report (Dhar, R., Reddy, V.B., and Weissman, S.M. (1978) J. Biol. Chem. 253, 612-620) presents the sequence of a portion of the simian virus 40 DNA that overlaps the region encoding the 5' end of the minor structural protein VP2. We report here the sequence of the remainder of the genes for minor structural proteins VP2 and VP3. The results indicate that the mRNA for the two proteins is read in the same phase and the initiation site for VP3 lies within the structural gene of VP2. The codons of the COOH-terminal amino acids of VP2 and VP3 are read in a second phase as the codons of the NH2-terminal amino acids of VP1.

Amino Acids↗
PubMed1975

Immune and antibody responses to an isolated capsid protein of foot-and-mouth disease virus.

The purified capsid proteins VP1, VP2, and VP3 of foot-and-mouth disease virus type A12 strain 119 emulsified with incomplete Freund's adjuvant were studied in swine and guinea pigs. Swine inoculated on days 0, 28, and 60 with 100-mug doses of VP3 were protected by day 82 against exposure to infected swine. Serums from animals inoculated with VP3 contained viral precipitating and neutralizing antibodies, but such serums recognized fewer viral antigenic determinants than did antiviral serums. Capsid proteins VP1 and VP2 did not produce detectable antiviral antibody in guinea pigs, and antiviral antibody responses in swine to a mixture of VP1, VP2, and VP3 were lower than the responses to VP3 alone. However, when swine were inoculated with VP1, VP2, and VP3 separately at different body sites, no interference with the response to VP3 was observed. Vaccine containing VP3 isolated from acetylethylenimine-treated virus appeared less protective for swine than vaccine containing VP3 from nontreated virus. Trypsinized virus, which contains the cleaved peptides VP3a and VP3b rather than intact VP3, produced approximately the same levels of antiviral antibody responses in guinea pigs as did virus. Conversely, an isolated mixture of VP3a and VP3b did not produce detectable antiviral antibody responses in guinea pigs. The VP3a-VP3b mixture did, however, sensitize guinea pigs to elicit such responses following reinoculation with a marginally effective dose of trypsinized virus.

Animals↗
PubMed1975

N-terminal amino acid sequences in the major capsid proteins of foot-and-mouth disease virus types A, O, and C.

Sequences of amino acids at the N-termini of virus proteins VP1, VP2, and VP3 were determined for foot-and-mouth disease virus types A12 strain 119, O1Brugge and C3Resende. In the polyacrylamide gel electrophoresis system used to purify the proteins, VP3 migrated faster than VP1 or VP2; and in the virion, VP3 could be cleaved by trypsin into VP3a and VP3b. The N-terminal amino acids for each of the virus types were glycine in VP1, aspartic acid in VP2, and threonine in VP3. No divergences in sequence across the virus types were indicated until at least the fourth position in VP1, and the third in VP3. For virus types A12, O1 and C3, the sequences were, respectively: for VP1 (Gly-ile-phe,pro,val---), (Gly,ile,phe---) and Gly-ile-phe,ala---); for VP2 (Asp,X,met---), (Asp---) and Asp-leu---); and for VP3 (Thr-thr-ala-thr---), (Thr-thr-ser---) and (Thr-thr---). Unresolved mixtures of VP3a and VP3b, from either A12 or O1 viruses, appeared to have the N-terminal amino acids threonine, which is presumed to be the same threonine as in uncleaved VP3 and serine, which is generated by the tryptic cleavage.

Amino Acid Sequence↗
PubMed1979

Topographical studies on poliovirus capsid proteins by chemical modification and cross-linking with bifunctional reagents.

Poliovirus capsid proteins comprise 15.1 lysines in VP1, 5.6 lysines in VP2, 11.7 lysines in VP3 and 5.5 lysines in VP4. Treatment with monofunctional reagent N-succinimidyl 2,3-3H-proprionate leads to the modification of 3.4 lysines in VP1, 0.6 lysines in VP2, 2.0 lysines in VP3 and 0.03 lysines in VP4. Chemical modification with the monofunctional reagent N-succinimidyl 3-(4-hydroxy,5-125I-iodophenyl)propionate results in a predominant labelling of VP1 and VP3, whereas VP2 is less accessible and VP4 is not modified. Cross-linking of poliovirus with bifunctional imidoesters, dimethyl suberimidate (DMS, 1.1 nm) and dimethyl adipimidate (DMA, 0.8 nm) leads to a new protein complex of mol. wt. which corresponds to the sum of VP1 and VP3. By cleavage with ammonia and electrophoresis on polyacrylamide gels in SDS, the proteins are identified as VP1 and VP3. This result gives evidence for a direct neighbourhood of VP1 and VP3 in the virus capsid. Treatment of the virus with the mono- and bifunctional reagents has no influence on the stability of the particle. The infectivity is reduced only by the bifunctional reagent.

Chemical Phenomena↗
PubMed1976

Cell-free translation of foot-and-mouth disease virus RNA into identifiable non-capsid and capsid proteins.

Foot-and-mouth disease virus (a member of the picornavirus group) RNA could be translated effectively in an S-30 extract from Ehrlich ascites tumour cells. This translation was inhibited by aurintricarboxylic acid, cycloheximide, puromycin and RNase. Cell-free products of translation were identified by disc gel electrophoresis and immunoprecipitation with specific antisera. Gel electrophoresis of the products without prior immunoprecipitation suggested the synthesis of some of the non-capsid proteins and capsid proteins VP1, VP2 and VP3 of the virus. Immunoprecipitations with antisera against whole virus and VP3 indicated the synthesis of VP3 and of at least two additional peptides of 100 000 and 56 000 daltons containing antigenic sites of VP3. Gel electrophoresis after immunoprecipitation with antiserum against virus infection-associated antigen indicated the synthesis of a different 56 000-dalton protein appearing to resemble non-capsid protein NCVP5. The amount of foot-and-mouth disease virus and VP3-specific peptides in the virus RNA-directed products were measured by immunoprecipitation.

Animals↗
PubMed1976

Structural proteins of polyoma virus: proteolytic degradation of virion proteins by exogenous and by virion-associated proteases.

A model has previously been proposed for the genetic relatedness of the structural proteins of polyoma virus, based upon similarities in the peptide maps of the major capsid protein VP1 with the virion proteins VP2 and VP3. Newer evidence suggests that this model is incorrect, and that protein VP1 is a product of one viral gene and that the multiple components of VP2 and VP3 are products of a second viral gene. Two-dimensional peptide maps of several preparations of polyoma purified separately from four separate infected-cell lysates has shown a variable content of VP1 peptides in proteins VP2 and VP3, with some preparations being free of detectable VP1 material in VP2 and VP3. An alternative explanation for the presence of VP1 peptides in the regions of VP2 and VP3 of some polyoma preparations involves the cleavage of proteins of polyoma virions during exposure to proteolytic enzymes in lysates of infected cells or to endogenous proteolytic activity of virions. Prolonged incubation of infected-cell lysates at 37 degrees C leads to an increase in the amount of 86,000-dalton dimer of VP1, a decrease in the relative amount of VP1, a decrease in or a loss of the lower band of VP2, and the appearance of a new major protein band of approximately 29,000 daltons. Two-dimensional peptide maps of the new 29,000-dalton protein show that it contains some VP1 peptides, indicating that this protein is derived from proteolytic cleavage of VP1. In addition, extensively purified polyoma virus contains a proteolytic activity that can be activated during disruption of the virus with 0.2 M Na2CO3-NaHCO3 (pH 10.6) in the presence of 5 X 10(-3) M dithiothreitol.

Dithiothreitol↗
PubMed1979

Intracellular localization of viral polypeptides during simian virus 40 infection.

African green monkey kidney cells infected by simian virus 40 were analyzed by immunofluorescence techniques for the nature and the time course of the appearance of viral polypeptides during infection. Reagents used in the study were anti-Vpl sera and affinity-purified anti-Vpl immunoglobulin G, anti-Vp3 sera, antivirus (anti-V) sera, and anti-tumor antigen sera. The results are summarized as follows. (i) Three types of staining, nuclear, perinuclear, and perinuclear accompanied by cytoplasmic staining, were observed in infected cells in reaction with anti-vpl antibody. In addition, a highly structured staining was observed at the periphery of nuclei of infected cells late in infection. (ii) In reaction with anti-Vp3 serum, the staining was confined within nuclei of cells throughout infection. (iii) Vp1 and Vp3 antigens seem to occupy different spacial regions of the nuclear area in cells. (iv) Vp1 and Vp3 antigens were expressed simultaneously during infection. (v) Centriolar staining observed early in infection paralleled the appearance of tumor (T-) antigen until 24 h after infection, after which time the frequency of positive centriolar staining decreased as infection progressed. (vi) T-antigen was first expressed at about 8 h after infection, and Vp1 and Vp3 antigens were first expressed at about 20 h after infection.

Animals↗
PubMed2026

Viral inhibition of the anaphase promoting complex enhances replication by elevating nucleotide pools.

The anaphase promoting complex/cyclosome (APC/C) is a large, ubiquitin ligase and a central regulator of cell cycle progression. By targeting key substrates for degradation during mitosis and G1 phase, the APC/C coordinates metabolic fluctuations that occur during the cell cycle. A diverse range of viruses have convergently evolved mechanisms to bind and inhibit the APC/C; however, a molecular understanding for these interactions has never been demonstrated. Here, we use chicken anemia virus (CAV), a small single-stranded DNA virus encoding only three proteins, to demonstrate the importance of viral APC/C inhibition during replication. We show that the Vp3 protein of CAV inhibits the APC/C, causing a dramatic mitotic arrest during infection. The mutant virus lacking Vp3 is defective for replication and can be rescued by APC/C inhibition. Metabolomic profiling during CAV infection revealed that Vp3 expression mediates a broad increase in nucleotide pools. Moreover, viral inhibition of the APC/C resulted in stabilization of enzymes required for nucleotide biosynthesis. These findings suggest that the APC/C is a general target of many viruses to elevate nucleotide levels and facilitate viral genome replication.

Anaphase-Promoting Complex-Cyclosome↗
PubMedDate not supplied

Dissociation and reassociation of poliovirus. II. Protein components obtained by urea treatment of the virus particle.

Dissociation of poliovirus by 9 M urea in 0.015 M NaCl at 25 degrees C resulted in the liberation of 35S RNA and of polypeptides sedimenting at 2S in sucrose gradients containing 9 M urea. However, a ribonucleopolypeptide (RNPP) complex sedimenting at 45S and oligomers of the viral polypeptides sedimenting at 7--8S were found in addition to the monomers sedimenting at 2S when the urea concentration was lowered to 5 M after the dissociation procedure. Ribonuclease treatment prevents the appearance of the RNPP-complex. The amount of the RNPP-complex decreased, when the dissociation was performed at higher ionic strength. Under these conditions small amounts of empty capsids were detected. Polyacrylamide gel electrophoresis showed that the RNPP-complex contained the polypeptide VP1. The oligomers (7--8S) contained the polypeptide VP3 and small amounts of VP2. The bulk of VP2 and some VP3 were found in the 2S position together with VP4. The molecular weight of the dissociation products in urea and phosphate buffer was determined by gel filtration to be about 30,000 for the monomeric polypeptides containing predominantly VP2 and about 70,000 for the oligomeric polypeptides containing predominantly VP3. Our results demonstrate that the oligomers and the RNPP-complex are not primary products obtained by dissociation of the virus particle by urea but are due to a reassociation of the polypeptides or of VP1 and RNA.

Centrifugation, Density Gradient↗
PubMed1977

Coupled transcription-translation of DNA injected into Xenopus oocytes.

A previous report from this laboratory showed that purified DNAs are transcribed after injection into the nucleus of Xenopus laevis oocytes (Mertz and Gurdon, 1977). Here we demonstrate that at least some of the RNA synthesized is translated within these injected cells to produce the correct protein products. Injection of Simian Virus 40 DNA into oocytes induces the synthesis of at least two proteins not normally synthesized in these cells. Using two-dimensional polyacrylamide gel electrophoresis and well characterized deletion mutants of SV40 that produce proteins of smaller size, we show that these two proteins are, indeed, the virus-coded proteins VP1 and VP3. Synthesis of VP1 and VP3 is inhibited by alpha-amanitin and, therefore, presumably mediated by a type II RNA polymerase. We also present evidence indicating that a histone-like protein is induced after the injection of a cloned plasmid DNA that codes for the Drosophila melanogaster histone proteins. This in vivo coupled transcription-translation system should be useful for identifying and mapping proteins coded by cloned eucaryotic DNAs.

Amanitins↗
PubMed1979

BK virus DNA sequence: extent of homology with simian virus 40 DNA.

The primary nucleotide sequence of three regions of BK virus (BKV) variant (MM) DNA has been determined. The region between map positions 0.715 and 0.900 includes the initiation points and partial coding sequences of the putative VP2 and VP3 proteins of BKV(MM), the amino acid sequences of which show over 80% homology with those of VP2 and VP3 of simian virus 40. The sequence of a potential leader protein X, 66 amino acids long for BKV(MM) and 62 long for simian virus 40, is also deduced. The regions between 0.595 and 0.398 and 0.310 and 0.175 include the coding sequence for the entire small t antigen and most of the large T antigen of BKV(MM). The DNA sequence within these regions comprises over 50% of the complete BKV(MM) genome and shows a 70% sequence homology with the corresponding regions of simian virus 40 DNA. This high degree of homology is at variance with the reported homology values of 11--20% estimated by hybridization measurements of heteroduplex analyses. Possible explanations for the discrepancy are discussed.

BK Virus↗
PubMed1975

The polypeptide structure of transmissible gastroenteritis virus.

The polypeptides of purified preparations of the coronavirus responsible for transmissible gastroenteritis of pigs have beem examined by polyacrylamide gel electrophoresis. Four major polypeptides, VPI (mol. wt. 200000), VP2 (50 000), VP3 (30000) and VP4 (28500) and two minor polypeptides, VPIa (105000) and VPIb (80500) have been reproducibly demonstrated in the virion, of which VPI, VP3 and VP4 contain carbohydrate. Treatment of the virion with the proteolytic enzyme bromelain removes the surface projections and VPI, thus identifying this glycopolypeptide as the major structural component of the projection.

Bromelains↗