Virion polypeptide composition of the human papovavirus BK: comparison with simian virus 40 and polyoma virus.

The polypeptide composition of labeled BK virus was compared with that of simian virus 40 (SV40) and polyoma virus by co-electrophoresis of disrupted virions in polyacrylamide gels containing approximately 73% of the capsid protein and had a molecular weight of 39,000. It was smaller than VP1 of SV40 and polyoma virus. The other polypeptides of BK virus were similar in molecular weight to those of SV40. A comparison of the proteins of BK virus and SV40 iodinated with chloramine T before and after disruption in alkaline buffer at pH 10.5 revealed differences between the two viruses in the number and distribution of tyrosines available for iodination. The tryptic peptides of VP1, VP3, VP4, and VP5 combined of SV40 were compared with those of the same polypeptides of BK virus. Among the 19 peptides of VP1 resolved, only two were common to both viruses. The analyses of VP4 and VP5, the histone-like proteins, however, showed more similarity between the viruses, with 6 of 15 resolved peptides in common. The tryptic digests of VP3 were completely different.     

Characterization of cricket paralysis virus-induced polypeptides in Drosophila cells

Cricket paralysis virus purified from Galleria mellonella larvae was shown to be similar to virus purified from Drosophila melanogaster cells. Cricket paralysis virus contained three major structural polypeptides of similar molecular weight (around 30,000), had a buoyant density of 1.344 g/ml, and had a capsid diameter of 27 nm. Twenty virus-induced polypeptides could be detected in CrPV-infected Drosophila cells. Two major polypeptides found in the infected cells corresponded to two structural viral polypeptides (VP1 and VP3), whereas the third major intracellular polypeptide was the apparent precursor of the third viral structural polypeptide (VP2). Three of the primary virus-induced polypeptides had molecular weights of 144,000, 124,000, and 115,000. These and other polypeptides were chased into lower-molecular-weight proteins when excess cold methionine was added after a short [³⁵S]methionine pulse. Although cricket paralysis virus has a number of characteristics in common with the mammalian enteroviruses, the extremely fast processing of high-molecular-weight polypeptides into viral proteins seems atypical. Also, no VP4 (8,000 to 10,000 molecular weight) has been found in the virus particles.     

Characteristics of structural proteins of infectious flacherie virus from the silkworm, Bombyx mori

Structural proteins and the characteristics of infectious flacherie virus (IFV) purified from the silkworm, Bombyx mori, are described. The purified IFV had four major structural proteins, which were detected only in high concentration gels of sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and a few minor ones. Molecular weights of the major proteins were 35,200 (VP 1), 33,000 (VP 2), 31,200 (VP 3), and 11,600 (VP 4), and numbers per virion were 62, 57, 54, and 31, respectively. Amino acid compositions of VP 1, VP 2, and VP 3 were similar to each other but that of VP 4 was somewhat different. By isoelectric focusing and two-dimensional electrophoresis, high resolution of the structural proteins was obtained with silver staining. The isoelectric points of the four major proteins were determined as 7.7(VP 1), 6.7(VP 2), 4.8(VP 3), and 5.5(VP 4). This work is the first report on insect picornaviruses that presents some discriminative properties of each viral protein that was compared to those of mammalian picornaviruses.     

Production of chicken anemia virus (CAV) VP1 and VP2 protein expressed by recombinant Escherichia coli

Chicken anemia virus (CAV) is an anemia agent of breeder and young chicks. This virus is the cause of economic losses across the chicken industry worldwide as a consequence of severe anemia and immunodeficiency among the birds. Two genes of CAV encoding the VP1 and VP2 proteins were cloned and expressed in Escherichia coli BL21 (DE3). A Western blot assay using His-tag antiserum was used to assess the expression level of the CAV viral proteins in E. coli. The results demonstrated that only full-length VP2 can be successfully expressed in E. coli, but not full-length VP1. A serial of N-terminus deletions of the VP1 protein, VP1Nd30, VP1Nd60 and VP1 Nd129, were created using PCR in order to improve VP1 expression. The results demonstrated that all three of these recombinant VP1 mutant proteins can be expressed in E. coli. VP1Nd129 protein demonstrates the highest expression level compared to the other two proteins. The specificity of Nd129-VP1 and VP2 protein were confirmed by mass spectrometry. By comparing the expression level of VP1Nd129 and VP2 protein after the addition of IPTG, the results indicated that the VP1Nd129 protein gave a higher level of protein expression than VP2. The highest yields of VP1Nd129 and VP2 were 26.2 and 15.5 mg/L, respectively, after IPTG induction with 0.1 mM IPTG for 6 h, respectively. The identification of the optimized conditions for production of the CAV viral proteins VP1 and VP2 will allow them to be used in the future as an antigen for the development of vaccines and diagnostic tests.     

Low molecular weight silk proteins in Galleria mellonella

Galleria cocoon proteins have been extracted by different solubilizing agents. Nine protein bands were observed by gel electrophoresis, with molecular weights ranging from 18 to 420 kD. Three silk proteins of 24, 29 and 30 kD were extracted only in the presence of β-mercaptoethanol, suggesting that they are covalently linked by disulfide bonds to the large fibroin. They are likely to be the products of the highly abundant mRNA of the posterior silk gland cells. In vitro translation analysis of this mRNA yielded 24, 29 and 30 kD proteins. Thus, as in Bombyx, the Galleria silk is composed of several subunits, including fibroin and low molecular weight polypeptides. However, the genes coding for fibroin or low molecular weight silk proteins in Bombyx and Galleria do not show nucleotide base homology.     

Protein components of two strains of granulosis virus of the armyworm, Pseudaletia unipuncta (Lepidoptera,Noctuidae)

A synergistic Hawaiian (GVH) and a nonsynergistic Oregonian (GVO) strain of a granulosis virus (GV) infect the armyworm, Pseudaletia unipuncta. The protein components of the enveloped virions and of the capsule (inclusion body) were compared between the two strains. When the enveloped virions of both strains were analyzed by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis, the protein patterns were similar except for minor peaks in the higher molecular weight region. On the other hand, the capsule proteins differed between the two strains when examined with immunoelectrophoresis and SDS-polyacrylamide gel electrophoresis. The capsule proteins of GVH were composed of two major proteins, one a structural protein and the other the protein of the synergistic factor. The capsule protein of GVO, however, had only the structural protein. The rocket immunoelectrophoresis and affinity chromatography indicated that the structural proteins of the two strains were partially dissimilar. The molecular weight of the structural protein of GVO was 29,100 ± 500 and that of GVH was 28,700 ± 500. The amount of synergistic factor in a GVH capsule was about 5% of the dissolved capsule components.     

Proteins of Epstein-Barr virus. I. Analysis of the polypeptides of purified enveloped Epstein-Barr virus.

Epstein-Barr virus (EBV) was purified from the extracellular fluid of HR-1 and B95-8 cell lines. The preparations of purified virus consisted of enveloped particles and had EBV-specific antigneic reactivity. Comparison of the amount of labeled protein in preparations of virus purified from cultures incubated in [35S]methionine with the amount of labeled protein in preparations obtained following a mixture of unlabeled virus with [35S]methionine-labeled cellular proteins indicated that less than 2% of the labeled protein in the purified virus preparation could be attributed to contamination with labeled cellular proteins. No extraneous membranous material was seen in thin sections of the purified virus preparations. Analysis of the polypeptides of purified enveloped EBV indicated the following. (i) Eighteen polypeptides could be resolved in Coomassie brilliant blue-stained electropherograms of extracellular virus purified from HR-1 and B95-8 cultures. (ii) Thirty-three polypeptides could be resolved in fluorograms of labeled EBV purified from B95-8 cultures and subjected to electrophoresis in acrylamide gels cross-linked with diallyltartardiamide. The molecular weight of the EBV polypeptides was estimated by co-electrophoresis with the polypeptides of purified herpes simplex virus and purified polypeptides of known molecular weight to range from 28 x 10(3) to approximately 290 x 10(3) (iii) The polypeptides of EBV could be grouped by their relative molar abundancy into three classes: VP6, 7, and 27 present in high abundance; VP1, 12, 20, 23, and 29 present in moderate abundance; and a third class of less abundant polypeptides, VP4, 5, 8, 9, 10, 11, 15, 16, 21, and 22. The remainder of the polypeptides could not be precisely quantitated. (iv) The polypeptides of purified EBV, although similar in number and in range of molecular weight to the polypeptides of purified herpes simplex virus, differ sufficiently from those of herpes simplex virus so as to preclude comparison of individual polypeptide components.     

Characterization of an extremely basic protein derived from granulosis virus nucleocapsids

Nucleocapsids were isolated from purified enveloped nucleocapsids of Plodia interpunctella granulosis virus by treatment with Nonidet P-40. When analyzed on sodium dodecyl sulfate-polyacrylamide gels, the nucleocapsids consisted of eight polypeptides. One of these, a major component with a molecular weight of 12,500 (VP12), was selectively extracted from the nucleocapsids with 0.25 M sulfuric acid. Its electrophoretic mobility on acetic acid-urea gels was intermediate to that of cellular histones and protamine. Amino acid analysis showed that 39% of the amino acid residues of VP12 were basic: 27% were arginine and 12% were histidine. The remaining residues consisted primarily of serine, valine, and isoleucine. Proteins of similar arginine content also were extracted from the granulosis virus of Pieris rapae and from the nuclear polyhedrosis viruses of Spodoptera frugiperda and Autographa californica. The basic polypeptide appeared to be virus specific because it was found in nucleocapsids and virus-infected cells but not in uninfected cells. VP12 was not present in polypeptide profiles of granulosis virus capsids, indicating that it was an internal or core protein of the nucleocapsids. Electron microscopic observations suggested that the basic protein was associated with the viral DNA in the form of a DNA-protein complex.     

Electron cryo-microscopy and image reconstruction of adeno-associated virus type 2 empty capsids

Adeno-associated virus type 2 empty capsids are composed of three proteins, VP1, VP2 and VP3, which have relative molecular masses of 87, 72 and 62 kDa, respectively, and differ in their N-terminal amino acid sequences. They have a likely molar ratio of 1:1:8 and occupy symmetrical equivalent positions in an icosahedrally arranged protein shell. We have investigated empty capsids of adeno-associated virus type 2 by electron cryo-microscopy and icosahedral image reconstruction. The three-dimensional map at 1.05 nm resolution showed sets of three elongated spikes surrounding the three-fold symmetry axes and narrow empty channels at the five-fold axes. The inside of the capsid superimposed with the previously determined structure of the canine parvovirus (Q. Xie and M.S. Chapman, 1996, J. Mol. Biol., 264, 497–520), whereas the outer surface showed clear discrepancies. Globular structures at the inner surface of the capsid at the two-fold symmetry axes were identified as possible positions for the N-terminal extensions of VP1 and VP2.     

Radioiodination of the envelope proteins of newcastle disease virus

Lactoperoxidase-catalyzed iodination selectively labels the two glycoproteins (VP1 and VP2) of Newcastle disease virus. The low-molecular-weight, nonglycosylated major viral protein, VP6, was not iodinated in the intact virus but was iodinated in disrupted virions, suggesting a localization on the inner, rather than the outer envelope surface. Studies on the distribution of virion proteins labeled with ¹²⁵I and ³H-isoleucine between detergent-soluble and detergent-insoluble fractions show that the virion proteins VP4, VP5, and VP6 are solubilized to a much lesser extent than are VP1 and VP2.     

Codon Optimization,Expression in Escherichia coli,and Immunogenicity of Recombinant Chinese Sacbrood Virus (CSBV) Structural Proteins VP1, VP2, and VP3

Chinese sacbrood virus (CSBV) is a small RNA virus family belonging to the genus Iflavirus that causes larval death, and even the collapse of entire bee colonies. The virus particle is spherical, non-enveloped, and its viral capsid is composed of four proteins, although the functions of the structural proteins are unclear. In this study, we used codon recoding to express the recombinant proteins VP1, VP2, and VP3 in Escherichia coli. SDS-PAGE analysis and Western blotting revealed that the target genes were expressed at high levels. Mice were then immunized with the purified, recombinant proteins, and antibody levels and lymphocyte proliferation were analyzed by ELISA and the MTT assay, respectively. The results show that the recombinant proteins induced high antibody levels and promoted lymphocyte proliferation. Polyclonal antibodies directed against these proteins will aid future studies of the molecular pathogenesis of CSBV.     

Stable association of viral protein VP1 with simian virus 40 DNA.

Mild dissociation of simian virus 40 particles releases a 110S virion core nucleoprotein complex containing histones and the three viral proteins VP1, VP2, and VP3. The association of viral protein VP1 within this nucleoprotein complex is mediated at least partially through a strong interaction with the viral DNA. Treatment of the virion-derived 110S nucleoprotein complex with 0.25% Sarkosyl dissociated VP2, VP3, and histones, leaving a stable VP1-DNA complex. The VP1-DNA complex had a sedimentation value of 30S and a density of 1.460 g/cm3. The calculated molecular weight of the complex was 7.9 x 10(6), with an average of 100 VP1 molecules per DNA. Agarose gel electrophoresis of the VP1-DNA complex demonstrated that VP1 is associated not only with form I and form II simian virus 40 DNAs but also with form III simian virus 40 DNA generated by cleavage with EcoRI.     

Identification of Two Novel Members of the Tentative Genus Wukipolyomavirus in Wild Rodents

Two novel polyomaviruses (PyVs) were identified in kidney and chest-cavity fluid samples of wild bank voles (Myodes glareolus) and common voles (Microtus arvalis) collected in Germany. All cloned and sequenced genomes had the typical PyV genome organization, including putative open reading frames for early regulatory proteins large T antigen and small T antigen on one strand and for structural late proteins (VP1, VP2 and VP3) on the other strand. Virus-like particles (VLPs) were generated by yeast expression of the VP1 protein of both PyVs. VLP-based ELISA and large T-antigen sequence-targeted polymerase-chain reaction investigations demonstrated signs of infection of these novel PyVs in about 42% of bank voles and 18% of common voles. In most cases only viral DNA, but not VP1-specific antibodies were detected. In additional animals exclusively VP1-specific antibodies, but no viral DNA was detected, indicative for virus clearance. Phylogenetic and clustering analysis including all known PyV genomes placed novel bank vole and common vole PyVs amongst members of the tentative Wukipolymavirus genus. The other known four rodent PyVs, Murine PyV and Hamster PyV, and Murine pneumotropic virus and Mastomys PyV belong to different phylogenetic clades, tentatively named Orthopolyomavirus I and Orthopolyomavirus II, respectively. In conclusion, the finding of novel vole-borne PyVs may suggest an evolutionary origin of ancient wukipolyomaviruses in rodents and may offer the possibility to develop a vole-based animal model for human wukipolyomaviruses.     

Analysis of HCF, the cellular cofactor of VP16, in herpes simplex virus-infected cells

Herpes simplex virus (HSV) immediate-early (IE) gene expression is initiated via the recruitment of the structural protein VP16 onto specific sites upstream of each IE gene promoter in a multicomponent complex (TRF.C) that also includes the cellular proteins Oct-1 and HCF. In vitro results have shown that HCF binds directly to VP16 and stabilizes TRF.C. Results from transfection assays have also indicated that HCF is involved in the nuclear import of VP16. However, there have been no reports on the role or the fate of HCF during HSV type 1 (HSV-1) infection. Here we show that the intracellular distribution of HCF is dramatically altered during HSV-1 infection and that the protein interacts with and colocalizes with VP16. Moreover, viral protein synthesis and replication were significantly reduced after infection of a BHK-21-derived temperature-sensitive cell line (tsBN67) which contains a mutant HCF unable to associate with VP16 at the nonpermissive temperature. Intracellular distribution of HCF and of newly synthesized VP16 in tsBN67-infected cells was similar to that observed in Vero cells, suggesting that late in infection the trafficking of both proteins was not dependent on their association. We constructed a stable cell line (tsBN67r) in which the temperature-sensitive phenotype was rescued by using an epitope-tagged wild-type HCF. In HSV-1-infected tsBN67r cells at the nonpermissive temperature, direct binding of HCF to VP16 was observed, but virus protein synthesis and replication were not restored to levels observed at the permissive temperature or in wild-type BHK cells. Together these results indicate that the factors involved in compartmentalization of VP16 alter during infection and that late in infection, VP16 and HCF may have additional roles reflected in their colocalization in replication compartments.     

Antigenic and protein sequence homology between VP13/14, a herpes simplex virus type 1 tegument protein, and gp10, a glycoprotein of equine herpesvirus 1 and 4.

Monospecific polyclonal antisera raised against VP13/14, a major tegument protein of herpes simplex virus type 1 cross-reacted with structural equine herpesvirus 1 and 4 proteins of Mr 120,000 and 123,000, respectively; these proteins are identical in molecular weight to the corresponding glycoprotein 10 (gp10) of each virus. Using a combination of immune precipitation and Western immunoblotting techniques, we confirmed that anti-VP13/14 and a monoclonal antibody to gp10 reacted with the same protein. Sequence analysis of a lambda gt11 insert of equine herpesvirus 1 gp10 identified an open reading frame in equine herpesvirus 4 with which it showed strong homology; this open reading frame also shared homology with gene UL47 of herpes simplex virus type 1 and gene 11 of varicella-zoster virus. This showed that, in addition to immunological cross-reactivity, VP13/14 and gp10 have protein sequence homology; it also allowed identification of VP13/14 as the gene product of UL47.     

Expression Analysis of Bombyx mori Bidensovirus Structural Proteins and Assembly of Virus-like Particles in Insect Cells

Bombyx mori bidensovirus (BmBDV) is a new designated species of the new genus Bidensovirus in the new family Bidnaviridae, which contains two single-stranded linear DNAs (VD1 and VD2) and causes the chronic densonucleosis disease of silkworm. Previous researches revealed that VD1-ORF3 encodes the major structural proteins VPs. In this work, through western blot, we found that VPs expressed from 48 h post-inoculation and kept increasing until 120 h post-inoculation in midgut of Bombyx mori. In order to further investigate the translation of vp gene, the ORFs (vp1 and vp2) of the VP started just up-stream of the first two candidate initiation codons were expressed in Sf9 cells by a baculovirus expression system. The expression products were purified by gradient density centrifugation and analyzed by Western blot and electron microscopy. The results showed that the expressions of vp1 yielded three proteins (VP1, VP1′, and VP2), which are the same with the viral VPs expression in midgut of Bombyx mori, and vp2 generated two VPs with the molecular weights of about 51 kDa (VP2) and 37 kDa. The observation by electron microscopy indicated that these VPs can auto-assemble into virus-like particles that could not be distinguished from virus particles. These findings will provide materials for studying the structure of BmBDV and be helpful in the studies on BmBDV-based disease in silkworms.     

Genetically determined nonsusceptibility of the silkworm,Bombyx mori,to infection with a densonucleosis virus (Densovirus)

A nonsusceptible and a highly susceptible strain of the silkworm, Bombyx mori, to peroral infection with a densonucleosis virus (DNV) were studied by probit analysis. Tests with the susceptible and nonsusceptible parent strains, their reciprocal F₁ hybrids, the F₂ hybrid, and the backcrossed hybrids to either of the parents demonstrated that the nonsusceptibility of the silkworm to DNV was inherited and controlled by a recessive gene which was not sex-linked.     

Structural Proteins of Adenovirus-Associated Virus Type 3

Three major structural proteins were found in adenovirus-associated virus (AAV) type 3H virions which were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The molecular weights of the polypeptides were determined to be approximately 66,000 (VP1), 80,000 (VP2), and 92,000 (VP3). The component having a molecular weight of 66,000 comprised about 80% of the total virion protein in the major AAV-3H particle, and the other two components comprised about 10% each. Proteins of the same molecular weight were found in the minor dense AAV-3H virion, but the 80,000- and 92,000-molecular-weight components were present at about one-half the concentration. The AAV-3H virion contains about 72 molecules of VP1 and 8 and 7 molecules of VP2 and VP3, respectively.     

Resolution of simian virus 40 proteins in whole cell extracts by two-dimensional electrophoresis: heterogeneity of the major capsid protein.

The major capsid protein (VP1) of simian virus 40 (SV40) has been analyzed by two-dimensional electrophoresis. This system separates protein according to isoelectric point by isoelectric-focusing, and according to molecular weight by sodium dodecylsulphate electrophoresis (O'Farrell, 1975). VP1 synthesis in infected CV-1 cells can be monitored directly by analysis of unfractionated whole cell extracts; the resolution of VP1 from cellular proteins allows its detection as early as 13 hr after infection. The two-dimensional separation of VP1 reveals that it is heterogeneous, consisting of one major protein (molecular weight 47,000 daltons and isoelectric point of approximately pH 6.8) and five minor protein components. The minor forms of VP1 are 10% of the total VP1 and differ from the major form of VP1 both in molecular weight (by approximately 500 daltons) and isoelectric point (ranging from approximately pH 6.7 to pH 6.9). Evidence is presented to show that two of the minor forms are phosphorylated derivatives of VP1, and it is further suggested that all the different forms of VP1 are the result of modifications of the primary product of translation. A temperature-sensitive mutant of the BC complementation group (BC11) of SV40 results in the synthesis of VP1 with an altered electrophoretic mobility; both the major form of VP1 and the minor forms are shifted in their isoelectric points. In addition to the specific case of SV40, two aspects of these studies should be generally significant to investigators studying eucaryotic gene expression by two-dimensional gel electrophoresis: first, the genetic origin of a protein can be determined by a temperature-sensitive mutation which causes a charge change in the resultant protein; and second, two or more protein spots on a two-dimensional separation may be the products of a single gene.