Structural proteins of Marek's disease virus

Marek's disease virus (MDV) was propagated in roller-bottle cultures of duck embryo fibroblasts and partially purified by sucrose gradient centrifugation. Analysis of viral protein by polyacrylamide gel electrophoresis revealed that at least eight proteins (designated VPI-VPVIII) were present in MDV. The VPI is the major viral protein. At least two viral proteins, VPII and VPIV, with glucosamine label could be detected. These two peaks may represent viral glycoprotein and may be associated with the viral envelope. No host cell proteins coelectrophoresed with any viral proteins. Similar electropherograms were obtained by coelectrophoresis of MDV and herpes simplex virus proteins and of MDV and pseudorabies virus proteins.     

Respiratory syncytial virus glycoproteins

The proteins of respiratory syncytial (RS) virus were analyzed by SDS-polyacrylamide gel electrophoresis. Eight virion structural proteins with molecular weights of 180,000, 89,000, 48,000, 42,000, 34,000, 28,000, 25,000, and 21,000 were identified. These proteins were given tentative designations of L (180,000), G (89,000), F1 (48,000), NP (42,000), P (34,000), M (28,000), Vp25 (25,000), and F2 (21,000). The 89,000-, 48,000-, and 21,000-dalton polypeptides were glycosylated and could be purified on lentil-lectin sepharose columns. All three glycoproteins could be immunoprecipitated from extracts of infected cells but not from uninfected cells, suggesting that they are viral specified. The host cell affected the apparent molecular weights of the largest and smallest glycosylated polypeptides possibly by differences in glycosylation. The 48,000- and 21,000-dalton glycopolypeptides were disulfide linked subunits of a 68,000-dalton glycoprotein that was seen on unreduced gels. The 68,000-dalton glycoprotein was thus similar to the fusion (F) protein of paramyxoviruses. Treatment of infected cultures with tunicamycin, a drug that blocks glycosylation, inhibited syncytial formation and resulted in over a 1000-fold reduction of extracellular infectious virus. Virions purified from tunicamycin-treated cells had reduced amounts of all three glycosylated proteins. No new forms of these proteins were conclusively identified, suggesting that unglycosylated forms of RS glycoproteins were not incorporated into virion membranes.     

Respiratory syncytial virus proteins

Respiratory syncytial (RS) virus grown in BS-C-1 cells was concentrated from the fluid of infected cultures by precipitation with polyethylene glycol (PEG) and banded by isopycnic centrifugation in sucrose or metrizamide density gradients. At least six virus-specified polypeptide bands, one of which was heterogeneous, could be resolved by continuous SDS-polyacrylamide gel electrophoresis (PAGE) and an additional band by discontinuous SDS-PAGE. The three predominant viral polypeptides were a glycopolypeptide of 48 × 10³ (VGP48), a nucleocapsid polypeptide of 41 × 10³ (VP41), and a polypeptide of 27 × 10³ molecular weight (VP27). Three minor viral polypeptides have been assigned the molecular weight of 38 × 10³ (VP38), 32 × 10³ (VP32) and 25 × 10³ (VP25). A minor glycopolypeptide of molecular weight 42 × 10³ (VGP42) may exist also. Partial purification was accompanied by the loss of high molecular weight glycopolypeptides; however, one high molecular polypeptide (P2) remained consistently associated with the presumptive polypeptides and may represent an eighth virus-specified polypeptide.VP27 can be obtained in relatively pure form by sedimentation of detergent-treated RS virus in a metrizamide gradient containing detergent.     

Epstein-Barr Virus induced proteins V: Comparison of EBV-specific polypeptides from different virus strains

EBV-associated polypeptides induced in different Epstein-Barr Virus (EBV)-producing cell lines by the tumor promotor TPA, and from purified EBV particles derived from the same lines were radioactively labeled and analyzed by immunoprecipitation with human VCA⁺MA⁺ sera. In virus-producing cells no significant differences in the molecular weight of³⁵S-methionine-labeled EBV-associated polypeptide patterns could be observed. The analysis of¹²⁵I-labeled polypeptides from purified virus particles of four different strains revealed that, in addition to common polypeptides, individual EBV strains contain strain-specific high molecular weight glycopolypeptides. These polypeptides, constituting part of the membrane antigen complex, are present in varying amounts. While P3HR-1 virus particles contain a major component of 250 000 and small amounts of 340 000 molecular weight polypeptides, QIMR-WIL virus particles have more 340 000 than 240 000 molecular weight polypeptides. Furthermore, in B95-8 particles and in particles from an EBV strain isolated from an African green monkey (AGM-EBV) respectively, large amounts of 360 000 and 250 000 polypeptides could be observed. Since these glycopolypeptides carry strain-, subgroup- and group-specific antigenic determinants, also found in virus strains produced in human and marmoset cells, it should be further investigated whether these differences in molecular weight are virus-strain- or cell-specific.     

Analysis of herpes simplex virus type 1 antigens exposed on the surface of infected tissue culture cells.

Crossed immunoelectrophoretic analysis of herpes simplex virus type 1 (HSV-1) antigens solubilized from infected tissue culture cells has previously identified five viral specific precipitates: the type-common Ag-11, Ag-8, and Ag-3, the type-specific Ag-6, and Ag-7, which may be type specific. In the present study, the antigens exposed at the surface of infected cells were analyzed by peroxidase-catalyzed ¹²⁵I labeling of the cell surface at different times postinfection. The antigens were detectable in the following sequence: Ag11 (5 hr postinfection), Ag-8 (8 hr postinfection) and Ag-6 and Ag-7 (between 8 and 18 hr postinfection). Ag-3 was not exposed at the surface of infected cells and was not present in the HSV-1 virion. Polypeptide analysis of the individual HSV-1 antigens after iodination of the surface of infected cells showed that mainly the glycopolypeptides of the protein complexes were present at the surface. No immunochemical cross-reactivity between the viral and the host cell antigens was demonstrated even though sodium dodecyl sulfate-polyacrylamide gel electrophoretic analysis indicated that polypeptides of equal molecular weight were present in Ag-8 and the host cell membrane.     

Cell-free synthesis of equine herpesvirus type 3 nucleocapsid polypeptides.

Rate-velocity centrifugation in sucrose gradients of nuclear lysates from cells infected with equine herpesvirus type 3 (EHV-3) demonstrated two major species of viral nucleocapsids that differed in buoyant density, sedimentation velocity, and DNA content. Nucleocapsids present in the bottom sucrose band (B capsids) comprised 62% of the total nucleocapsid protein and possessed a higher density and seven times more DNA than nucleocapsids isolated from the top sucrose band (A capsids). Analysis of B-capsid proteins in SDS-polyacrylamide gels revealed the presence of five major polypeptides with molecular weights of 151,000, 59,000, 48,000, 40,000, and 30,000. In vitro synthesis of EHV-3 nucleocapsid polypeptides was demonstrated in a cell-free translation system consisting of (1) 150,000 g supernatant from rabbit reticulocytes; (2) preincubated ribosomes from the cells of a continuous equine cell line (ETCC); (3) factors dissociated from infected ETCC cell ribosomes with high salt; (4) RNA extracted from the cytoplasm of cells infected with EHV-3. EHV-3 nucleocapsid polypeptides were selected from the total in vitro synthesized products by indirect immunoprecipitation with specific antiviral rabbit serum and antiglobulin goat serum. Polyacrylamide-gel electrophoretic analysis of the cell-free products synthesized in response to poly(A)-rich RNA from EHV-3 infected cells and precipitated with immune serum prepared against purified viral nucleocapsids indicated that four of the five major nucleocapsid polypeptides were made in vitro.     

Purification of vaccinia virus-induced thymidine kinase activity from [35S]methionine-labeled cells.

Vaccinia virus thymidine kinase (TK) activity was purified from the cytosol of [³⁵S]methionine-labeled, virus-infected LM(TK-) mouse fibroblast cells. The purification procedure entailed (i) selective binding of TK activity to a Sepharose 4B-5′-amino-5′-deoxythymidine gel and elution of the enzyme from the enzyme-gel complex, (ii) preparative polyacrylamide gel electrophoresis (PAGE), and (iii) glycerol gradient centrifugation of the purified vaccinia virus TK activity. [³⁵S]Methionine-labeled cytosol fractions from uninfected cells were also mixed with nonlabeled vaccinia virus TK and purified by the same procedure to determine whether cellular proteins copurified with vaccinia virus TK. The purified vaccinia virus TK activity was adsorbed to an immunoadsorbent, made by coupling immunoglobulins (IgG) from antivaccinia virus TK rabbit antisera with CNBr-activated Sepharose 413. After elution of the labeled polypeptides from the immunoadsorbent, the eluates, as well as samples from earlier purification steps, were analyzed by SDS-polyacrylamide slab gel electrophoresis and autoradiography. Radioactive vaccinia virus TK that had been partially purified by analytical disc PAGE and by isoelectric focusing in polyacrylamide gels was also analyzed by SDS-polyacrylamide slab gel electrophoreses and autoradiography. The results of these experiments suggest that vaccinia virus TK consists of two subunits with molecular weights of about 40,000–42,000. Induction of the 40,000- to 42,000-dalton polypeptide was also observed in cells infected by vaccinia virus mutant 1004B, suggesting that this mutant induces the formation of an enzymatically inactive TK polypeptide.     

Immune response to herpes simplex virus infections: virus-specific antibodies in sera from patients with recurrent facial infections.

Radioimmunoprecipitation assays were used to identify antibodies against a number of herpes simplex virus type 1-specific antigens in serum samples from individuals with recurrent facial herpes virus infections and from seropositive individuals without recurrent infections. Individuals with recurrent infections contributed three sequential serum samples each: immediately after the appearance of lesions, 3 weeks later, and 3 months later. Antibodies against at least 18 viral polypeptides were present in all positive sera: these included antibodies against the major nucleocapsid polypeptide (approximate molecular weight, 150,000) and against two glycopolypeptides with molecular weights of 115,000 to 130,000. No significant differences were observed between the serum samples in regard to their virus-specific antibody composition. The high-molecular-weight glycopolypeptides were partially purified and used in quantitative titration experiments. All sera tested were equally reactive with this material. It was concluded that under the experimental conditions an individual's susceptibility to recurrent herpetic infections could not be correlated with quantitative or qualitative changes in the levels of virus-specific antibodies.     

Virion polypeptides and structure of SV 40

Summary Five virion polypeptides, VPI to VPV, have been identified in purified SV40 virus by polyacrylamide gel electrophoresis of which two, VPI and VPII, have been located in the virus capsid. The number of molecular subunits in VPI and VP II calculated from experimental data suggest that they represent the hexons and pentons, respectively, of a 72 unit icosahedral capsid structure.     

Studies on the formation of the influenza virus envelope

An investigation of the insertion of fowl plague virus-specified polypeptides into the plasma membrane of infected chick cells and their subsequent incorporation into virus particles is reported. Virus-specified haemagglutinin and neuraminidase glycopolypeptides and matrix polypeptide were incorporated into the plasma membrane of infected cells, and, although these polypeptides accumulated during infection to become major components of the plasma membrane, there was no apparent displacement of any host cell membrane protein as a result of virus infection. The haemagglutinin precursor and neuraminidase polypeptides were synthesized in close association with the rough endoplasmic reticulum and were at least partially glycosylated while associated with this structure. These polypeptides were subsequently transferred via the smooth endoplasmic reticulum to the plasma membrane where the precursor haemagglutinin polypeptide was cleaved to yield the two haemagglutinin polypeptides. Analyses of haemagglutinating and nonhaemagglutinating fractions of plasma membrane preparations from infected cells indicated that the virus haemagglutinin was localized in discrete regions of the membrane whereas the neuraminidase was more generally distributed throughout the membrane. The matrix protein appeared to be synthesized in close association with the plasma membrane and inserted directly into regions of the plasma membrane which contain virus haemagglutinin, and it is suggested that this process represents the final stage in the formation of the virus envelope prior to virus assembly.     

Structural polypeptides of the hamster strain of equine herpes virus type 1: products associated with purification

Equine herpes virus purified from viremic serum of infected hamsters, either in sucrose or potassium tartrate gradients, can be further separated into an enveloped form (EF) and a membranous top component (MTC) by centrifugation in high salt-Tris-EDTA gradients. The polypeptides of sucrose purified virions, MTC, EF virions and nucleocapsids (purified in Renografin-76 density gradients) were analyzed by discontinuous sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The EF virions were comprised of 28 polypeptides ranging in molecular weight from 276,000 to 16,000, 12 of them larger than 100,000. Sucrose purified virions and MTC were composed of a larger number of polypeptides, 50 and 38 respectively. Purified nucleocapsids contained five major structural proteins and six minor ones, these 11 nucleocapsid proteins ranged in molecular weight from 147,000 to 20,000.     

Identification of serotype ii infectious bursal disease virus proteins

Polyacrylamide gel electrophoresis (PAGE) of serotype II IBDV (OH and MO strains) purified from infected Vero cells resolved a previously undetected major viral polypeptide, VP2. The molecular weight (MW) of VP2 was different between the two strains of serotype II. It was 43.5 kDa in strain OH and 44 kDa in strain MO. This was higher than the MW of VP2 in SAL strain of serotype I IBDV which was 41 kDa. VPX (50 kDa), VP3 (33 kDa) and VP4 (30.5 kDa) were similar in both serotype II virus strains but were also of higher MW than VPX (48 kDa), VP3 (32 kDa) and VP4 (30 kDa) of SAL virus. VP1 (80 kDa) had the same MW in both serotypes.     

Coat protein and protease activity as in vitro translation products of potato carlavirus M RNA

Genomic-size RNA isolated from purified potato carlavirus M (PVM) was translated in both the reticulocyte and the wheat germ cell-free, messenger-dependent systems. The PVM RNA translated the same set of major products in both in vitro systems. The Mr values of the most prominent polypeptides observed consistently were 185,000 (P185), 147,000 (P147), 94,000 (P94), 87,000 (P87), 72,000 (P72), 67,000 (P67), 52,000 (P52), 46,000 (P46), 35,000 (P35) and 25,000 (P25). Relatively low amounts of a translation product of Mr 200,000 (P200) were often detectable in both systems. The P35 polypeptide displayed the same molecular weight and one-dimensional peptide map as the virus coat protein (CP), and was precipitated by antibodies raised against PVM and PVM CP. The kinetics of appearance of the in vitro synthesized polypeptides suggested that primary translation products of high molecular weight undergo post-translational proteolytic cleavage.     

A comparison of the polypeptides of four measles virus strains

The polypeptides of measles virions from four different sources have been analyzed and compared by polyacrylamide gel electrophoresis in both phosphate buffer and discontinuous Tris buffer systems. A similar pattern of six polypeptides was found with the four different virus strains. Only one polypeptide (G) was found to contain detectable amounts of carbohydrate label; this was the largest virion polypeptide with an estimated molecular weight of 80,000. Slight differences in electrophoretic mobilities of the nucleocapsid protein subunits (NP) were found among the different strains; with estimated molecular weights in the 60,000 to 62,000 range. Another polypeptide with a molecular weight of ～ 70,000 has been found to be associated with the nucleocapsids isolated from infected cells. The three remaining polypeptides identified have estimated molecular weights of 55,000, 42,000, and 37,000, and the smallest of these has been designated M, by analogy to other paramyxoviruses. The differences between the present results and previous findings with measles virus, and the possible relationship of measles virus polypeptides to those of other paramyxoviruses, have been discussed.     

Synthesis of Drosophila X virus proteins in cultured Drosophila cells

The genome of Drosophila X virus (DXV) is made up of two segments of dsRNA with molecular weights of 2.3 X 10(6) (A) and 2.2 X 10(6) (B). Agarose gel electrophoresis of RNA fragments produced by S1 nuclease digestion of partially denatured, purified segment A and B indicated that the two genome segments have different nucleotide sequences. The dose-response curve of virus infectivity was linear, indicating that the two genome segments reside in the same particle. The virions contained five polypeptides (VPs) that fell into three molecular weight size classes; large, 110K; medium, 49K and 45K; and small, 34K and 27K. Virus infection of Drosophila cells induced the synthesis of five infected cell polypeptides (ICPs); 110K, 67K, 49K, 34K, and 27K. Pulse-chase experiments and peptide mapping revealed that four of these (110K, 67K, 34K, and 27K) were primary gene products and that ICP 49 was generated by post-translational cleavage of ICP 67. The major capsid protein, VP 45, was cleaved from ICP 49; however, this cleavage was incomplete because both polypeptides were present in purified virus. The results suggest that the strategy for protein synthesis of DXV differs from that of other dsRNA viruses.     

Charge heterogeneity in polypeptides of negative strand RNA viruses

We surveyed charge heterogeneity in the polypeptides of three enveloped RNA viruses, Sendai virus, influenza virus (WSN strain), and vesicular stomatitis virus (VSV). Isoelectric focusing in polyacrylamide gel was followed by electrophoresis in a second dimension after denaturation of the polypeptides with sodium dodecyl sulfate. Nucleocapsid polypeptides P and NP of Sendai virus exhibited charge heterogeneity which may correspond to various extents of post-translational phosphorylation (R. A. Lamb and P. W. Choppin, 1977). The synthesis of Sendai virus polypeptides in infected cells. 111. Phosphorylation of polypeptides. Virology81, 382–397. Nucleocapsid polypeptide N of VSV was more homogeneous, whereas the NP polypeptide of influenza virus appeared to be too basic (isoelectric point greater than 8.0) to be resolved in the isoelectric focusing system employed. Glycosylated envelope polypeptides of all three viruses separated into multiple (3 to 8) acidic species with isoelectric points in the range of 4 to 5 for VSV glycopolypeptide G and 5 to 6 for the glycopolypeptides of Sendai virus and influenza virus. Although some of the heterogeneity in VSV glycopolypeptide G may stem from variations in content of N-acetyl neuraminic acid (NANA), a different basis for the heterogeneity is suggested by failure of extensive neuraminidase treatment to abolish it. Moreover, NANA is removed from the glycopolypeptides of Sendai virus and influenza virus by virus-specified neuraminidases. A possible relationship between content of amino sugars (other than NANA) and charge heterogeneity was suggested by the finding that the amount of radioactive glucosamine incorporated biosynthetically into G of VSV and HN of Sendai virus was greater in the more electropositive species. The functional consequences of this variety of post-translational modifications remain to be determined.     

Structural polypeptides of a canine parvovirus: Study by immunoadsorption and sequential analysis of infected cells

Summary The sequential appearance of three polypeptides, molecular weights (MW) 67,000, 70,000, 85,000 in cells infected with canine parvovirus (CPV) was studied by sodium dodecyl sulphate polyacrylamide gel electrophoresis. The viral origin of three polypeptides was confirmed by immunoadsorption using a specific antiserum, and polypeptides of similar MW were also demonstrated in purified CPV virions.A further polypeptide of MW 50,000 was also present in both infected cell extracts and purified virus. However the viral origin for this polypeptide could not be confirmed by immunoadsorption.With 2 Figures     

Isolation and analysis of virus-specific ribonucleoprotein of tobacco mosaic virus-infected tobacco

A ribonucleoprotein fraction (vRNP) of a characteristic buoyant density greater than the buoyant density of tobacco mosaic virus (TMV) particles has been isolated from infected tissue by Cs2SO4 density gradient centrifugation. The vRNP particles appear to be TMV specific because they are synthesized in the presence of actinomycin D and have RNAs identified as genomic and I-class subgenomic (apparent Mr 1.1-1.3 x 106 and 0.60.8 x 10(6)) RNAs by their electrophoretic mobility and hybridization to plasmid-bearing RNA sequences. Polypeptides of apparent molecular weights 17,500 (TMV coat), 31,000, 37,000, and 39,000 were major constituents of vRNP. Of the minor polypeptides, those of apparent molecular weights 70,000, 68,000, 55,000, and 25,000 had electrophoretic mobilities similar to mobilities of polypeptides found in a ribonucleoprotein preparation from uninoculated plants. vRNP from common TMV-infected plants, but not from plants infected with a mutant that did not form native coat protein, reacted with immunoglobins against TMV and TMV coat protein. Common TMV and its vRNP differed in the extent of reactivity toward the two immunoglobins, in electron microscopic appearance, and in the higher sensitivity of vRNP to ribonuclease.     

Characterization of the polypeptides synthesized in cells infected with a temperature-sensitive mutant derived from an HVJ (Sendai virus) carrier culture

Summary The intracellular synthesis of virus-specific polypeptides in cells infected with the wild-type virus of HVJ (HVJ-W) (haemagglutinating virus of Japan—the Sendai strain of parainfluenza 1 virus) and with a temperature-sensitive(ts) mutant (HVJ-pB) derived from an HVJ carrier culture has been analysed by polyacrylamide gel electrophoresis. At the permissive temperature (32° C), all of the known virus structural polypeptides were identified in cells infected with each strain of virus and in addition to the non-structural polypeptides B and C, another polypeptide at the region with a molecular weight of 26,000 to 27,000 (26 to 27K) could be detected in infected cells. At the non-permissive temperature (38° C), the synthesis of the polypeptide M was markedly restrained in cells infected with HVJ-pB, while other major virus polypeptides were present in approximately comparable amounts to cells infected with the wild-type virus. A non-structural polypeptide with a molecular weight of 105K was dominant ints mutant infected cells at higher temperatures and disappeared after temperature-shift from 38° to 32° C. The production of the non-structural polypeptides B and 27K was also temperature-sensitive. The molecular weights of the polypeptides B, M and 27K in HVJ-pB infected cells were larger than those of the corresponding polypeptides in HVJ-W infected cells. The synthesis of the M protein in HVJ-pB infected cells started just after lowering the incubation temperature and the newly made M protein was successfully incorporated into virus particles.With 4 Figures     

Immunoprecipitable polypeptides specified by varicella-zoster virus

Polypeptides encoded by varicella-zoster virus (VZV) in infected cell cultures have been identified by radioimmune precipitation techniques. Detergent-solubilized extracts of VZV-infected cells were reacted with highly specific VZV antisera raised in strain-2 guinea pigs immunized with sonicates of syngeneic virus-infected cells. Fractionation of the immunoprecipitates in acrylamide slab gels demonstrated an average of 16 polypeptides, which ranged in molecular weight from 32,000 to å200,000. These included the three major immunogenic glycoproteins (gp 62, gp 98, and gp 118) and a prominent higher molecular weight nonglycosylated polypeptide at 155,000. One of the [³⁵S]methionine-labeled polypeptides comigrated with purified actin. Not all polypeptides were visible in any one particular fluorogram, but comparative analysis of polypeptide profiles derived from electrophoreses performed with different gel concentrations and different crosslinkers (methylene-bisacrylamide and N,N-diallyltartardiamide) clearly established a consistent and reproducible pattern of radioactive bands. A low background of radio-activity was nonspecifically precipitated by the antigen-antibody-protein A complexes; however, with the exception of a common band comigrating with actin, the electrophoretic profiles representing virus-specific and nonspecific immunoprecipitates were easily distinguished.