Major Structural Polypeptides Research Articles

Phosphoproteins of Spring Viremia of Carp virus

Tryptic peptide analyses of the structural polypeptides of Spring Viremia of Carp virus (SVCV) have established that its two principal species of virion phosphoprotein (NS1, NS2) are related to each other. The NS polypeptides can be distinguished by tryptic peptide analyses from the other major structural polypeptides of SVCV, namely L, G, N, and M. Limited proteolysis of these two viral NS polypeptides, labeled in vitro by [γ- 32P]ATP in reactions catalyzed by the virion protein kinase, or in vivo, have shown that the labeled oligopeptides derived from them can be differentiated from each other, suggesting that they differ with respect to their phosphorylation sites. For both in vitro-and in vivo-labeled NS1 and NS2 species the major phosphorylated amino acid is phosphoserine.

Cellular synthesis and modification of murine hepatitis virus polypeptides.

Mouse L fibroblasts infected with mouse hepatitis virus, MHV3, and radiolabelled with 35S-methionine, contained, in addition to the three major structural polypeptides, p24, p56 and p180, two additional ones, p22 and p50. Of these total five polypeptides, only three, p22, p56 and p180, were labelled in infected cells during a 2 min 35S-methionine pulse and are, therefore, presumed to be immediate translation products. Pulse-chases and chymotryptic peptide mapping experiments showed apparent precursor-product relationships between p56 and p50 and between p22 and p24. Protein synthesis in infected cells was synchronized at the initiation stage by pre-exposure to hypertonic medium. Using a 0.5 min pulse-10 min chase sequence, to limit incorporation of 35S-methionine to stretches of approx. 100 amino acids adjacent to translational initiation sites, it was found that all three polypeptides, p22, and p56 and p180 contained radiolabel. It is thus apparent that translation of the three major structural proteins (or precursors) is initiated independently rather than at a single site as in the cases of other positive-strand RNA viruses such as polio or Semliki Forest virus.

Autographa californica nuclear polyhedrosis virus phosphoproteins and synthesis of intracellular proteins after virus infection

Polypeptides and phosphoproteins isolated from nuclear and cytoplasmic fractions of TN-368-10 cells infected with Autographa californica nuclear polyhedrosis virus (AcMNPV) were analyzed by polyacrylamide gel electrophoresis. Likewise, AcMNPV extracellular virus and alkali-released virus were compared. ACMNPV extracellular virus possessed at least nine phosphoproteins while the alkali-released virus had about 14 phosphoproteins. The major structural polypeptide of AcMNPV, polyhedrin, was phosphorylated. Intracellular proteins of infected TN-368-10 cells were pulse-labeled with [35S]methionine and separated into nuclear and cytoplasmic fractions. At least 36 virus-induced polypeptides were detected in infected cells, and several polypeptides were found as early as 5 hr postinfection. The patterns of synthesis and appearance of infected cell polypeptides were complex, but polypeptide differences between nuclear and cytoplasmic fractions were detected. Twenty phosphoproteins labeled with 32P were also detected in the two fractions, and at least six had electrophoretic mobilities similar to virus-associated phosphoproteins.

The Polypeptides Induced in Drosophila Cells by a Virus of Heteronychus arator

SUMMARY The virus isolated from the black beetle, Heteronychus arator, is an icosahedral ribovirus with a diam. of 30 nm and contains one major structural polypeptide of mol. wt. 40000. The virus can be grown in Drosophila melanogaster cells and also titred in this line using a plaque assay method. Infected Drosophila cells pulsed with 35S-methionine in the presence of actinomycin D induced three virus polypeptides of mol. wt. 110000, 40000 and 8000. Short pulses, chases with excess non-radioactive methionine and pre-pulse treatments with protease inhibitors failed to demonstrate processing of virus-induced polypeptides.

A comparative study of the polypeptides of three iridescent viruses by N-terminal analysis, amino acid analysis, and surface labeling

Polyacrylamide gel analysis of the structural proteins of three types of iridescent viruses (2, 6, and 9) demonstrated that the purified virions had one major and more than 20 minor polypeptides. Surface labeling procedures performed on pure intact virions, using 125I in the presence of lactoperoxidase and chloramine T (at low iodine concentrations), demonstrated that the major and two or three minor polypeptides were located on the outside. The major structural polypeptide was isolated from each virus type by preparative polyacrylamide gel electrophoresis. Amino acid analysis indicated that this protein was very similar in the three iridescent viruses. The three polypeptides had an identical N terminal (proline). While the major polypeptide of each virus has a slightly different molecular weight as determined by polyacrylamide gel electrophoresis, the similarities in iodine labeling, N terminals, and amino acids suggests a common function for this protein.

Immunological relationships between homologous structural polypeptides of measles and canine distemper virus.

The major structural polypeptides of measles and canine distemper virus (CDV) were isolated by SDS-polyacrylamide slab gel electrophoresis. Rabbit hyperimmune sera directed against the isolated HA, P, NP, F and M polypeptides were prepared. In addition, rabbit hyperimmune sera directed against purified native internal components of measles virus and against purified virions of measles and CDV, untreated or treated with trypsin, were prepared. These sera were used to study the immunological relationships between measles and CDV in immune precipitation tests with 35S-methionine-labelled extracellular virions and intracellular virus polypeptides and by use of component fixation and mixed haemadsorption tests. An immunological relationship between the M polypeptides was demonstrated with sera against both native partially denatured antigens. A reciprocal immunological cross-reactivity between the NP polypeptides was demonstrated by the use of sera directed against native antigens. However, antisera directed against the NP polypeptides isolated from gels did not show any cross-reaction, indicating that they lacked antibodies against the shared antigenic determinants. The immunological cross-reactivity between envelope components of the two viruses was analysed. A close reciprocal immunological relationship between the F polypeptides was demonstrated by immune precipitation of labelled polypeptides. Immunological cross-reactivity between the HA polypeptides of the two viruses could not be demonstrated by use of the same technique neither with sera directed against denatured antigen nor with sera against native antigen when extracellular denatured 35S-methionine-labelled purified virions were used in immune precipitation. A slight cross-reactivity could, however, be demonstrated with sera against native antigens in immune precipitation tests with 35S-methionine-labelled intracellular antigens. It is concluded that all the major structural polypeptides of measles and CDV show varying degrees of reciprocal immunological cross-reactions. The M polypeptides of the two viruses appear most closely related, whereas the HA polypeptides show the most pronounced differences.

Structural polypeptides of canine distemper virus.

The structural polypeptides of two strains of canine distemper virus and the Lec strain of measles virus were analysed by SDS-polyacrylamide-slab-gel electrophoresis. One strain of canine distemper virus derived from a live vaccine (Convac, Dumex), contained six major structural polypeptides with mol.wt. of 85, 78, 59, 43, 41 and 34 x 10(3). The 85K polypeptide was glycosylated. It was interpreted to be equivalent ot the 79K glycoprotein of the measles hemagglutinin. The second strain, a rapidly growing variant of the Onderstepoort strain of canine distemper virus characterized by extensive syncytium forming cytopathic effects in tissue culture, contained the 5, 43, 41 and 34K polypeptides, but the 85 and 78K polypeptides were not present in detectable amounts. The 43K polypeptide was identified as cellular actin by limited proteolysis. By use of monospecific rabbit hyperimmune sera against each of the major structural polypeptides of measles virus, the 59, 41 and 34K structural polypeptides could be identified as nucleocapsid protein (NP), fusion (F) polypeptide, and the membrane (M) polypeptide, respectively. In neutralization tests with rabbit hyperimmune sera against each of the two strains, this Onderstepoort strain, which contained reduced amounts of the hemagglutinin glycoprotein, gave higher neutralization titers than the vaccine strain.

Biochemical characterization of Rift Valley fever virus

Rift Valley fever virus (RVFV) polypeptides were shown to share similar biochemical properties with members of the family Bunyaviradae. Electrophoretic analysis of RVFV revealed one nonglycosylated and two glycosylated major proteins with molecular weights of 25,000, 56,000, and 65,000, respectively. In addition, a 100,000 MW glycoprotein was found. The 25,000 MW protein was identified as the major nucleocapsid protein. The virion density in CsCl was 1.21 g/ml, while that of the nucleocapsid was 1.29 g/ml. The number and molecular weights of major structural polypeptides of several diverse RVFV isolates were identical. The presence of three RNA segments, large, medium, and small, with molecular weights of 2.7, 1.7, and 0.6 × 10 6, was demonstrated. The close relationships of RVFV proteins and RNA with reported molecular weights of some members of the Phlebotomus fever (PHL) group viruses were compatible with the serological cross-reactivity among these viruses. These findings support the classification of RVFV with the PHL group viruses in the family Bunyaviradae.

Alignment of the peptides derived from acid-catalyzed cleavage of an aspartylprolyl bond in the major internal structural polypeptide of avian retroviruses.

The major internal structural polypeptide (p27) of Rous sarcoma virus (RSV), and the analogous polypeptide (P27(0)) OF Rous-associated virus-O (RAV-O), an endogenous virus released spontaneously by some chicken cells) have been cleaved selectively at a single aspartylprolyl peptide bond to yield two fragments. The NH2- and COOH-terminal amino acid sequences of p27 and p27(0) and their mild acid-cleavage fragments have been determined. These results show the existence of an identical cleavage site and a similar NH2- and COOH-terminal amino acid sequence in both the polypeptides. Furthermore they indicate that the difference in the molecular weights of p27 and p27(0) results from an insertion of amino acids in the COOH-terminal peptide of p27(0) rather than a shift in the scission site of the precursor molecule.

Purification of Theiler's murine encephalomyelitis virus and analysis of the structural virion polypeptides: correlation of the polypeptide profile with virulence.

Theiler's murine encephalomyelitis viruses (TMEV) are separable into two groups based on their biological behavior: those highly virulent isolates which are unable to cause persistent infection and the less virulent isolates which regularly produce persistent central nervous system infection in mice. Two highly virulent and five less virulent TMEV were found to have the same buoyant density (1.34 g/ml) on isopycnic centrifugation and virion structure by electron microscopy. Negatively stained virus particles purified in Cs(2)SO(4) gradients appeared to have icosahedral symmetry and measured 28 nm in diameter. Mature virions were found to possess three major structural polypeptides, VP1, VP2 and VP3, in the range of 25,000 to 35,000 daltons, and a smaller fourth major polypeptide, VP4, of 6,000 daltons on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The precursor of VP2 and VP4, VP0, which is a minor polypeptide of mature picornavirus particles, was also identified. However, a slight but consistent difference in several of the capsid polypeptides between the highly virulent and less virulent TMEV was found. VP1 was slightly larger (34,000 versus 33,500 daltons) and VP2 was slightly smaller (31,000 versus 32,000 daltons) for the highly virulent strains compared to the same polypeptide species in the less virulent viruses. VP0 was also slightly smaller (35,500 versus 36,000 daltons) for the highly virulent isolates compared to their less virulent counterparts. Finally, trypsin which was used initially in our purification procedure resulted in preferential cleavage of a 2,000-molecular-weight fragment or fragments from VP1 of only the less virulent isolates.

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 [(35)S]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.

Measles virus-specified polypeptides in infected cells.

The synthesis of wild-type measles virus-specified polypeptides in Vero cells in pulse-chase experiments, in cells with synchronized protein synthesis by high salt concentration, and in the presence of proteolytic enzyme inhibitors was analyzed by polyacrylamide slab-gel electrophoresis. Six major (L, G, 2, NP, 5 and M) structural polypeptides were identified in infected cells. The results of pulse-chase experiments suggested that most of the structural polypeptides were synthesized at their final length. Polypeptide M was found to be sensitive to trypsin. In TLCK-treated cells its molecular weight was about 1000--2000 daltons higher than in untreated cells. A minor virus-specific polypeptide with a molecular weight of about 23,000 was found as a very faint and diffuse band. In addition, three nonstructural polypeptides with molecular weights of 65,000, 38,000 and 18,000 were also detected. The experiments with proteolytic enzyme inhibitors and with synchronized protein synthesis suggested that the polypeptide with a molecular weight of 65,000 might be a precursor of the structural polypeptide 5.

Characterization of purified structural proteins of murine mammary tumor virus

The major structural polypeptides of a type-B oncornavirus, murine mammary tumor virus (MuMTV), were isolated to homogeneity by ion-exchange and molecular sieving chromatography. Viral component proteins included the major envelope glycoproteins (gp68, gp55, and gp34) as well as the internal proteins (p28, p18, p14, and p12). Characterizations of these viral proteins included molecular weights, N-terminal amino acids, and quantitative amino acid analyses.

Restriction Maps of Five Autographa californica M NPV Variants, Trichoplusia ni M NPV, and Galleria mellonella M NPV DNAs with Endonucleases Sma I, Kpn I, Bam HI, Sac I, Xho I, and Eco RI

The restriction sites of Autographa californica nuclear polyhedrosis virus (AcMNPV) E2 DNA were mapped for the endonucleases SmaI, KpnI, BamHI, SacI, XhoI, and EcoRI. The restriction maps of four other AcMNPV variants, Trichoplusia ni (TnMNPV), and Galleria mellonella (GmMNPV) genomes were determined and compared to the endonuclease cleavage maps of AcMNPV E2 DNA. The viral structural polypeptides of AcMNPV variants S3, E2, S1, M3, and R9 were the same when analyzed by polyacrylamide gel electrophoresis. The major structural polypeptides of GmMNPV and TnMNPV had the same pattern in polyacrylamide gels as did AcMNPV structural polypeptides. GmMNPV and TnMNPV had several minor structural protein differences as compared with AcMNPV. AcMNPV variants, TnMNPV, and GmMNPV were distinct but with very similar genomes and protein structures.

Topography of a flexible ribonucleoprotein helix: protein-protein contacts in Sendai virus nucleocapsids

Contacts among the three polypeptide species in the flexible helical nucleocapsids of a paramyxovirus were examined with bifunctional protein cross-linking reagents. Polypeptides L and P, minor components of Sendai virus nucleocapsids implicated in viral RNA polymerase activity, were efficiently cross-linked into large complexes, indicating that they enjoy abundant contacts with neighboring protein molecules in the helix. Less reactivity was found in the case of the major structural polypeptide, NP; about half of all molecules of NP formed large cross-linked complexes, most of the rest remaining as monomers along with a small proportion of homodimers and low-order oligomers. Marked heterogeneity in the cross-linking reactivity of NP molecules, which may reflect the conformational quasi-equivalence inherent in a flexible helix, was indicated by the production of several conformers of homodimers and other low-order oligomers of NP, and by failure of the kinetics of NP cross-linking to conform to a simple statistical model of random polmerization. The validity of the statistical model was shown by cross-linking experiments with the rigid helical virus, tobacco mosaic virus.

Synthesis of Tacaribe viral proteins

The synthesis of Tacaribe virus-specific proteins in infected BHK-21 cells has been analyzed by polyacrylamide slab gel electrophoresis and fluorography. The two major structural polypeptides of the virion were observed above the host cell background by pulse labeling with amino acid or sugar precursors. In addition to the virion glycoprotein (G), another major virus-specific glycoprotein (MW = 70,000) was detected in infected cells. The nucleoprotein (N) was first detected in infected cells around 24–34 hr postinfection (pi). It appeared to be synthesized as a primary gene product, since no larger precursor was detected by short pulse labeling. The rate of synthesis of N increased until 48 hr pi followed by a slight decrease by 72 hr pi. The structural and nonstructural glycoproteins were detected around 48 hr pi and their rate of synthesis increased up to 60 hr pi. The nucleocapsid protein, the minor nonglycosylated virion protein (P), as well as the structural and nonstructural glycoprotein could be specifically precipitated from infected cells using serum from rabbits hyperimmunized with Tacaribe virions. These results as well as pulse-chase experiments experiments suggest that the nonstructural glycoprotein may be a precursor of the virion glycoprotein.

Proteins of equine herpesvirus type 3 I. Polypeptides of the purified virion

Enveloped virions of equine herpesvirus type 3 (EHV-3) were purified from the extracellular fluids of infected horse embryo fibroblast cell cultures (KyED) by sequential banding in dextran-10 and potassium tartrate density gradients. The preparations of purified virus consisted of enveloped herpesvirus particles with little extraneous cellular material demonstrable by electron microscopy or by overt addition of labeled cellular proteins prior to purification. Structural polypeptides of the purified EHV-3 virions were analyzed by electrophoresis in SDS-polyacrylamide slab gels cross-linked with N,N′-diallytartardiamide. Thirty-four polypeptides, ranging in molecular weight from 14 × 10 3 to 220 × 10 3 were resolved in Coomassie brilliant blue-stained electropherograms of the purified virions. Ten of these proteins were larger than 100 × 10 3 and two were larger than 200 × 10 3. A 148 × 10 3 capsid protein was a major structural polypeptide of the EHV-3 virion. Coelectrophoresis of the proteins of EHV-3 virions with those of the genetically unrelated equine herpes-virus type 1 (EHV-1) revealed a similarity in size range and number of the virion structural proteins; however, the molecular weights and proportional composition of the majority of EHV-3 polypeptides differed significantly from those of the virion proteins of EHV-1.

Protein structure of lymphocytic choriomeningitis virus: Identification of the virus structural and cell associated polypeptides

Lymphocytic choriomeningitis virus (LCMV) has been found to contain 3 major structural polypeptides. The largest of these was shown to be a nonglycosylated nucleoprotein (NP) of approximately 63,000 daltons which was found internally in the virion and was associated with a dense ribonucleoprotein complex. Two glycopeptides, termed GP-1 and GP-2, of approximately 54,000 and 35,000 daltons, respectively, were shown to be localized on the virion surface by proteolytic digestion of intact virus. Peptide maps of GP-1 and GP-2 indicated that they were independent polypeptides and that GP-2 was not a cleavage product of GP-1. Furthermore, the tryptic peptide map of NP was distinct from the two glycopeptides. Pulse-labeling studies of the intracellular synthesis of LCMV specific polypeptides in infected BHK-21 cells demonstrated synthesis of the nucleoprotein by 6 hr after infection at a multiplicity of infection of 10, corresponding to the beginning of log phase of viral replication. In cytosol extracts of LCMV-infected cells enhanced by immune precipitation an additional apparently nonstructural glycopeptide with a molecular weight of 74–75,000 was observed. This cell associated glycopeptide, designated GP-C, was readily detectable after 24–48 hr of infection, and appeared to accumulate with increasing time of incubation. While the relationship of GP-C to the virion glycopeptides GP-1 and GP-2 has not been determined, immune precipitation studies have shown that GP-C does not share antigenic determinants with NP. The probable relationships between the polypeptides of lymphocytic choriomeningitis virus both to the pathogenesis of virus induced immune diseases and to the structure of other arenaviruses have been discussed.

Amino-terminal amino acid sequence of the major structural polypeptides of avian retroviruses: sequence homology between reticuloendotheliosis virus p30 and p30s of mammalian retroviruses.

The major structural polypeptides, p30 of reticuloendotheliosis virus (REV) (strain T) and p27 of avian sarcoma virus B77, have been compared with regard to amino acid composition. NH2-terminal amino acid sequence, and immunological crossreactions. The amino acid composition of the two polypeptides is distinct, and a comparison of the first 30 NH2-terminal amino acids of REV p30 with that for the first 25 of B77 p27 yields only three homologous residues. In competition radioimmunoassays the polypeptides show no crossreactivity. A comparison of the amino acid composition and NH2-terminal amino acid sequence of REV p30 with those reported for several mammalian retrovirus p30s shows remarkable similarities. Both REV and mammalian p30s contain a large number of polar residues in their amino acid composition and show approximately 40% homology in the first 30 NH2-terminal amino acids. No crossreactivity could be observed, however, in competition radioimmunoassays between Rauscher murine leukemia virus p30 and that of REV. The observations reported here suggest a close evolutionary relationship between REV and the mammalian retroviruses.

The relative hydrophobicity of oncornaviral structural proteins

The interaction of the major structural polypeptides from the murine mammary tumor virus (MuMTV) and Rauscher murine leukemia virus (R-MuLV) with alkyl-agarose derivatives containing hydrocarbon arms of various lengths was examined. Both R-MuLV and MuMTV polypeptides were selectively adsorbed to octylimino (C 8)-agarose and decylimino (C 10)-agarose columns. Elution of proteins was accomplished using buffers containing 8.5 M ethylene glycol. In certain cases, polypeptides could only be removed from the columns by the addition of detergent to ethylene glycol-containing buffers. By examining the chromatographic behavior of polypeptides using (C 8)- and (C 10)-agarose columns and the conditions required for the elution of adsorbed proteins, we were able to determine the relative potential for hydrophobic interaction (degree of hydrophobicity) for each viral protein. The most hydrophobic polypeptide of MuMTV was found to be the glycoprotein gp34, while p15(E) and the gag precursor prp67 appeared to be the most hydrophobic of the R-MuLV proteins. The least hydrophobic polypeptides of MuMTV were p23 and p16, while those of R-MuLV were the two major internal proteins, p10 and p12. Four degrees of apparent hydrophobicity, from least hydrophobic to strongly hydrophobic, could be discerned by this procedure. The major core proteins (MuMTV p28, R-MuLV p30) of both viruses were slightly hydrophobic, while their major glycoproteins were moderately hydrophobic. The degree of hydrophobicity of oncornaviral polypeptides, as determined by hydrophobic chromatography, appeared to bear some relation to the different subviral components with which the proteins were associated. Our results indicate that hydrophobic chromatography may provide a new parameter for the characterization of oncornaviral polypeptides. This procedure should also prove applicable to the study of polypeptides from other viral systems.