Characterization of three species of nucleocapsids of equine herpesvirus type-1 (EHV-1).

Three distinct species of nucleocapsids of equine herpesvirus type-1 (EHV-1) were isolated from infected L-M cell nuclei. The particles were classified on the basis of their densities in Renografin gradients as Light (L; ? = 1.237 g/cc), Intermediate (I; ? = 1.244 g/cc), and Heavy (H; ? = 1.258 g/cc). Analysis of the three nucleocapsid species, radioactively labeled with an ³H-labeled or ¹⁴C-labeled amino acid mixture, by discontinuous SDS-polyacrylamide-gel electrophoresis revealed significant and reproducible differences in their structural protein compositions. H nucleocapsids were comprised of six major proteins (I, II, III, IV, IVa, and V) with respective molecular weights of 148,000, 59,000, 46,000, 37,000, 30,000, and 18,000; these proteins comprised 63.7, 9.3, 6.0, 10.8, 5.3, and 4.1%, respectively, of total protein. These six proteins were also present in I nucleocapsids, however nucleocapsid protein IVa (MW, 30,000) was present only as a minor component and comprised less than 1% of total protein. L nucleocapsids were comprised of only four of these major structural proteins, as proteins III (MW, 46,000) and IVa (MW, 30,000) were absent. In addition, several minor proteins, each comprising less than 1% of total nucleocapsid protein, were present in each nucleocapsid species.Electrophoretic analysis of nucleocapsids labeled with [³H]arginine indicated that proteins extremely rich in arginine are not present in these three species. Phosphoproteins both of enveloped virions and of nucleocapsids were identified by electrophoretic analysis of particles radioactively labeled with inorganic phosphate (H₃³²PO₄). Twelve virion structural proteins were phosphorylated in vivo; five of these, including the major virion phosphoprotein VP 10 (MW, 127,000), were envelope specific proteins. Four of these 12 viral proteins were also phosphorylated in nucleocapsids, however the pattern of phosphorylation of nucleocapsid proteins varied among the three species. The two major phosphoproteins of nucleocapsids were proteins III and IVa which are absent in L nucleocapsids; protein V is phosphorylated only in H nucleocapsids.Analysis of the DNA content of the three nucleocapsid species indicated that preparations of H nucleocapsids contain more DNA than do those of the I and L species. Electron microscopic analysis of the nucleocapsids supported these results, as L and I nucleocapsids lack a dense inner nucleoid structure characteristic of the H species.     

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.     

Equine cytomegalovirus: structural proteins of virions and nucleocapsids

Enveloped virions and nucleocapsids of equine cytomegalovirus (ECMV; equine herpesvirus type 2) have been purified from the supernatants and the nuclear extracts of infected rabbit kidney (RK) cells, respectively, and their structural protein compositions have been analyzed. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis revealed that ECMV nucleocapsids were composed of nine proteins (average molecular weights = 148K, 52K, 49.5K, 46K, 43.5K, 38.5K, 27K, 20K, and 18K), which together constituted 89% of the total nucleocapsid protein on the basis of incorporated 3H-labeled amino acids. The 148K protein comprised 47.3% of the total protein and thus appeared to be similar in molecular weight and proportional composition to the major capsid proteins of other herpesviruses. Purified virions were composed of 37 proteins whose average molecular weights ranged from 14K to greater than 200K. Three intense glycoprotein bands (83K, 78K, and 73.5K) as well as four less intensely labeled glycoproteins were detected in [3H]glucosamine-labeled virion preparations. At least 14 structural proteins were readily detected in extracts of infected cells which had been [35S]methionine labeled late in infection, and 11 of these were immunoprecipitated by rabbit antiserum against purified virions. The protein composition of ECMV differs substantially from those of equine herpesvirus type 1 and type 3 as well as from those of other herpesviruses.     

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 × 10³ to 220 × 10³ were resolved in Coomassie brilliant blue-stained electropherograms of the purified virions. Ten of these proteins were larger than 100 × 10³ and two were larger than 200 × 10³. A 148 × 10³ 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.     

Human parainfluenza virus 3: Purification and characterization of subviral components,viral proteins and viral RNA

A simple method was established that allowed large quantities of human parainfluenza 3 (PF3) virions to be isolated from tissue culture cells. The purity of the virus was sufficient for biochemical analysis of virion proteins. The density of PF3 virions was 1.18–1.20. Purified virions contained seven viral proteins with estimated molecular weights of: L, 180000; P, 83000; HN, 69000; NP, 66000; f₀, 60000; F₁, 51000; and M, 38000, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions. There were three phosphoproteins, P, NP and M, and two glycoproteins, HN and F (includes f₀ and f₁). F_1,2, the activated, cleaved, fusion glycoprotein (60000 Da), consisting of two disulfide-linked subunits, f₁ and F₂, was seen only under nonreducing conditions. Because of its small size (approximately 9000 Da) F₂ could be seen only on gels with high acrylamide concentrations. As in other enveloped viruses, cellular actin (43 000 Da) was present in purified virions. Several minor bands migrating between NP and M represented breakdown products of NP.Solubilization of the virion membrane in low salt buffer with non-ionic detergent resulted in the loss of HN and F. In high salt buffer, the M protein was also removed. Nucleocapsids isolated by CsCl centrifugation contained L, P, NP and small amounts of M. Nucleocapsids isolated in the presence of the ionic detergent, sarcosyl, contained only the NP protein. The density of nucleocapsids was 1.29–1.30. Genomic 50S RNA isolated from nucleocapsids had an estimated molecular weight of 5 × 10⁶.     

Protease degradation of Autographa californica nuclear polyhedrosis virus proteins

The Autographa californica nuclear polyhedrosis virus (Ac-NPV) occlusion bodies produced in vivo contained a protease which degraded occlusion body matrix protein and polypeptides of enveloped nucleocapsids during alkaline dissolution. The protease activity was required for complete dissociation of occlusion body matrix protein from the viral membrane during alkali liberation of the virus from occlusion bodies. In addition protease activity was required for the subsequent removal of the viral membrane from the nucleocapsids by Nonidet P40 treatments. Protease activity was not detected in occlusion bodies isolated from tissue culture cells or heat-treated (80 degrees , 45 min) occlusion bodies isolated from insect larvae. When occluded Ac-NPV virions were purified in the absence of protease activity, 26- polypeptides with molecular weights ranging from 115,000 to 15,000 were identified. The occlusion body matrix protein had an apparent molecular weight of 33,000.     

Phosphoproteins of adenovirus 2

Analysis of [³²P]orthophosphate-labeled extracts of adenovirus 2 (AD2)-infected HeLa cells revealed three major adenovirus-specific phosphorylated species, of molecular weights (MW) 100,000, 72,000, and 33,000. The 72,000 MW species became detectable as a phosphorylated entity at about 15 hr after infection, the 33,000 and 100,000 MW species, at about 20–30 hr after infection. A variety of chemical and enzymatic tests indicated that each of the ³²P-labeled species was a phosphoprotein, with the label esterified to serine and threonine residues. In the 100,000 and 33,000 MW proteins, the ³²P was associated primarily with serine residues; the 72,000 MW protein contained approximately equimolar amounts of phosphoserine and phosphothreonine. Digestion of each protein with trypsin, followed by two-dimensional fingerprint analysis, suggested that the 72,000 MW protein contains two, or possibly three, sites for phosphorylation and that the 100,000 MW protein may be multiply phosphorylated. Two of the phosphoproteins exhibited an apparent increase in molecular weight during a pulse/chase labeling experiment (32,000 to 34,000 MW; 70,000 to 74,000 MW) which may reflect the post-translational phosphorylation of those proteins. None of the three major phosphoproteins is a structural component of purified adenovirions. Subcellular fractionation of infected cell lysates with the detergents NP-40 and Tween 40-sodium deoxycholate revealed that the 100,000 MW protein is located primarily in the cytoplasm, the 72,000 MW protein primarily in the nucleus, and the 33,000 MW protein in the nuclear membrane. The subcellular distribution of each protein was identical whether the proteins were labeled with [³²P]orthophosphate or with radioactive amino acids. Analysis of the tryptic peptides of ¹⁴C-labeled amino acid-labeled proteins showed that many of the ¹⁴C-labeled tryptic peptides of the 33,000 MW protein were also present in the 100,000 MW protein. Why two such closely related proteins are sequestered in disparate regions of the infected cells is not known. Although ³²P-labeled Ad2 structural proteins could not be visualized above the background of host phosphoproteins in a crude lysate, electrophoretic analysis of purified adenovirus virions revealed one major phosphorylated protein, of molecular weight 66,000 (Protein III_a). With long exposures of the fluorograms, it was possible to detect nine additional phosphorylated virion products. Four of these (molecular weights 54,000, 39,000, 29,000 and 19,000) were present at low levels in the virion and have not previously been described. Subfractionation of the virions with pyridine revealed that two of these previously undescribed proteins (54,000 and 19,000 MW) are located in the DNA-containing core of the virion particle. The other five phosphoproteins [core V (48,500 MW), VI (24,000 MW), VII (18,500 MW), IX (12,000 MW), and XI (6000 MW)] were present in greater quantities, but only a small fraction of each of the molecules was phosphorylated.     

High resolution two-dimensional gel electrophoresis of structural proteins of baculoviruses of Autographa californica and Porthetria (lymantria) dispar

The structural polypeptides of baculoviruses of Autographa californica (AcMNPV) and Porthetria dispar (PdMNPV) were analyzed by two-dimensional (2-D) gel electrophoresis. Purified proteins were solubilized in urea-NP40 mix and separated by isoelectric focusing in the first dimension; electrophoresis in the presence of sodium dodecyl sulfate (SDS) separated proteins by molecular weight in the second dimension. Eighty-one acidic polypeptides ranging in molecular weight from 13,500 to 86,000 Da were resolved in AcMNPV enveloped virions. The predominant polypeptide had a molecular weight of 41,500 and was considered to be the major capsid protein. Nucleocapsids from AcMNPV were resolved into 64 polypeptides. At least 11 of the polypeptides, including most of the high molecular weight proteins, that were not resolved in nucleocapsids were considered to be envelope proteins. For PdMNPV enveloped virions, there were 95 acidic polypeptides ranging in molecular weight from 13,500 to 85,500. The predominant polypeptide had a molecular weight of 46,500 Da. Polyhedral proteins (polyhedrin) isolated from protease-inactivated polyhedra and separable into a single major polypeptide (approx. 31,000) on one-dimensional SDS-polyacrylamide gel electrophoresis were resolved into six polypeptides for both viruses. All six polyhedrin polypeptides had the same molecular weight, but their isoelectric points ranged from pH 5.3 to 5.9 for AcMNPV and from pH 5.7 to 6.2 for PdMNPV. These six polypeptides were also detected when protease-inactivated or noninactivated whole polyhedra were analyzed directly by 2-D electrophoresis. It is assumed that not all the observed baculovirus polypeptides were unique species. Some proteins, especially the polyhedrin polypeptides, appeared to be related and had altered mobilities as a consequence of post-translational modifications.     

Localization of P, NP, and M proteins on Sendai virus nucleocapsid using immunogold labeling

The distribution of NP, P, and M proteins on Sendai virus nucleocapsids purified from cells and virions were studied by immunogold staining using monoclonal antibodies. NP molecules were found uniformly along the entire length of both cytosol and virion derived nucleocapsids. This observation is in accord with the earlier proposals that NP molecules maintained the structural integrity of the nucleocapsid. The distribution of P in nucleocapsids derived from the cytosol differed from the distribution in those originating from virions. In nucleocapsids derived from the cytosol, P molecules occurred in 4 to 10 discreet clusters at varying locations along the length of the nucleocapsid. In contrast, on nucleocapsids derived from virions, P molecules were uniformly distributed over the entire length of the nucleocapsid. These observations suggest that the distribution of P depends on the functional state of the nucleocapsid. The occurrence of P clusters at different locations on intracellular nucleocapsids indicates that P is a mobile molecule; this suggestion is consistent with P's role in viral RNA synthesis. The distribution of the matrix (M) protein also depended on where the nucleocapsids were derived from. Large quantities of M protein were found along the entire length of nucleocapsids derived from the cytosol, while in virion nucleocapsids, many fewer molecules of M were observed. The large amounts of M on the nucleocapsids originating from the cytosol supports the hypothesis that M protein mediates the recognition between the nucleocapsid and the envelope glycoproteins.     

Unusual features of protein interaction in human immunodeficiency virus (HIV) virions

Summary The treatment of HIV-1 virions with ionic and nonionic detergents (NP 40, ocytlglucoside, Na deoxycholate) resulted in an effect unusual for enveloped viruses: instead of solubilization of glycoproteins, the core protein p24 was solubilized while envelope glycoproteins with other structural proteins were found in subviral particles. These date are consistent with a model of HIV structural organization in which glycoproteins are included in the matrix formed by the protein p17 and suggest that p24 is neither involved in the matrix nor closely bound to any viral proteins.     

Identification of two major virion protein genes of white spot syndrome virus of shrimp

White Spot Syndrome Virus (WSSV) is an invertebrate virus, causing considerable mortality in shrimp. Two structural proteins of WSSV were identified. WSSV virions are enveloped nucleocapsids with a bacilliform morphology with an approximate size of 275 x 120 nm, and a tail-like extension at one end. The double-stranded viral DNA has an approximate size 290 kb. WSSV virions, isolated from infected shrimps, contained four major proteins: 28 kDa (VP28), 26 kDa (VP26), 24 kDa (VP24), and 19 kDa (VP19) in size, respectively. VP26 and VP24 were found associated with nucleocapsids; the others were associated with the envelope. N-terminal amino acid sequences of nucleocapsid protein VP26 and the envelope protein VP28 were obtained by protein sequencing and used to identify the respective genes (vp26 and vp28) in the WSSV genome. To confirm that the open reading frames of WSSV vp26 (612) and vp28 (612) are coding for the putative major virion proteins, they were expressed in insect cells using baculovirus vectors and analyzed by Western analysis. A polyclonal antiserum against total WSSV virions confirmed the virion origin of VP26 and VP28. Both proteins contained a putative transmembrane domain at their N terminus and many putative N- and O-glycosylation sites. These major viral proteins showed no homology to baculovirus structural proteins, suggesting, together with the lack of DNA sequence homology to other viruses, that WSSV may be a representative of a new virus family, Whispoviridae.     

Structural polypeptides of the American (VR-2332) strain of porcine reproductive and respiratory syndrome virus

Summary The structural polypeptides of the isolate VR-2332 of porcine reproductive and respiratory syndrome virus were analyzed in sucrose gradient-purified virions. The virus had an average density of 1.15 g/cm³ and contained, by SDS-PAGE, three major polypeptides with apparent molecular weights of 15, 19 and 26–30 kDa, which were designated as nucleocapsid (N), matrix (M) and envelope (E), respectively. The predominant structural protein was N. N-glycosidase F digestion only affected E whereas O-glycosidase or endoglycosidase H digestion had no effect, suggesting that the viral glycoproteins contain only complex N-linked carbohydrates.     

Neutralizing antibodies detect a disulfide-linked glycoprotein complex within the envelope of human cytomegalovirus

The specificity of neutralizing antibodies for human cytomegalovirus (CMV) envelope proteins was studied by comparing the reactivity of human CMV immune sera with that of a group of CMV-specific monoclonal antibodies. Characterization of this group of monoclonal antibodies revealed that six antibodies bound intact virions, and four of these antibodies neutralized infectious virus in vitro. All of the monoclonal antibodies, as well as human immune sera, precipitated three virion glycoproteins of estimated molecular weights of 160-K 116K, and 55K. Human immune sera also precipitated proteins of estimated molecular weights of 200K, 145K, 100K, 66K, and 34K. The three envelope glycoproteins detected by both the neutralizing monoclonal antibodies and immune human sera were shown to exist as a covalently linked, disulfide-bonded protein complex within virions. This result provided an explanation for the reactivity with multiple proteins of such highly specific reagents as monoclonal antibodies. Furthermore, these findings suggested that determinant(s) detected by CMV-neutralizing antibodies were expressed by this complex of envelope proteins.     

Identification of a nucleocapsid protein as a specific serological marker of human herpesvirus 6 infection.

Enveloped whole virions and nucleocapsids of human herpesvirus 6 (HHV-6) strain Z29 were purified from supernatant fluids of infected human cord blood lymphocytes by filtration through polyvinylpyrrolidone-treated filters, banding on a Nycondenz step gradient, and centrifugation through two successive continuous sucrose gradients. More than 20 proteins ranging in molecular weight from less than 30,000 to more than 200,000 were identified in preparations of purified whole virions labeled with [35S]methionine and [35S]cysteine. Immunogenic virion proteins of HHV-6 were identified in immunoblot assays with human immune sera, immune sera generated from mice immunized with purified whole virions or purified nucleocapsids, and a monoclonal antibody generated from a mouse immunized with purified nucleocapsids. The sera and the monoclonal antibody reacted strongly with a 101-kilodalton protein in the immunoblots, suggesting that the protein is a component of the nucleocapsid. Human sera lacking HHV-6-specific antibodies and seropositive for one or more of the other human herpesviruses failed to react with this protein, indicating that it is a specific serologic marker for HHV-6 infection.     

Electrophoretic analysis of the proteins of different alphavirus strains

Proteins of various alphavirus strains: Venezuelan equine encephalomyelitis, Semliki Forest, and Sindbis, were studied by a high resolution polyacrylamide gel electrophoresis. In addition to structural C, E1 and E2 proteins, the infected cells were found to contain a number of nonstructural polypeptides: B, 83 kD (analogue of nsP2), 75 kD (nsP3), PE2, precursor of E2 and a product of modification of nucleocapsid protein C27. In virions, in addition to the main structural polypeptides, protein components with molecular weights about 100 kD were found which, most likely, were aggregates of E1/E2 glycoproteins, and 40 kD, a product of E1 protein degradation. The alphavirus strains under study differed both in the electrophoretic mobility of the above-mentioned virus-specific polypeptides and in the stability of B and PE2 proteins in the infected cells. The intracellular stability of polyprotein B depended considerably also on the host cell.     

Structural components of mouse mammary tumor virus. I. Polypeptides of the virion.

Mouse mammary tumor virus (mMTV) obtained from MJY-alpha cell cultures and analyzed by polyacrylamide gel electrophoresis possesses four major polypeptides and six to eight minor components. Three of the major proteins, with estimated molecular weights of 60,000, 52,000 and 37,000, are glycoproteins, as demonstrated by labeling with [³H]glucosamine. Labeling with [³⁵S]sulfate revealed significant amounts of sulfate associated with the 60,000 (gp60) and 52,000 (gp52) glycoproteins, whereas sulfate was minimally incorporated into the 37,000 (gp37) dalton glycoprotein. At least three glycopeptide species containing both [³H]glucosamine and [³⁵S]sulfate labels were obtained after extensive Pronase digestion of mMTV, indicating that [³⁵S]sulfate is covalently linked to the carbohydrate component of mMTV glycoproteins. Variations in the polypeptide pattern of mMTV were observed when virions were grown and labeled under different culture conditions. The amount of gp60 decreased substantially, with an apparent increase in a minor polypeptide at 33,000 daltons, if virions were harvested from stationary cultures or if culture medium was not changed daily during viral harvests. When virions containing gp60 were incubated with medium from stationary cultures or with stationary cell layers, the level of gp60 decreased with a corresponding increase in the amount of radioactivity at 33,000 daltons. Cleavage of gp60 and gp52 was demonstrated by treatment of purified mMTV virions with trypsin or alpha-chymotrypsin (1–150 μg/ml) for 1 min to 3 hr. Virions were morphologically unaltered after incubation with either protease; however, both gp60 and gp52 were absent from the polypeptide patterns. The data suggest that gp52 is cleaved to form 33,000, 22,000, and possibly 37,000 dalton glycoproteins which remain associated with virions. Other mMTV virion polypeptides were resistant to treatment with protease.     

Baculovirus structural polypeptides

The structural polypeptides of eight insect baculoviruses were studied using vertical slab polyacrylamide gel electrophoresis. All viruses revealed a complex but unique composition of 15 to 25 bands with molecular weights ranging from 15,000 to 160,000. Since certain baculoviruses have more than one nucleocapsid per viral envelope (multiples), comparisons were made of the multiples and singles (enveloped single nucleocapsids) for each virus. Quantitative and qualitative differences were documented to exist in polypeptide composition. Where possible, the envelopes of certain baculoviruses were selectively removed in order to identify the major capsid proteins. Autographa californica MNPV (NPV: nuclear polyhedrosis virus) capsids contained two major polypeptides, VP18.5 and VP37. Rachiplusia ou MNPV capsids contained several polypeptides of which VP16, VP17, VP18, VP30, and VP36 were considered major constituents. Anticarsa gemmatalis MNPV contained one major capsid protein, VP29, and several minor polypeptides. Major capsid proteins of Heliothis zea SNPV were VP16, VP28, and VP63; as were VP16, VP17, and VP31 of Trichoplusia ni granulosis virus (GV).