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.     

Membrane (M) protein of HVJ (Sendai virus): its role in virus assembly.

The role in virus assembly of membrane or matrix (M) protein of HVJ (Sendai virus) was investigated. When virions were disrupted with alkali Tween 20, nucleocapsids with a sheathlike cover on the surface and a width of approx 33 nm were often obtained, and this sheathlike structure was composed of M protein. Selective aggregation of viral components in vitro was examined. Each viral component was isolated separately from virions by using detergents, and they were mixed with each other in various combinations. The structures which reaggregated were examined for their protein composition, and the results indicate that the nucleocapsid protein does not combine with viral glycoproteins to form a coaggregate unless M protein is present, which suggests that the M protein in vivo may be a mediator for the association of nucleocapsid with the modified areas of plasma membrane which will become viral envelope.     

Functions of a protein kinase activity associated with purified capsids of the granulosis virus infecting Plodia interpunctella

Activation of a protein kinase associated with purified capsids of the granulosis virus of Plodia interpunctella resulted in release of the DNA from the nucleocapsid as determined by electron microscopy. Heat treatment of the virions (65 degrees for 10 min) inactivated the kinase and prevented this uncoating event. The basic viral core protein, VP12, is the predominant phosphate acceptor for the protein kinase and was the only DNA-binding protein present in nucleocapsids. VP12 binding to 32P-nick-translated granulosis virus DNA was determined by the hybridization of the nick-translated DNA to nucleocapsid proteins transferred electrophoretically to nitrocellolose after separation by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Profiles obtained when nick-translated DNA was added to sucrose gradients in the absence and presence of VP12 substantiated the DNA-binding capability of VP12. Comparison of the DNA-binding capability of phosphorylated and nonphosphorylated VP12 using sucrose gradient sedimentation provided evidence that phosphorylation of the basic protein reduced its capability to bind DNA. We propose the endogenous protein kinase activity of the granulosis virus may function in two ways: release of the DNA from the nucleocapsid (uncoating), and decondensation of the DNA due to phosphorylation of the basic core protein, VP12.     

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.     

Interaction of white spot syndrome virus VP26 protein with actin

VP26 protein, the product of the WSV311 gene of white spot syndrome virus (WSSV), is one of major structural proteins of virus. In this study, when purified virions were treated with Triton X-100 detergent, VP26 protein was present in both the envelope and the nucleocapsid fraction. We have rationalized this finding by suggesting that VP26 protein might be located in the space between the envelope and the nucleocapsid. By using a fluorescent probe method, we have investigated the interaction between VP26 protein and some proteins of host cells. Three major VP26-binding proteins were purified from crayfish hemocytes by affinity-chromatography, in which the protein with an apparent molecular mass of 42 kDa was identified as actin by mass spectrometry (MS). Moreover, the association of VP26 protein with actin microfilaments was confirmed by coimmunoprecipitation.     

Studies of the molecular anatomy of the L-M cell strain of equine herpes virus type 1: proteins of the nucleocapsid and intact virion

The structural components of purified enveloped virions and of purified nucleocapsids of the tissue culture strain (L-M cell) of equine herpes virus type 1 (EHV-1L) were analyzed by discontinuous sodium dodecyl sulfate polyacryalmide gel electrophoresis. Enveloped virions were comprised of 28 structural proteins of average molecular weights ranging from 270,000 to 16,000. Twelve of the proteins exhibited molecular weights of greater than 100,000, and six of these were above 200,000. Utilizing radioactively labeled compounds (³H-glucosamine and ³H-choline), four glycoproteins, four lipoproteins, and nine glycolipoproteins were shown to be present in the virions. Purified nucleocapsids, isolated from nuclear extracts of infected L-M cells, contained five major structural proteins with average molecular weights of 148,000, 59,000, 46,000, 36,000, and 18,000. These five proteins comprised greater than 96% of total nucleocapsid protein, on the basis of radioactivity. The 148,000 MW protein (VP 9) accounted for approximately 65% of the total nucleocapsid protein and was the major structural protein of both nucleocapsids and intact virions. None of these proteins corresponded to glycoproteins or lipoproteins present in enveloped virions, indicating that glycoproteins and lipoproteins are components of the envelope. The remaining 4% consisted of eight structural proteins ranging from 140,000 to 30,000 MW and were judged to be minor structural components, reproducibly present in all preparations of nucleocapsids.     

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.     

Electron microscopic observation on a non-occluded baculo-like virus in shrimps

Summary.  A severe disease of farm-raised shrimp, Penaeus chinensis has spread throughout Chinese coasts since 1993. Recently, a baculo-like virus has been diagnosed as the causative agent for this epidemic disease. The electron microscopic observation of the virus is described. Thin sections of hepatopancreatic and hypodermic tissue of diseased P. chinensis showed many rod-shaped, enveloped, baculo-like virions in hypertrophied nuclei of infected cells. The virion was filled with a highly electron dense core. No occlusion bodies have been found. Negative stained intact virions, purified from infected tissues by centrifugation on sucrose discontinuous gradients, demonstrated that the viral envelops had been broken, but the cylindrical nucleocapsids could be observed clearly. The nucleocapsid of average 62 nm × 314 nm was composed of a helix system of capsomers, giving rise to an open stacked ring structure and repeating approximately every third ring. The number of repeating unit was 13 to 15. We propose that the virus described here could be designated as Non-Occluded Shrimp Virus (NOSV) or Penaeus chinensis Baculo-Like Virus (PcBLV). Received January 15, 1997 Accepted June 5, 1997     

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.     

Topography of phosphate residues in Sendai virus proteins

Phosphorylation is a common post-translational modification of Sendai virus proteins, occurring in all species except L. Phosphate residues in the envelope proteins FIN, F, and M were completely removed by the action of bacterial alkaline phosphatase on virions disrupted with nonionic detergent, whereas intact virions were entirely resistant to the enzyme. These results suggest that phosphate residues are located in domains of the native envelope proteins that lie within the virion. The phosphate residues in nucleocapsid proteins P and NP were also removed efficiently when intact nucleocapsids were treated with the phosphatase. Indeed, most of the phosphates in P and NP were located in the limited external domains that were cleaved from these protein species by controlled proteolysis. Phosphorylation of these domains may, therefore, regulate nucleocapsid functions and/or mediate electrostatic interactions with the nascent virus envelope during virion assembly.     

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.     

Phosphoproteins, structural components of rhabdoviruses

Polypeptides derived from purified rabies, vesicular stomatitis, and Kern Canyon viruses, which were labeled with ³H-amino acids and ³²P-phosphate, were subjected to electrophoretic fractionation in order to determine which of the structural proteins is phosphorylated. In the rabies virion, the nucleocapsid protein (N protein) was found to be phosphorylated, whereas the other three virus proteins were not. N protein of free viral nucleocapsids isolated from rabies virus-infected cells was also phosphorylated. The phosphorylation of the N protein of rabies virus is confined to a relatively small terminal segment of the polypeptide chain which can be specifically cleaved off by treatment of the nucleocapsid with trypsin. In vesicular stomatitis virus the minor NS protein component is the only phosphoprotein. In Kern Canyon virus the envelope glycoprotein and the N protein are both phosphorylated, but to a lesser extent than either the N protein of rabies virus or the NS protein of vesicular stomatitis virus. All three rhabdoviruses contain a virion-bound protein kinase. Free nucleocapsids derived from rabies virus-infected cells and purified by equilibrium centrifugation in CsCl solution also contain a protein kinase. Intracellular nucleocapsids of vesicular stomatitis or Kern Canyon viruses do not exhibit protein kinase activity.     

Early events in the infection of human B lymphocytes by Epstein-Barr virus: the internalization process

The early events in the infection of normal B lymphocytes and B lymphoblastoid cells by Epstein-Barr virus (EBV) were examined by electron and immunoelectron microscopy and by infectivity and inhibition studies. Purified EBV remained on the cell surface at 4 degrees and appeared as 250-nm ovoid particles in contact with the cell membrane through 50-nm envelope projections. Internalization of EBV in normal B lymphocytes into large (300-500 nm) uncoated vacuoles was initiated within 2 to 5 min at 37 degrees. At this stage approximately 1/3 of cell-associated virus was located in cellular invaginations while another 1/3 was in cell vacuoles. Direct fusion of EBV with the outer cell membrane was not observed. Instead, viral deenvelopment and nucleocapsid transit into the cytoplasm occurred from the large endocytic vesicles within 15 to 30 min at 37 degrees and did not involve lysosomal enzymes. During this time, the viral envelope became amorphous and its separation from the nucleocapsid was evident. After 60 to 90 min at 37 degrees, viral nucleocapsids were visualized in close proximity to the cell nucleus. Weak bases such as chloroquine, methylamine, and ammonium chloride retarded viral deenvelopment and fusion inside the endocytic vacuoles, resulting in abrogation of viral infectivity and accumulation of intact virions within cell vacuoles. These studies indicate that EBV enters normal B lymphocytes by a different endocytic pathway than the clathrin-receptosome-lysosome pathway utilized by many other ligands, including a number of viruses, to enter cells. In contrast to the pathway of entry into normal B lymphocytes, EBV entered B lymphoblastoid cells by direct fusion with the outer cell membrane within 2 to 5 min at 37 degrees.     

Topological organization of Sindbis virus capsid protein in isolated nucleocapsids

Sindbis virus nucleocapsids were isolated from mature virions by a two-step purification method. Detergent-treated virions were sedimented in sucrose gradients and the nucleocapsid peaks chromatographed on RNase-free Sephadex G-200. The purified nucleocapsids displayed several morphologies when examined in the electron microscope. These morphologies, and the results of double-angle shadowing, suggest that the core of this enveloped virus has the shape of a regular icosahedron with a triangulation number of 4. Peptide mapping of capsid protein obtained from nucleocapsids that had been radioiodinated by a variety of means, indicated that of the four tyrosine residues in the protein, only Tyr180 was exposed at the surface of the icosahedral structure. The other three residues were not exposed on the outer surface of the nucleocapsid shell, nor on the surface of capsid protein itself, implying that they were buried within the folded protein.     

Association of the Sendai virus C protein with nucleocapsids

Summary The subcellular localization of the nonstructural protein C of Sendai virus was investigated by means of indirect immunofluorescence microscopy of Sendai virus-infected cells, using an antiserum specific for C protein. In infected cells, C protein was detected exclusively in the cytoplasm as granular fluorescence, which coincided very well with the distribution of nucleocapsid protein NP and phosphoprotein P, which were also detected with specific antisera. This suggested that these proteins are present together in inclusions, probably forming nucleocapsids. In contrast, when the NP and C proteins were individually expressed in COS cells by transfection with expression plasmids containing cDNA for these proteins, their distribution patterns in the cytoplasm were found to be quite different from each other. Protein-blot analyses of purified virions revealed the presence of a significant amount of the C protein in virions, which indicated that C protein is integrated into virions. Under conditions in which most of the envelope-associated proteins, such as HN, F, and M, were removed from the virions by a detergent, the C protein remained tightly associated with the nucleocapsids — about 40 molecules per nucleocapsid.     

Morphology and infectivity of virus that persistently caused infection in an AGS cell line

A recent report has indicated that proteins and genes of simian virus 5 (SV5) are detected in a human gastric adenocarcinoma (AGS) cell line, which is widely provided for oncology, immunology, and microbiology research. However, the production of infective virions has not been determined in this cell line. In this study, the morphology and infectivity of the virus particles of the AGS cell line were studied by light and electron microscopy and virus transmission assay. The virus particles were approximately 176.0 ± 41.1 nm in diameter. The particles possessed projections 8–12 nm long on the surface and contained a nucleocapsid determined to be 13–18 nm in width and less than 1,000 nm in length. The virus was transmissible to the Vero cell line, induced multinuclear giant cell formation, and reproduced the same shape of antigenic virions. In this study, the persistently infected virus in the AGS cell line was determined to be infective and form reproducible virions, and a new morphological feature of SV5 was determined.     

Measles,canine distemper and respiratory syncytial virions and nucleocapsids. A comparative study of their structure,polypeptide and nucleic acid composition

Measles, canine distemper (CDV) and respiratory syncytial (RS) virus nucleocapsids were isolated and purified from infected cell cytoplasm, and virions were processed from the extracellular fluid of infected cell monolayers. Ultrastructurally the nucleocapsids possessed diameters of 17–18 nm with no differences noted between the RS nucleocapsids and other nucleocapsids examined. The nucleocapsids exhibited sedimentation values of from 200–300S and possessed buoyant density values of 1.29–1.30 gm/cc in CsCl. Direct chemical measurements and/or 280/260 nm absorption ratios, showed measles, CDV and RS nucleocapsids to contain 5% RNA. RNA, phenol extracted from [³²P]-labelled measles, CDV and RS nucleocapsids was found to be RNase A sensitive, while being DNase resistant. Base composition studies of the [³²P]-labelled RNA from these three viral nucleocapsids yielded values of 20–22 mole % for cytosine 21–23 mole % for guanine, 26–28mole % for adenine and 29–31 mole % for uridine. Uridine was always found to be the nucleotide in greatest abundance. Analysis by SDS-polyacrylamide gel electrophoresis (PAGE) of measles and CDV nucleocapsid obtained from infected cells dispersed without enzymes revealed them to contain a single polypeptide with molecular weights of 61000 and 54000 daltons, respectively. The polypeptide patterns of [³H]-fucose and [¹⁴C]-amino acid labelled measles and CDV virions were examined by SDS-PAGE and each was revealed to possess at least six polypeptides. At least one of the measles and two of the CDV polypeptides contained carbohydrate. These results demonstrate that measles, CDV and RS viruses have a number of properties in common with the better characterized members of the paramyxovirus group (NDV, Sendai and SV5).Presented at the workshop on molecular and pathogenetic aspects of measles virus, 9./11. April 1974, Belfast, Northern Ireland.     

Characterization of a coronavirus: I. Structural proteins: Effects of preparative conditions on the migration of protein in polyacrylamide gels

Coronavirus A59 possesses four size classes of structural proteins which have apparent molecular weights measured by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) of 23,000 (GP23), 50,000 (VP50), 90,000 (GP90), and 180,000 (GP180). VP50 is the only structural protein which is completely unaffected by protease treatment of intact virions. This species is the most highly labeled by polar amino acids such as glutamic acid and arginine and it is probably associated with the viral nucleocapsid. GP90, GP180, and GP23 are membrane-associated proteins. However, after protease treatment of virions, only 20% of the GP23 molecule is digested, whereas all of the GP90 and GP180 are removed. GP90 and GP180 appear to comprise most of the prominent layer of characteristic projections on the external surface of the viral envelope. The major portion of GP23 is presumed to lie within the lipid envelope, protected from protease digestion. GP23 and the protease resistant portion, p¹18, exhibit anomalous behavior on SDS-PAGE. After heating to 100° in SDS the electrophoretic mobility of these polypeptides is altered and several new forms of lower mobility are produced. β-Mercaptoethanol and dithiothreitol exaggerate the effects of heating.     

Isolation and comparative study of the nucleocapsids of measles and canine distemper viruses from infected cells

Nucleocapsids were isolated and purified from cells infected with measles and canine distemper virus (CDV). Electron microscopy of negatively stained nucleocapsids revealed the mean outside diameter was approximately 17-18 nm in each case and was not significantly different from similar measurements obtained with SV5 nucleocapsids and tobacco mosaic virus (TMV). All nucleocapsids also revealed central hollow cores which ranged from 4.3 to 5.2 nm. Direct chemical determinations showed that both CDV and measles virus nucleocapsids contained about 5% RNA. UV absorption spectra of measles and CDV nucleocapsids were typical for nucleoproteins and 280 260 nm ratios gave estimates of 5% RNA in each case. Buoyant density determinations in CsCl with both unlabeled and 32P-labeled nucleocapsids gave figures of 1.29-1.30 g/cc. Sedimentation analysis by analytical and sucrose gradient centrifugation revealed considerable heterogeneity, with nucleocapsids ranging from about 200-300 S. This heterogeneity was shown by electron microscopy to be due to the highly fragmented nature of the nucleocapsids. Analysis of CDV and measles nucleocapsids by SDS-polyacrylamide gel electrophoresis revealed a single major polypeptide when infected cells were harvested in the absence of proteolytic enzymes. These had estimated molecular weight of 54,000 (CDV) and 61,000 (measles). However, nucleocapsids prepared from cells harvested with the aid of trypsin or pronase contained two polypeptides (38,000 and 24,000 for measles; 27,000 and 22,500 for CDV), while similarly prepared SV5 nucleocapsid contained only a single polypeptide (44,500). These differences in polypeptide composition were correlated with differences in morphologic appearance of the nucleocapsids. Nucleocapsids harvested from cells in the absence of proteolytic enzymes had a loosely coiled, flexible appearance, while those harvested from cells treated with trypsin or pronase had a tightly coiled, rigid appearance. RNA was extracted from 32P-labeled measles and CDV nucleocapsids. Treatment with RNase A converted most of the 32P to a TCA-soluble form and DNase had no effect. Base compositions were determined on the 32P-labeled RNA and both viruses were rich in uracil, the molar ratios for the four bases were similar for each virus and were not significantly different from values reported in the literature for the better-characterized members of the Paramyxovirus group (NDV, Sendai and SV5).