Structural proteins of fixed rabies virus in the suckling mouse brain

Structural proteins of fixed rabies virus grown in the suckling mouse brain were analysed by SDS-polyacrylamide gel electrophoresis (PAGE). The relative content of G protein to L, N and M1 proteins of brain-grown virus was much lower when compared with that of the virus grown in chick embryo fibroblasts. The results indicate that nucleocapsid complexes, consisting of L, N and M1 proteins were the predominant antigens accumulating in the brain.     

Expression of Immunogenic Puumala Virus Nucleocapsid Protein in Transgenic Tobacco and Potato Plants

Transgenic plants, expressing recombinant proteins, are suitable alternatives for the production of relevant immunogens. In the present study, the expression of Puumala virus nucleocapsid protein in tobacco and potato plants (Nicotiana tabacum and Solanum tuberosum) and its immunogenicity was investigated. After infection of leaf discs of SR1 tobacco and tuber discs of potato cv. Desiree with the Agrobacterium strain LBA4404 (pAL4404, pBinAR-PUU-S) containing the 1302 bp cDNA sequence of S-RNA segment of a Puumala virus, transgenic tobacco and potato plants expressed the Puumala virus nucleocapsid protein under control of the cauliflower 35S promoter. The recombinant proteins were found to be identical to the authentic Puumala virus nucleocapsid protein as analyzed by immunoblotting. Expression of the nucleocapsid protein was investigated over four plant generations (P to F4) and found to be stable (1 ng/3 g dried leaf tissue). Transgenic tobacco plants were smaller compared to controls. The transformed potato plants were morphologically similar to control plants and produced tubers as the control potatoes. The S-antigen was expressed at a level of 1 ng protein/5 g and 1 ng protein/4 g dried leaf and root tissues, respectively, and remained stable in the first generation of vegetatively propagated potato plants. The immunogenicity of the Puumala virus nucleocapsid protein expressed in Nicotiana tabacum and Solanum tuberosum was investigated in New Zealand white rabbits. They were immunized with leaf extracts from transgenic tobacco and potato plants, and the serum recognized Puumala virus nucleocapsid protein. Transgenic plants expressing hantaviral proteins can thus be used for the development of cost-effective diagnostic systems and for alternative vaccination strategies.     

Association of influenza virus proteins with cytoplasmic fractions

Cytoplasmic extracts of chick embryo fibroblasts infected with fowl plague virus were separated into fractions containing smooth membranes, rough membranes, free ribosomes and polysomes, and a soluble fraction. Viral proteins were analyzed in these fractions by polyacrylamide gel electrophoresis. The hemagglutinin glycoproteins were found to be associated with rough and smooth membranes, and pulse-chase experiments revealed that the large glycoprotein HA migrates from rough to smooth membranes where it is cleaved into glycoproteins HA₁ and HA. The nonglycosylated envelope polypeptide M was located predominantly in smooth membranes, the nucleocapsid protein NP in a fraction of intermediate density, and the nonstructural polypeptide NS in the fractions containing ribosomes. A large amount of protein P was found in the soluble fraction.If glycoprotein synthesis was inhibited by glucosamine or deoxyglucose the predominant virus-specific component located on smooth membranes was protein HA₀ thought to be the unglycosylated or incompletely glycosylated polypeptide of glycoprotein HA. Like HA, HA₀ migrated from rough to smooth membranes where it was also cleaved into two fragments. These data show that polypeptide HA₀ has a high affinity for membranes and they further underline the close relationship between proteins HA and HA₀.     

Chemical crosslinking of bacteriophage phi 6 nucleocapsid proteins

phi 6 is a lipid-containing dsRNA bacteriophage of Pseudomonas syringae. Its nucleocapsid (NC) has common features with Reoviridae core particles. We report here the crosslinking of phi 6 NC proteins with cleavable 12-A span chemical crosslinker, dithiobis(succinimidyl propionate). The crosslinked complexes were analyzed in two-dimensional polyacrylamide gels or by using monoclonal antibodies to uncleaved protein complexes in one-dimensional protein gels. The NC surface protein (P8) forms a series of multimeric homopolymers. The phi 6 lytic enzyme, protein P5, is associated with P8 on the NC surface. The interior NC proteins P1 and P4, associated with the virus polymerase activity, are also in contact with the P8 shell. A P1 + P4 complex is also formed. Only one of the NC proteins (P7) did not easily form complexes with the other NC proteins. These results indicate a very closely packed P8 surface lattice with specific contacts to the internal NC proteins.     

Envelope proteins of vesicular stomatitis virions: accessibility to iodination

The glycoprotein and, to a lesser extent, the matrix membrane protein of intact vesicular stomatitis virions were specificially iodinated by oxidation with lactoperoxidase or chloramine T. The virion envelope provided an effective barrier against iodination of nucleocapsid proteins. Selective removal of glycoprotein by trypsin or Triton X-100 exposed the membrane matrix protein to somewhat more extensive iodination but the nucleocapsid N protein became only slightly more accessible to iodination; the nucleocapsid L and NS proteins remained unlabeled.     

Assembly of nucleocapsids with cytosolic and membrane-derived matrix proteins of vesicular stomatitis virus.

During budding of vesicular stomatitis virus (VSV), the viral matrix (M) protein binds the viral nucleocapsid to the host plasma membrane and condenses the nucleocapsid into the tightly coiled nucleocapsid-M protein (NCM) complex observed in virions. In infected cells, the viral M protein exists mostly as a soluble molecule in the cytoplasm, and a small amount is bound to the plasma membrane. Despite the high concentrations of M protein and intracellular nucleocapsids in the cytoplasm, they are not associated with each other except at the sites of budding. The experiments presented here address the question of why M protein and nucleocapsids associate with each other only at the plasma membrane but not in the cytoplasm of infected cells. An assay for exchange of soluble M protein into NCM complexes in vitro was used to show that both cytosolic and membrane-derived M proteins bound to virion NCM complexes with affinities similar to that observed for virion M protein, indicating that both cytosolic and membrane-derived M proteins are competent for virus assembly. However, neither cytosolic nor membrane-derived M protein bound to intracellular nucleocapsids with the same high affinity observed for virion NCM complexes. Cytosolic M protein was able to bind intracellular nucleocapsids, but with an affinity approximately eightfold less than that observed in virion NCM complexes. Membrane-derived M protein exhibited little or no binding activity for intracellular nucleocapsids. These data indicate that intracellular nucleocapsids, and not intracellular M proteins, need to undergo an assembly-initiating event in order to assemble into an NCM complex. Since neither membrane-derived nor cytosolic M protein could initiate high-affinity binding to intracellular nucleocapsids, the results suggest that another viral or host factor is required for assembly of the NCM complex observed in virions.     

Identification and location of the structural glycoproteins of a tissue culture-adapted turkey enteric coronavirus

Summary The Minnesota strain of turkey enteric coronavirus (TCV) was grown on a human rectal tumor (HRT-18) cell line in the presence of radiolabeled amino acids and glucosamine to analyse virion structural proteins. In addition to the 52,000 unglycosylated nucleocapsid protein, three major glycoprotein species were found to be associated with the viral envelope. A predominant glycosylated protein with a molecular weight of 22–24,000 represented the transmembrane matrix protein. Larger glycoproteins with apparent molecular weights of 180–200,000 (gp 200), 120–125,000 (gp 120) and 95–100,000 (gp 100) were associated to the characteristic large bulbous projections (peplomers) located at the surface of the virion. The gp 100 and gp 120 species apparently arose from a proteolytic cleavage of gp 200, as suggested by digestion studies with trypsin and chymotrypsin. An additional large glycoprotein with mol.wt. of 140,000 (gp 140), that behaved as a disulfide-linked dimer of a 66,000 molecule, was found to be associated to granular projections located near the base of the large peplomers. Digestion studies with trypsin, bromelain and pronase demonstrated that gp 140 was related to the hemagglutinating activity of the virus. An inner membranous sac or tongue-shaped structure could be visualized in the interior of the viral particles following treatment with pronase. In contrast, trypsin or chymotrypsin treatments resulted in evaginations (budding) on the virus surface. Progeny viral particles produced in TCV-infected cell cultures in the presence of tunicamycin lacked both types of surface projections, as demonstrated by electron microscopy and electrophoresis. The matrix protein also appeared to be reduced to its unglycosylated form, concomitant with a considerable loss of its antigenicity. Thus, with respect to its morphological and biochemical characteristics, TCV resembles viruses belonging to the group of mammalian hemagglutinating coronaviruses, but differs in that both types of envelope glycoproteins are N-glycosylated as in case of the avian infectious bronchitis virus.     

Protease treatment and chemical crosslinking of a Flavivirus: Tick borne encephalitis virus

Summary Tick-borne encephalitis virus was treated with pronase or thermolysin. The resulting particles were banded in sucrose gradients and analyzed for polypeptide composition. Both enzymes caused a reduction in particle density from 1.19 to 1.15–1.16 g/cm³. No loss of viral lipid or nucleic acid could be observed. SDS-polyacrylamidegel electrophoresis showed that only the core protein V₂ was unchanged whereas the envelope proteins V₃ and V₁ had disappeared from their original positions in the PAGE profile. Instead a new peptide(s) with molecular weight of 4000–6000 was found in which hydrophobic amino-acids were enriched.Crosslinking by dimethyl-3.3-dithiobispropionimidate (DTBP) made the virus resistent to solubilization of the envelope proteins by TX-100. This could be interpreted by the formation of a dense envelope protein network around the nucleocapsid preventing its liberation by TX-100. Some data however indicate that direct crosslinking of at least one of the envelope proteins with the core cannot be excluded.With 5 Figures     

Heterogeneity of envelope polypeptides among strains of venezuelan encephalitis virus.

Comparative virion polypeptide analysis of 29 strains of purified Venezuelan encephalitis virus by electrophoresis side by side on discontinuous, slab polyacrylamide gels revealed heterogeneity in electrophoretic mobility and apparent numbers of polypeptides. All strains had two common proteins: a capsid protein of 36,000 molecular weight, and an envelope glycoprotein of 50,000–51,000 molecular weight. Another glycoprotein ranged in molecular weight between 51,000 and 55,000. Eight strains yielded an additional glycoprotein peak in the range of 56,000–58,000; two others showed a small glycoprotein peak at 45,000–46,000. These polypeptide patterns were not influenced by the cell species (chick, hamster, monkey) in which the virus was cultured.     

Structural polypeptides of canine distemper virus

Summary 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 34×10³. The 85K polypeptide was glycosylated. It was interpreted to be equivalent to 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 59, 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.With 7 Figures     

MHC class II-restricted T-cell hybridomas recognizing the nucleocapsid protein of avian coronavirus IBV.

Mice were immunized with purified infectious bronchitis virus (IBV), strain M41. Spleen cells, expanded in vitro by stimulation with M41, were immortalized by fusion to obtain T-cell hybridomas, and two major histocompatability complex (MHC) class II (I-E)-restricted T-cell hybridomas were selected with specificity for IBV. Both hybridomas selectively recognized the internal nucleocapsid protein. The responses to 12 different strains of IBV varied markedly. This demonstrates antigenic variation of the nucleocapsid protein in addition to the known variation of the surface glycoprotein S.     

In situ cross-linking of vesicular stomatitis virus proteins with reversible agents.

Several reversible cross-linking agents were tested for their ability to cross-link the proteins of intact vesicular stomatitis virus (VSV). Results were analyzed by two-dimensional acrylamide gel electrophoresis in the sodium dodecyl sulfate-discontinuous buffer system. Formaldehyde cross-linking was found to be reversible by heat, and gave results similar to the reagent dimethyl-3,3′-dithio-bisproprionimidate (DTBP), cleavable by reduction of its internal disulfide bond. These two reagents caused the formation of cross-linked polypeptides with molecular weights consistent with the species N:M, G:M, and G:N, where N, M, and G stand for the VSV nucleoprotein, membrane protein, and glycoprotein, respectively. Other agents tested included o-phenanthroline in the presence of Cu²⁺ and O₂, and the heavy metal ions Cu²⁺ and Ni²⁺, all reversible by β-mercaptoethanol. These agents gave similar results: No cross-linking of G proteins was observed, and of the possible heterodimers, only N:M was found. NS, the phosphopolypeptide of VSV, was found in complexes corresponding in molecular weight to homodimer and homotetramer species, possibly indicating a tetrameric configuration of the native NS protein. The apparent molecular weights of cross-linked polypeptides were found to be about 20% greater than for linear polypeptides of the same molecular weight, when the mobility in acrylamide gels of a number of cross-linked species from native proteins, enzymes, and viral nucleocapsids was compared to the mobility of linear polypeptides. The accessibility of sulfhydryl groups in native and SDS-disupted VSV was assayed by reaction with radioactive iodoacetic acid; it was found that the G protein contained no free sulfhydryl groups, explaining its failure to react with o-phenanthroline/Cu²⁺ and heavy metal ions. Removal of the G proteins from the virus with nonionic detergent prior to cross-linking with DTBP showed the coordinate loss of both the G and the N spots corresponding in apparent molecular weight to the G:N heterodimer. This result provides evidence that the glycoprotein of VSV may be capable of interacting in some manner with the core nucleoprotein.     

Respiratory syncytial virus polypeptides: their location in the virion.

Purified respiratory syncytial (RS) virus contains, in addition to the six to seven polypeptides previously reported (S. Levine, 1977, J. Virol., 21, 427–431), a large polypeptide (VPO), MW > 160,000. Treatment of purified virus with trypsin removes the major glycoproteins, VP1 and 2. Treatment of purified virus with 2% Triton X-100 in HBSS (equivalent to 0.15 M NaCl) solubilizes the glycoproteins VP1 and 2 and a nonglycosylated protein, VP5, MW 28,000, which suggests that VP5 is an M protein. Treatment with 2% Triton X-100 in 0.4 M NaCl solubilizes all the virion proteins except VPO and VP3, which are also not solubilized in 0.8 M NaCl. The results suggest that VP3, MW 44,000, is the major nucleocapsid protein, and that VPO is not a superficial contaminant of the virus preparation, but instead is closely associated with the nucleocapsid. Only VP3 is present in nucleocapsids isolated from RS virus-infected cells by isopycnic centrifugation in CsCl.     

Radioimmunoassay of the major group specific protein of endogenous baboon type C viruses: relation to the RD-114-CCC group and detection of antigen in normal baboon tissues

The major group specific (gs) protein of the endogenous baboon type C virus M7 was purified to homogeneity by gel filtration and isoelectric focusing. The protein has a molecular weight of approximately 33,000, as determined by electrophoresis in polyacrylamide gels containing sodium dodecyl sulfate, and an isoelectric point (pI) of 7.1, different from the pIs of similarly purified gs proteins from six other mammalian type C viruses. Detergent disrupted M7 virus, whether grown in canine thymus or human rhabdomyosarcoma cells, fully displaced radiolabeled M7 gs protein from antigen-antibody complexes in a competitive radioimmunoassay. No antigenic differences were detected among the gs proteins of five independent isolates of baboon type C viruses grown in various cultured cell lines. The gs proteins of six independently isolated feline viruses of the RD-114/CCC group were antigenically related to, but could be distinguished from, the gs proteins of baboon type C viruses. No significant cross-reactions were observed in the radioimmunoassay for M7 gs protein using several other type C viruses, including two previously isolated from a woolly monkey and a gibbon ape. Group specific antigen was found in normal baboon testicular and splenic tissues using the M7 radioimmunoassay; no gs antigen could be detected in these same tissues using a radioimmunoassay for the gs protein of the woolly monkey type C virus. No gs antigen was found in baboon liver or in the tissues of several other primates.     

Evidence for a single structural polypeptide in foot-and-mouth disease virus

Summary Foot-and-mouth disease virus (FMDV) degraded at low ionic-strength as well as purified viral protein (E _{276 m} ^1% =11.1; max._{276 m}/min._{250 m} =3.0) were examined for heterogeneity by polyacrylamide gel electrophoresis. These preparations, each 8M in urea, 1% in sodium dodecylsulfate and 0.14M in mercaptoethanol (ME), all gave rise to a single, fast-moving protein zone when electrophoresed in 6% acrylamide gels not containing urea. In smaller pore-size gels (7.5 and 10%), slower-migrating zones were also present. When urea was present in the 6% gels, slower zones were always present when the protein was reduced with 0.14M ME. However, only the fast zone was seen after treatment with 4M ME. These findings indicate that the slower-migrating zones are due to discrete classes of aggregated protein and that FMDV structural protein is comprised of an electrophoretically homogeneous polypeptide.     

The synthesis of Sendai virus polypeptides in infected cells. III. Phosphorylation of polypeptides.

Phosphorylated and unphosphorylated forms of the membrane polypeptide (M) and the nucleocapsid polypeptide (NP) of Sendai virus have been identified in both infected cells and virions. Polypeptide B, found previously in infected cells, has been shown to be a phosphorylated form of M by peptide mapping, by conversion of M to B by phosphorylation in both pulse-chase experiments in infected cells and in vitro by a virion-associated protein kinase and, conversely, by conversion of B to M through the loss of phosphate in cell lysates. Although very little of the phosphorylated form was found in virions, B and M were found in similar proportions in infected cells after a 30-min pulse, suggesting that the nonphosphorylated form is preferentially incorporated into virions or that phosphate is removed during the maturation process. The finding of phosphorylation of M and NP in cells suggests that this may play a role in virus replication or assembly. Two phosphorylated forms of the nucleocapsid polypeptide (NP_P1 and NP_P2) have been found, but the unphosphorylated NP is the predominant form in both cells and virions. The similarity of these three polypeptides, except for their phosphate content, has been shown by peptide mapping. The membrane polypeptides B and M were separated by electrophoresis on polyacrylamide gels in the absence of urea, but in the presence of 4 M urea they comigrated. In contrast to these results with polypeptides B and M, polypeptides NP, NP_P1 and NP_P2 were resolved in gels in the presence of 4 M urea but comigrated in its absence. Thus, the behavior of viral phosphopolypeptides in different polyacrylamide-gel systems varies depending on the polypeptides. Possible biological roles of phosphorylation of the virus polypeptides are discussed.     

Myelin glycosphingolipid/cholesterol-enriched microdomains selectively sequester the non-compact myelin proteins CNP and MOG

Plasma membranes are complex arrays of protein and lipid subdomains. Detergent-insoluble, glycosphingolipid/cholesterol-enriched micro-domains (DIGCEMs) have been implicated in protein sorting and/or as sites for signaling cascades in the plasma membrane. We previously identified the presence of DIGCEMs in oligodendrocytes in culture and purified myelin and characterized a novel DIGCEM-associated tetraspan protein, MVP17/rMAL (Kim et al. (1995) Journal of Neuroscience Research 42, 413–422). We have now analyzed the association of known myelin proteins with DIGCEMs in order to provide a better understanding of their roles during myelin biogenesis. We used four well-established criteria to identify myelin DIGCEM-associated proteins: insolubility in a non-ionic detergent Triton X-100 at low temperature (4°C), flotation of the insoluble complexes to low density fractions in sucrose gradients, and TX-100 solubilization at 37°C, or at 4°C following treatment with the cholesterol-binding detergent saponin. We demonstrate that these proteins fall into four distinct groups. Although all tested proteins could be floated to a low-density fraction, proteolipid protein (PLP), myelin basic protein (MBP) and myelin associated glycoprotein (MAG) were solubilized by the detergent extraction, and connexin32 (Cx32) and oligodendrocyte-specific protein (OSP) met only some of the criteria for DIGCEMs. Only the non-compact myelin proteins 2,3-cyclic nucleotide 3-phosphodiesterase (CNP) and myelin/oligodendrocyte glycoprotein (MOG) satisfied all four criteria for DIGCEM-associated proteins. Significantly, only 40% of CNP and MOG were selectively associated with DIGCEMs. This suggests that they may have both non-active soluble, and functionally active DIGCEM-associated, forms in the membrane, consistent with current views that DIGCEMs provide platforms for bringing together and activating components of the signal transduction apparatus. We therefore propose that CNP and MOG may have unique roles among the major myelin proteins in signaling pathways mediated by lipid-protein microdomains formed in myelin.     

Assembly of Sendai virus: M protein interacts with F and HN proteins and with the cytoplasmic tail and transmembrane domain of F protein

Sendai virus matrix protein (M protein) is critically important for virus assembly and budding and is presumed to interact with viral glycoproteins on the outer side and viral nucleocapsid on the inner side. However, since M protein alone binds to lipid membranes, it has been difficult to demonstrate the specific interaction of M protein with HN or F protein, the Sendai viral glycoproteins. Using Triton X-100 (TX-100) detergent treatment of membrane fractions and flotation in sucrose gradients, we report that the membrane-bound M protein expressed alone or coexpressed with heterologous glycoprotein (influenza virus HA) was totally TX-100 soluble but the membrane-bound M protein coexpressed with HN or F protein either individually or together was predominantly detergent-resistant and floated to the top of the density gradient. Furthermore, both the cytoplasmic tail and the transmembrane domain of F protein facilitated binding of M protein to detergent-resistant membranes. Analysis of the membrane association of M protein in the early and late phases of the Sendai virus infectious cycle revealed that the interaction of M protein with mature glycoproteins that associated with the detergent-resistant lipid rafts was responsible for the detergent resistance of the membrane-bound M protein. Immunofluorescence analysis by confocal microscopy also demonstrated that in Sendai virus-infected cells, a fraction of M protein colocalized with F and HN proteins and that some M protein also became associated with the F and HN proteins while they were in transit to the plasma membrane via the exocytic pathway. These studies indicate that F and HN interact with M protein in the absence of any other viral proteins and that F associates with M protein via its cytoplasmic tail and transmembrane domain.