Structural proteins of La Crosse virus.

Preparations of La Crosse virus, a member of the California encephalitis group of bunyaviruses, were found to possess three major virion proteins. Two of the proteins were glycosylated (G1 and G2) and were located on the surface of the virus particles. These two glycoproteins were present in equimolar amounts and possessed apparent molecular weights of 120 X 10(3) and 34 X 10(3). Virion nucleocapsids, isolated by a nonionic detergent and salt treatment, contained another major protein, N (molecular weight = 23 X 10(3)). A large, but minor, protein species L (molecular weight = 180 X 10(3)) was also found in virus preparations. The approximate number of protein molecules per virion has been determined. Electron microscopy of purified La Crosse virus indicated that the virus particle (mean diameter, 91 nm) is enveloped and possesses irregular surface projections (length, 10 nm).     

Virion RNA species of the arenaviruses Pichinde, Tacaribe, and Tamiami.

The principal RNA species isolated from labeled preparations of the arenavirus Pichinde usually include a large viral RNA species L (apparent molecular weight = 3.2 X 10(6)), and a smaller viral RNA species S (apparent molecular weight = 1.6 X 10(6)). In addition, either little or considerable quantities of 28S rRNA as well as 18S rRNA can also be obtained in virus extracts, depending on the virus stock and growth conditions used to generate virus preparations. Similar RNA species have been identified in RNA extracted from Tacaribe and Tamiami arenavirus preparations. Oligonucleotide fingerprint analyses have confirmed the host ribosomal origin of the 28S and 18S species. Such analyses have also indicated that the Pichinde viral L and S RNA species each contain unique nucleotide sequences. Viral RNA preparations isolated by conventional phenol-sodium dodecyl sulfate extraction often have much of their L and S RNA species in the form of aggregates as visualized by either electron microscopy or oligonucleotide fingerprinting of material recovered from the top of gels (run by using undenatured RNA preparations). Circular and linear RNA forms have also been seen in electron micrographs of undenatured RNA preparations, although denatured viral RNA preparations have yielded mostly linear RNA species with few RNA aggregates or circular forms.     

Synthesis and Cleavage of Influenza Virus Proteins

The NWS strain of influenza virus grows rapidly in and kills the MDCK dog kidney cell strain. Within 1 to 2 hr, the virus inhibits host cell protein synthesis and for 3 to 4 hr more it directs the synthesis of influenza virus proteins at a rate about twice that of uninfected cell synthesis. The rates of virus ribonucleic acid (RNA) and protein synthesis reach a maximum within the first few hours after infection and then drop. Plaque assays exhibit a linear dose-response, indicating that only one virion is necessary for productive infection. We have confirmed earlier reports regarding the fragmented nature of the RNA genome of purified influenza virions. However, high resolution gel electrophoresis indicated that each size class of viral RNA is heterogenous, so that there are at least 10 and probably more fragment sizes of RNA in these virions. Repeated attempts to detect infectivity in preparations of extracted viral RNA were completely negative (over a 10(8)-fold loss of infectivity after extraction). Even infection of the "infectious" RNA-treated cells with intact, related, influenza viruses failed to support infectivity of the isolated RNA or to rescue a host range genetic marker of the RNA. Purified influenza virions exhibit only three major protein peaks based on separation according to molecular weights. These three major virion proteins are the only major virion proteins synthesized in infected cells. This is true throughout the infectious cycle from several hours after infection until the cells are dying. However, the molecular weight of these virion proteins differs slightly depending upon the cell type in which the virus is grown. No host membrane proteins are incorporated into the virions as they bud through the cell membrane. Pulse-chase labeling early after infection or prolonged chase experiments indicate that influenza virus proteins are cleaved from one or more precursor polypeptides. In fact, each of the three major peaks seems to be a heterogeneous mixture of polypeptides in various stages of cleavage. Peptide analysis confirms that the three major peaks share common peptides, but the exact precursor product relationships are not clear. There may be one or several precursor proteins. Also there could be overlapping messenger RNA molecules of varying length giving rise to polypeptides of various sizes and overlapping sequences. Late in infection, amino acid labeling shows a preponderance of internal nucleocapsid protein synthesis, indicating that either this protein is much more stable to cleavage in infection or it is made from a more stable messenger. There is no obvious relationship between virion RNA fragments and viral protein sizes, so these fragments may be artifacts.     

Structural components of influenza C virions.

The genome RNA species of influenza type C virions were analyzed by polyacrylamide gel electrophoresis. The pattern obtained was found to resemble those of other influenza viruses. Six RNA species were resolved, with estimated sizes ranging from 0.37 X 10(6) to 1.25 X 10(6) daltons. The internal ribonucleoproteins of influenza C virions were found to sediment heterogeneously in glycerol velocity gradients as demonstrated previously with influenza A/WSN virus. The ribonucleoproteins possessed diameters of 12 to 15 nm, with lengths ranging from 30 to 100 nm. Of the three major virion polypeptides (molecular weights, 88,000, 66,000, and 26,000), only the largest is glycosylated. Similar polypeptide species were present in influenza C virions of five different strains. All three major proteins of influenza C virions possess electrophoretic mobilities distinguishable from those of the major polypeptides of influenza A/WSN. The 66,000-dalton protein is associated with the ribonucleoprotein components. Two additional glycosylated polypeptides, with estimated molecular weights of 65,000 and 30,000, were detected in virions grown in embryonated eggs, but not in virus particles obtained from chicken embryo fibroblasts.     

Ribonucleic Acid and protein synthesis in chick embryo cells infected with fowl plague virus

Fowl plague virus comprised four major protein components and several minor ones, two strains of the virus giving similar results. One of the components was identified as the nucleocapsid protein. Synthesis of the virion proteins could readily be detected in infected cells 3 hr after infection. The two subcellular fractions associated with viral ribonucleic acid (RNA) polymerase activity (nuclei and ribosomal pellet) were associated with the protein of the nucleocapsid and a second virion protein of unidentified function. Measurement of viral RNA and protein synthesis in cells infected with preparations of ultraviolet irradiated virus showed that the capacity to synthesise the RNA and protein species of highest molecular weight was lost most quickly, suggesting that the pieces of viral RNA function independently.     

Antigens of Pichinde virus I. Relationship of soluble antigens derived from infected BHK-21 cells to the structural components of the virion.

Antigens detected by the complement-fixation (CF) test were prepared from BHK-21 cells infected with Pichinde virus.The preparations contained two antigens demonstrable by immunodiffusion. The antigen present in abundance was heat stable, Pronase resistant, and had a molecular weight of 20,000 to 30,000 as estimated by gel filtration. Polyacrylamide gel electrophoresis of purified antigen demonstrated two low-molecular-weight polypeptides. An identical antigenic determinant was found by disrupting purified virus with Nonidet P-40; however, none of the viral polypeptides co-migrated with the polypeptides derived from purified CF antigen. Pronase digestion of disrupted virus did not alter antigenicity but degraded the viral peptides to sizes similar to those associated with the major CF antigen. These observations suggest that the major CF antigen of Pichnide virus is a cleavage product of the structural proteins of the virus.     

Studies on Particle Components of Cacao Swollen Shoot Virus

Virions of the cacao swollen shoot virus (CSSV) strain 1A were purified and used for studies of its particle components. CSSV virions had a buoyant density of 1.365 g/cm³ in buffered CsCl. Following SDS polyacrylamide gel electrophoresis (PAGE), CSSV-specific proteins were identified in electroblot immunoassays (EBIA) with cross-absorbed polyclonal antibodies and especially well with monoclonal antibodies (MABs) to CSSV-1A. Based upon EBIA experiments with a selected MAB, CSSV virions appeared to have one capsid protein species with a relative molecular mass (M), of about 43 kd that was shown to be not glycosylated. However, this protein is sensitive to proteolytic degradation as degradation products ranging from 37 to 33 kd were found in addition to the 43 kd protein. Studies on the viral genome of CSSV revealed that CSSV virions contain a DNA of about 7. 5 kbp. Nucleic acid probes obtained by cloning parts of the viral genome yielded specific hybridization reactions with extracts and preparations from plants infected with strain 1A of CSSV but not with those from non-inoculated plants. One clone of 738 bp was sequenced and shown to contain a motif similar to the putative RNA binding domain of pararetroviruses. Based upon particle morphology and properties of the virion components, CSSV can be grouped with other nonenveloped bacilliform viruses for which the name badnaviruses has been proposed.     

Proteins of Norwalk virus.

The proteins of the Norwalk virus were studied by polyacrylamide gel electrophoresis. Highly purified specifically immunoprecipitated virions appeared to contain a single primary structural protein with a molecular weight of 59,000. In addition, a soluble Norwalk viral protein with a molecular weight of 30,000 was identified in fecal specimens containing Norwalk virus. The protein structure of the virion is similar to that of the Calciviridae family.     

Segmented genome and nucleocapsid of La Crosse virus.

La Crosse (LAC) virions purified by velocity and equilibrium gradient centrifugation contained three single-stranded RNA species. The three segments had sedimentation coefficients of 31S, 25S, and 12S by sodium dodecyl sulfate-sucrose gradient centrifugation. By comparison with other viral and cellular RNA species, the LAC viral RNAs had molecular weights of 2.9 x 10(6), 1.8 x 10(6), and 0.4 x 10(6). Phenol-sodium dodecyl sulfate-extracted LAC virion RNA was not infectious for BHK-21 cell cultures under conditions in which Sindbis viral RNA was infectious. Treatment of LAC virus with the nonionic detergent Triton X-100 and salt released three nucleocapsid structures, each containing one species of virion RNA. The nucleocapsids had sedimenation coefficients of 115S, 90S, and 65S. Negative-contrast electron microscopy of the nucleocapsids indicated that they were convoluted, supercoiled, and apparently circular. They had a mean diameter of 10 to 12 nm and modal lengths of 200, 510, and 700 nm (some were even longer). By chemical and enzymatic analysis of purified viral RNA, one type of 5' nucleotide (pppAp) present in the proportion of one per RNA segment was identified. After periodate oxidation, each virion RNA species was labeled by reduction with [3H]sodium borohydride. Taken together, these results suggest that although the nucleocapsids appear as closed loops, the viral RNA has free 5' and 3' ends and is, therefore, not circular.     

Biochemical studies on the Phlebotomus fever group viruses (Bunyaviridae family).

Analyses of the virion polypeptides and genomes of several Phlebotomus fever group viruses, Karimabad, Punta Toro, Chagres, and the sandfly fever Sicilian serotype viruses, have established that they are biochemically similar to the accepted members of the Bunyaviridae family. Like snowshoe hare virus (a member of the California serogroup of the Bunyavirus genus of the Bunyaviridae family), Karimabad, Punta Toro, Chagres, and the sandfly fever Sicilian serotype viruses all have three viral RNA species, designated large (L), medium (M), and small (S). Oligonucleotide fingerprint analyses of Karimabad and Punta Toro virus RNA species indicated that their L, M, and S RNA species are unique. By polyacrylamide gel electrophoresis it was determined for Karimabad virus that the apparent molecular weights of its L, M, and S RNA species are 2.6 X 10(6), 2.2 X 10(6), and 0.8 X 10(6), respectively. For Punta Toro virus, the apparent molecular weights of its L, M, and S RNA species are 2.8 X 10(6), 1.8 X 10(6), and 0.75 X 10(6), respectively. The major internal nucleocapsid (N) protein of Karimabad virus was found to have a molecular weight of 21 X 10(3). A similar polypeptide size class was identified in preparations of sandfly fever Sicilian serotype, Chagres, and Punta Toro viruses. The Karimabad virus glycoproteins formed the external surface projections on virus particles and could be removed from virus preparations by protease treatment. The glycoproteins in an unreduced sample could be resolved into two size classes by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. They had apparent molecular weights of 62 X 10(3) and 50 X 10(3) in continuous polyacrylamide gels. When Karimabad virus preparations were reduced with 1% beta-mercaptoethanol, prior to resolution by continuous polyacrylamide gel electrophoresis, all the viral glycoprotein was recovered in a single size class, having an apparent molecular weight of 62 X 10(3). Two or three major virion polypeptides have been identified in preparations of Punta Toro, Chagres, and sandfly fever Sicilian serotype viruses.     

In Vitro Synthesis of Proteins by Membrane-Bound Polyribosomes from Vesicular Stomatitis Virus-Infected HeLa Cells

Membrane-bound polysomes from vesicular stomatitis virus (VSV)-infected HeLa cells synthesize predominantly three proteins in an in vitro protein synthesizing system. These three proteins have different molecular weights than the viral structural proteins, i.e., 115,000, 88,000, and 72,000. Addition of preincubated L or HeLa cell S10 or HeLa cell crude initiation factors stimulates amino acid incorporation and, furthermore, alters the pattern of proteins synthesized. Stimulated membrane-bound polysomes synthesize predominantly viral protein G and lesser amounts of N, NS, and M. In vitro synthesized proteins G and N are very similar to virion proteins G and N based on analysis of tryptic methionine-labeled peptides. Most methionine-labeled tryptic peptides of virion G protein contain no carbohydrate moieties, since about 90% of sugar-labeled peptides co-chromatograph with only about 10% of methionine-labeled peptides. Sucrose gradient analysis of the labeled RNA present in VSV-infected membrane-bound polysomes reveals a relative enrichment in a class of viral RNA sedimenting slightly faster than the total population of the 13 to 15S mRNA, as compared to a VSV-infected crude cytoplasmic extract. A number of proteins, other than the viral structural proteins, are synthesized in the cytoplasm of five lines of VSV-infected cells. One of these proteins has the same molecular weight as the major in vitro synthesized protein, P(88). In vitro synthesized protein P(88) does not appear to be a precursor of viral structural proteins G, N, or M based on pulse-chase experiments and tryptic peptide mapping. Nonstimulated membrane-bound polysomes from uninfected HeLa cells synthesize the same size distribution of proteins as nonstimulated VSV-infected membrane-bound polysomes.     

Translational strategy of Solanum nodiflorum mottle virus RNA: synthesis of a coat protein precursor in vitro and in vivo.

Solanum nodiflorum mottle virus RNA (Mr = 1.5 X 10(6)) was translated in vitro in a wheat embryo extract. Four major products were synthesized: 2 related proteins of molecular weight 100K (P100) and 67K (P67), a protein of molecular weight 38K (P38), and a methionine-lacking protein of molecular weight 28K (P28). P38 was synthesized by a minor RNA component (Mr approximately 0.4 X 10(6)) and comigrated with the only viral product detected in SNMV-infected N. clevelandii protoplasts. Antiserum raised against purified SNMV virions precipitated both in vitro- and in vivo-synthesized P38, suggesting that it is either a precursor to or an intact form of SNMV coat protein whose apparent molecular weight in purified virus preparations is 30K.     

Subgenomic mRNA of Aura alphavirus is packaged into virions.

Purified virions of Aura virus, a South American alphavirus related to Sindbis virus, were found to contain two RNA species, one of 12 kb and the other of 4.2 kb. Northern (RNA) blot analysis, primer extension analysis, and limited sequencing showed that the 12-kb RNA was the viral genomic RNA, whereas the 4.2-kb RNA present in virus preparations was identical to the 26S subgenomic RNA present in infected cells. The subgenomic RNA is the messenger for translation of the viral structural proteins, and its synthesis is absolutely required for replication of the virus. Although 26S RNA is present in the cytosol of all cells infected by alphaviruses, this is the first report of incorporation of the subgenomic RNA into alphavirus particles. Packaging of the Aura virus subgenomic mRNA occurred following infection of mosquito (Aedes albopictus C6/36), hamster (BHK-21), or monkey (Vero) cells. Quantitation of the amounts of genomic and subgenomic RNA both in virions and in infected cells showed that the ratio of genomic to subgenomic RNA was 3- to 10-fold higher in Aura virions than in infected cells. Thus, although the subgenomic RNA is packaged efficiently, the genomic RNA has a selective advantage during packaging. In contrast, in parallel experiments with Sindbis virus, packaging of subgenomic RNA was not detectable. We also found that subgenomic RNA was present in about threefold-greater amounts relative to genomic RNA in cells infected by Aura virus than in cells infected by Sindbis virus. Packaging of the Aura virus subgenomic RNA, but not those of other alphaviruses, suggests that Aura virus 26S RNA contains a packaging signal for incorporation into virions. The importance of the packaging of this RNA into virions in the natural history of the virus remains to be determined.     

Proteins induced in tissue culture by four isolates of Theiler''s murine encephalomyelitis virus.

The proteins specified by four Theiler's murine encephalomyelitis virus isolates in infected BHK-21 cells were studied. Their processing, sensitivity to trypsin, and the changeover after viral infection from synthesis of cellular proteins to synthesis of viral proteins were determined by one- and two-dimensional gel electrophoreses. The molecular weights and isoelectric points of the structural and nonstructural proteins of DA and WW isolates, which represent the less virulent subgroup of Theiler's murine encephalomyelitis virus, and of GDVII and FA isolates, which represent the virulent subgroup, were found to be the same. The sensitivity of DA and GDVII isolates to trypsin, as purified virions, and in infected cell extracts was similar. The shut-off of cellular protein synthesis in cells infected with the same two isolates and the changeover to the synthesis of viral proteins appeared to have the same pattern. These findings are interesting since the two subgroups of Theiler's murine encephalomyelitis virus differ in their pathogenicity, intracellular development in infected BHK-21 cells, and RNA composition, as determined by RNase T1 fingerprinting analysis.     

Ribonucleic Acid Species of Intracellular Nucleocapsids and Released Virions of Vesicular Stomatitis Virus

Infection of chicken embryo cells with vesicular stomatitis (VS) virus resulted in variable production of three classes of intracellular viral ribonucleocapsids with sedimentation coefficients of approximately 140S, 110S, and 80S, as well as three corresponding classes of released virions designated B, LT, and T. Intracellular nucleocapsids of each class contained three proteins of which the major N protein was firmly bound, and the minor L and NS1 proteins were readily dissociated with 0.5 m NaCl. The ribonucleic acid (RNA) species extracted from B, LT, and T virions, and from corresponding intracellular nucleocapsids, contained RNA species with approximate molecular weights of 3.2 x 10(6), 2.0 x 10(6), and 10(6), respectively, as determined by polyacrylamide gel electrophoresis. These values are roughly equivalent to sedimentation coefficients of 42S, 28S, and 23S for each of the virion and nucleocapsid RNA species. Cells infected at high multiplicity with undiluted passage VS virus gave rise primarily to virions and nucleocapsids containing 23S RNA, whereas cells productively infected with purified B virions produced predominantly B and LT virions and nucleocapsids. At late stages in the productive cycle of infection, more virions containing 42S RNA were produced, but the intracellular pool of nucleocapsids containing 28S and 23S RNA remained relatively constant. Additional studies by more refined techniques are required to test the hypothesis that nucleocapsids containing 28S and 23S RNA are precursors of the 42S RNA in infectious VS-B virions and that production of defective T and LT virions results from failure of ligation of the RNA precursors.     

Characterization of bovine viral diarrhea virus proteins.

Virus-specific proteins were examined in cultured cells infected with bovine viral diarrhea virus. By using antisera obtained from virus-infected animals, three major virus-specific polypeptides with molecular weights of 115,000 (115K), 80K, and 55K were observed. Minor proteins of 45,000 and 38,000 daltons were also noted. Tryptic peptide mapping indicated that the 115K and the 80K polypeptides were structurally related. The 55K protein was glycosylated and appeared not to be related to the 115K and 80K proteins. Pulse-chase experiments failed to demonstrate any procursor-product relationship among any of these proteins, and all three polypeptides were found in purified virion preparations. The significance of these findings with respect to the replication of bovine viral diarrhea virus is discussed.     

In Vitro Translation of Uukuniemi Virus-Specific RNAs: Identification of a Nonstructural Protein and a Precursor to the Membrane Glycoproteins

We isolated the virus-specific RNA species from Uukuniemi virus-infected chicken embryo cells and fractionated them by sucrose gradient centrifugation. In addition to three RNA species cosedimenting with the three viral RNA segments L (29S), M (23S), and S (17S), a fourth major RNA species, sedimenting at about 12S (S2), was found early in the infection. Annealing experiments indicated that the cytoplasmic L and M RNA species consisted of both plus and minus strands, with the plus strands in slight excess. Most of the S1 RNA was of negative polarity, whereas S2 was of positive polarity. The S2 RNA specifically annealed to the virion S RNA segment, indicating that it is transcribed from this segment. In vitro translation of the individual RNA species in micrococcal nuclease-treated cell-free reticulocyte extracts showed that an mRNA cosedimenting with the virion M RNA directed the synthesis of a virus-specific 110,000-dalton polypeptide (p110). This polypeptide could be immunoprecipitated with antiserum prepared against purified virions. When translation was carried out in the presence of dog pancreas microsomes, p110 was absent. Instead, an immunoprecipitable polypeptide band, with a molecular weight of about 70,000 and migrating between the virion surface glycoproteins G1 and G2, was observed. It is thus likely that the glycoproteins are synthesized as a precursor (p110), which during translation is cleaved roughly in the middle to yield G1 and G2. The 12S RNA species directed the synthesis of the nucleocapsid protein and a novel polypeptide with an apparent molecular weight of about 30,000. The latter was not precipitated with antivirion serum and was absent from lysates programmed with the corresponding RNA fraction from a mock-infected extract. Since, in addition, it was not found in purified virions and was present in the cytoplasm of infected cells but not in uninfected cells, it probably represents a nonstructural polypeptide.     

Synthesis of Saint Louis Encephalitis Virus Ribonucleic Acid in BHK-21/13 Cells

Infection of baby hamster kidney cells (BHK-21/13) with Saint Louis encephalitis (SLE) virus depressed the rate of protein and ribonucleic acid (RNA) synthesis until viral RNA synthesis began 6 hr postinfection (PI). Virus-directed RNA synthesis was subsequently inhibited until 12 hr PI when virion maturation began. The rate of protein synthesis reached a peak 6 hr PI and was subsequently depressed until just before the onset of virion maturation. Density gradient analysis of phenol-extracted RNA from actinomycin-treated infected cells indicated that, at 6 to 8 hr and again at 12 to 20 hr PI, three species of viral-specific RNA were synthesized. The most rapid sedimenting form (43S) was ribonuclease-sensitive and had a base composition similar to the RNA isolated from mature virions. The 20S RNA species was ribonuclease-resistant and had a sedimentation coefficient and base composition similar to the replicative form associated with other arbovirus infections. The 26S RNA was ribonuclease-resistant (0.2 mug/ml, 0.1 m NaCl, 25 C, 30 min) and had a nucleotide base composition closer to the 20S form than to the values for 43S RNA. Five-minute pulse labeling of infected cultures during the period viral RNA synthesis was maximal resulted in labeling of only the 20S to 22S RNA fractions. With pulse-labeling periods of 10 min, both the 20S and 26S RNA species were radioactive. Periods of radioactive labeling of as long as 15 min were required before the 43S form was radioactively labeled. These results suggest that the 20S and 26S RNA may be intermediate forms in the synthesis of 43S viral RNA.