Genome complexities of the three mRNA species of snowshoe hare bunyavirus and in vitro translation of S mRNA to viral N polypeptide.

The genome complexities of the principal intracellular viral complementary RNA species of the snowshoe hare bunyavirus have been analyzed by duplex analyses involving hybridization of complementary RNA to individual 32P-labeled viral RNA species (large, L; medium, M; and small, S), recovery of nuclease-resistant duplexes, and determination of the oligonucleotide fingerprints of the protected 32P-labeled viral sequences. The result for the M RNA (which codes for the glycoproteins G1 and G2; J. R. Gentsch and D. H. L. Bishop, J. Virol. 30:767-770, 1979) indicates that there is a single polycistronic M mRNA. Similar results were obtained for the L and S RNA species. In vitro translation studies with the S complementary RNA species of snowshoe hare virus as well as melted purified S duplexes substantiate earlier genetic and molecular studies (J. R. Gentsch and D. H. L. Bishop, J. Virol. 28:417-419, 1978; J. Gentsch, D. H. L. Bishop, and J. F. Obijeski, J. Gen. Virol. 34-257-268, 1977), which indicate that S mRNA codes for the virion nucleocapsid protein N.

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.

Unique peptide maps of the three largest proteins specified by the flavivirus Kunjin.

Tryptic digests of four polypeptides found in Kunjin virus-infected Vero cells, NV5, NV4, V3, and NV3, were compared by peptide mapping. The polypeptides to be analyzed were labeled with radioactive methionine and separated by electrophoresis through polyacrylamide gels containing sodium dodecyl sulfate. Because infection of Vero cells by Kunjin virus does not inhibit host cell protein synthesis, radioactively labeled viral polypeptides prepared from infected cells migrate coincidentally during sodium dodecyl sulfate-gel electrophoresis with some of the labeled host proteins. Thus, the genuine viral methionine-containing peptides in tryptic digests of viral proteins have been identified by co-analyzing polypeptides from [3H]methionine-labeled uninfected cells and [35S]methionine-labeled infected cells and determining the 35S/3H ratio in the peptides resolved in two dimensions on thin-layer chromatography plates. The peptide map of NV3 demonstrated that it is host coded, whereas NV5, NV4, and V3 have unique peptide maps and, therefore, account for approximately one-half of the coding potential of Kunjin virus RNA.

Comparisons of the peptide maps of Kunjin virus proteins smaller than the envelope protein.

We analyzed the maps of [35S]methionine-labeled tryptic peptides of the Kunjin virus-specified proteins NV2 1/2, NV2, V2, NV1 1/2, NV1, and V1. The peptides of NV1 1/2 are identical to those of V2, except for one peptide contained only in the latter. The maps of each of the other proteins are unique and, consequently there is no evidence of any one protein being derived from another by proteolytic cleavage. The tryptic peptide maps of the above polypeptides were also compared with those of the larger Kunjin proteins, NV5, NV4, and V3. The resolution of the peptides of NV2 1/2 and NV1 is adequate to exclude any relationships with NV5 and NV4, but a possible relationship with V3 remains, although it seems unlikely in the light of other evidence. Since the peptide map of V1 is comprised of only two methionine-containing peptides similar in mobilities to two peptides found in the maps of NV5, NV4, and V3, a precursor, if any, of V1 has not been positively identified. The peptides of NV2 and V2 (NV1 1/2) are not contained in digests of NV5, NV4, or V3, and therefore, like the latter, NV2 and V2 (NV1 1/2) are independent products of translation from the positive-strand genome.

The virus particle nucleic acids and proteins of four bunyaviruses.

The structural polypeptides of five bunyaviruses, snowshoe hare, Lumbo and La Crosse viruses (members of the California encephalitis subgroup of bunyaviruses), Bunyamwera and Main Drain viruses (members of the Bunyamwera subgroup of bunyaviruses), have been compared by polyacrylamide-SDS gel electrophoresis. Each virus was found to possess three major structural polypeptides, two glycoproteins (G1 and G2), and one nucleocapsid protein (N). Although the sizes of the G1 polypeptides (mol. wt. approx. 115 X 10(3)) and G2 polypeptides (mol. wt. approx. 38 X 10(3)) of the five viruses were found to be essentially similar, the sizes of the N polypeptides of the various viruses differed (mol. wt. range 19 to 24 X 10(3)). The RNA genomes of four bunyaviruses (snowshoe hare, La Crosse, Bunyamwera and Main Drain) have also been compared. Each virus has three RNA species of mol. wt. approx. 3 X 10(6), 1-9 X 10(6) and 0-4 X 10(6). Minor size differences were observed for the smallest RNA species of the four viruses (mol. wt. range 0-34 to 0-50 X 10(6)). For snowshoe hare virus the RNA segments hav a 5' sequence of pppAp...which suggests that the RNA is linear and not circular.

Composition of the Zaysan virion.

The sedimentation coefficient of Zaysan virion ribonucleic acid (RNA), estimated by sucrose density gradient centrifugation, was 42 S. Pancreatic ribonuclease digested the viral RNA to acid-soluble fragments. Two viral structural proteins with apparent molecular weight of 50,000 and 30,000 daltons were identified by polyacrylamide gel electrophoresis. A dense, RNA-rich particle, containing the smaller polypeptide, was isolated after mild detergent treatment of Zaysan virus.

Surface structure of Uukuniemi virus.

Uukuniemi virus, grown in chicken embryo fibroblasts, has been studied by electron microscopy using negative staining, thin sectioning, and freeze-etching techniques. The spherical virus particle measures about 95 nm in diameter. Its envelope consists of a 5-nm thick membrane covered by 8- to 10-nm long surface projections. These are composed of two polypeptides species of about the same size. Both of them can be removed by digestion with the proteolytic enzyme thermolysin except for a small fragment. The enzyme-treated particles are smooth surfaced and extremely deformable. The glycopolypeptides are clustered to form hollow cylindrical morphological units, 10 to 12 nm in diameter, with a 5-nm central cavity. Both negative staining and freeze-etching suggest that these units are penton-hexon clusters arranged in a T = 12, P = 3, icosahedral surface lattice. The membrane to which the surface subunits are attached is probably a lipid bilayer as evidenced by its double-track appearance in thin sections and the tendency of the freeze fracturing to occur within it. The strand-like nucleoprotein appears from thin-sectioning results to be to a large part located in a zone underneath the membrane.

The proteins of Murray Valley encephalitis virus.

Proteins specified by Murray Valley encephalitis virus were labelled during virus growth in Vero and in PS cells, and separated by polyacrylamide gel electrophoresis. The purified virus particle contains three proteins (V-1, V-2 AND V-3) whereas the slow sedimenting haemagglutinin or virus sub-particle lacks the core protein V-2 but contains NV-2, a non-structural protein. Seven non-structural proteins in addition to V-2 and V-3 were identified in infected cells. Electrophoretic profiles by virus-specified proteins in both cell lines were almost identical after elimination by a double-label technique of the background of continuing host-cell protein synthesis. Glucosamine was incorporated into the envelope protein V-3 and NV-2. From 26 to 46h post-infection in Vero cells, the proportion and amounts of virus-specified proteins remained constant and they were non-equimolar; incorporation of labelled leucine into V-2 was much greater than incorporation into NV-2, whereas in cells infected with Kunjin (a related flavivirus) this ratio of incorporation was reversed. At 21 to 25h, the synthesis of V-2 was less prominent but there was an enhanced synthesis of NV-X. Apart from V-I, NV-I, NV-4 and NV-5, all proteins are larger than the corresponding Kunjin virus proteins and together represent about 400 x 10-3 daltons of polypeptide synthesis, which is close to the maximum coding content of the flavivirus genome.

Structural polypeptides of California encephalitis virus: BFS-283.

The polypepetides of California encephalitis virus (BFS-283) were analyzed by polyacrylamide gel electrophoresis (PAGE). Four polypeptides were detected in virions grown in both BHK-21 and LLC-MK2 cell cultures with molecular weights of 17,500. 30,000, 38,000, and 82,000 (VP-1, VP-2, VP-3, and VP-4, respectively). Viral proteins 2, 3, and 4 were glycoproteins and appeared to be associated with the envelope of the virus. Treatment of virions (rho=1.18 g/cm3) with then non-ionic detergent, NP-40, allowed detection of a RNA-rich fraction (rho=1.26/cm3) with contained the smallest polypeptides (VP-1).

Structural components of Oriboca virus.

Analysis of purified Oriboca virions by neutral, sodium dodecyl sulfate polyacrylamide-gel electrophoresis indicated the presence of three structural polypeptides designated V-1, V-2, and V-3 on the basis of their relative electrophoretic mobilities in 8% gels. Polypeptides V-2 and V-3 are glycopeptides associated with the virion envelope as demonstrated by the preferential incorporation of labeled glucosamine into the polypeptides and by release of the polypeptides from the intact virion by the nonionic detergent NP-40. Polypeptide V-1 is the protein component of the nucleoprotein core of Oriboca virus as evidenced by the specific incorporation of uridine into the nucleoprotein, its release from the intact virion by NP-40 treatment, and its separation by both rate-zonal and isopycnic density gradient centrifugation from both the intact virion and envelope components. Molecular weights have been tentatively assigned to the polypeptides by extrapolation from the structural polypeptides of Sindbis virus when both are run in the same gel. Polypeptide V-1 has an apparent molecular weight of 20,000 to 23,000; V-2, 30,000 to 32,000; and V-3, 83,000 to 85,000.

Comparison of the virion polypeptides of group B arboviruses.

The virion polypeptides of eight group B arboviruses were compared by polyacrylamide gel coelectrophoresis. All contained three polypeptides, V-1, V-2, and V-3. The mosquito-borne viruses had electrophoretically similar small membrane polypeptides (V-1); the tick-borne viruses had a V-1 of significantly decreased mobility.

Analysis of arbovirus ribonucleic acid forms by polyacrylamide gel electrophoresis.

Viral ribonucleic acid (RNA) from Semliki Forest virus- and Sindbis virus-infected cells was analyzed by electrophoresis on polyacrylamide gels. In contrast to earlier results obtained by sucrose density gradient centrifugation, all of the known viral RNA forms (i.e., the 42S, 26S, replicative form, and replicative intermediate) were very clearly separated. The high resolution of the electrophoretic method permitted the identification of two new single-stranded RNA species. In addition, the replicative form was shown to be heterogeneous and to consist of at least two forms. The results suggested that the replicative forms occur in vivo although in relatively small amounts.