Characterization of RNA from equine infectious anemia virus.

The genome of equine infectious anemia virus, a nononcogenic retrovirus, has been characterized by velocity sedimentation, electrophoresis in polyacrylamide gels, buoyant density in CS2SO4, and susceptibility to nuclease digestion. The nucleic acid of purified virus was resolved by sedimentation analysis into a fast-sedimenting genome component, which comprises about two-thirds of the virion RNA, and a slow-sedimenting RNA, which is probably comprised of host-derived tRNA and a trace amount of 5S RNA. The fast-sedimenting RNA had a sedimentation coefficient of 62S and a molecular weight of 5.4 X 10(6) to 5.6 X 10(6), as determined by sedimentation velocity and electrophoretic mobility. Upon heat denaturation, [3H]uridine-labeled 62S RNA dissociated into material comprised of 90 to 95% single-stranded species, sedimenting predominantly at 34S, with a molecular weight of 2.7 X 10(6) to 2.9 X 10(6) and 5 to 10% 4S RNA. The 62S RNA was predominantly single-stranded but contained double-stranded regions, as indicated by partial resistance to RNase IA and SI nuclease and by a lower buoyant density in CS2SO4 than that of the single-stranded 34S RNA derived by heat denaturation. These data indicated that the viral genome consisted of two 34S subunits of single-stranded RNA held in a high-molecular-weight complex with 4S RNA by a mechanism involving a small degree of base pairing. Thus, the structure of equine infectious anemia virus RNA is similar to that of other retroviruses.     

Structure of the intracellular defective viral RNAs of defective interfering particles of mouse hepatitis virus.

The intracellular defective RNAs generated during high-multiplicity serial passages of mouse hepatitis virus JHM strain on DBT cells were examined. Seven novel species of single-stranded polyadenylic acid-containing defective RNAs were identified from passages 3 through 22. The largest of these RNAs, DIssA (molecular weight [mw], 5.2 X 10(6)), is identical to the genomic RNA packaged in the defective interfering particles produced from these cells. Other RNA species, DIssB1 (mw, 1.9 X 10(6) to 1.6 X 10(6)), DIssB2 (mw, 1.6 X 10(6)), DIssC (mw, 2.8 X 10(6)) DIssD (mw, 0.82 X 10(6)), DIssE (mw, 0.78 X 10(6)), and DIssF (mw, 1.3 X 10(6)) were detected at different passage levels. RNase T1-resistant oligonucleotide fingerprinting demonstrated that all these RNAs were related and had multiple deletions of the genomic sequences. They contained different subsets of the genomic sequences from those of the standard intracellular mRNAs of nondefective mouse hepatitis virus JHM strain. Thus these novel intracellular viral RNAs were identified as defective interfering RNAs of mouse hepatitis virus JHM strain. The synthesis of six of the seven normal mRNA species specific to mouse hepatitis virus JHM strain was completely inhibited when cells were infected with viruses of late-passage levels. However, the synthesis of RNA7 and its product, viral nucleoprotein, was not significantly altered in late passages. The possible mechanism for the generation of defective interfering RNAs was discussed.     

Generation of defective interfering particles of Semliki Forest virus in a clone of Aedes albopictus (mosquito) cells.

Serial undiluted passage of Semliki Forest virus in a clone of Aedes albopictus cells resulted in a marked decrease in infectious virus yields due to the generation and accumulation of defective interfering particles. Virus from the third passage had a high particle/infectivity ratio and interfered specifically with homologous but not heterologous standard virus replication. Two RNA species of molecular weights 0.78 X 10(6) and 0.61 X 10(6) were the major RNA components of purified passage 4 virus. These RNA species were also the predominant virus RNA species detected in cells infected with passage 3 virus. Synthesis of standard virus RNA and virus-specified protein was much reduced in passage 3 virus-infected cells. Interference with standard virus replication and the synthesis of large amounts of defective interfering RNA were also observed in chicken embryo cells infected with passage 3 virus from mosquito cells.     

Isolation and characterization of canine distemper virus-specific RNA

Ten species of virus-specific RNA were detected in Vero cells infected with the FXNO strain of canine distemper virus (CDV). The largest RNA was the genome-sized RNA and the nine smaller species were polyadenylated RNAs. Similar results were obtained for nine other strains of CDV. The molecular weights of these ten RNAs were determined to be 4.61 X 10(6), 2.46 X 10(6), 1.52 X 10(6), 1.32 X 10(6), 1.19 X 10(6), 1.07 X 10(6), 0.77 X 10(6), 0.65 X 10(6), 0.58 X 10(6), and 0.48 X 10(6). By in vitro translation of the polyadenylated RNAs in a rabbit reticulocyte lysate system, three different proteins which probably correspond to H, NP, and M were synthesized from the fraction containing RNAs 7, 8, 9, and 10.     

High molecular weight RNAs from Rous sarcoma virus and Moloney murine leukemia virus contain two subunits.

The molecular weights of the large genomic RNAs from Rous sarcoma and Moloney murine leukemia viruses were determined by a combination of sedimentation coefficients and retardation coefficients from gel electrophoresis. Six RNA standards, ranging from 0.7 X 10(6) to 5.3 X 10(6) daltons, were employed. Studies in the presence of varying concentrations of Mg2+ showed that the method provided valid molecular weights for RNAs of differing amounts of ordered structure. The molecular weight (X 10(-6)) of the high molecular weight RNA complexe from Rous sarcoma virus was 7.6 (+/-0.3) and from murine leukemia virus was 6.9 (+/-0.3). The molecular weights (X 10 (-6) of their Subunits were 3.3 (+/-0.1) and 2.8 (+/-0.2), respectively. Hence, the large complexes consisted of two, not three or more, subunits plus small associated RNAs. The high molecular weight RNA from cloned Rous sarcoma virus was heterogenous in molecular weight although the apparent molecular radius was constant; stuides were performed on subfractions of the RNA as well as on RNA from virus harvested at various time intervals. The preparations with lowest molecular weight approached a mass equal to twice that of the subunit, with hydrodynamic properties approaching those expected of normal single-stranded RNA.     

Rubella virus 40S genome RNA specifies a 24S subgenomic mRNA that codes for a precursor to structural proteins.

We have analyzed the structure of the rubella virus genome RNA and the virus-specific RNA species synthesized in B-Vero cells infected with rubella virus. A single-stranded, capped, and polyadenylated RNA species sedimenting at 40S in a sucrose gradient was released from purified virions treated with sodium dodecyl sulfate. This RNA species migrated with an Mr of about 3.8 X 10(6) in an agarose gel after denaturation with glyoxal and dimethyl sulfoxide. Infected cells labeled with [3H]uridine in the presence of actinomycin D contained, in addition to the 40S RNA, a single-stranded polyadenylated 24S RNA species as shown by sucrose gradient analysis. In a Northern blot analysis, this RNA hybridized to a cDNA probe derived from the 3' portion of the genomic 40S RNA. In vitro translation of the 24S RNA species yielded a 110,000-dalton polypeptide, in addition to some smaller products which were immunoprecipitated with an antiserum prepared against the structural proteins E1, E2a, E2b, and C. Since the sum of the molecular weights of the nonglycosylated envelope proteins and the capsid protein has been estimated to be about 116,000 (C. Oker-Blom et al., J. Virol. 46:964-973, 1983), these results suggest that the 24S RNA species represents a subgenomic mRNA coding for a precursor (p110) to the structural proteins of rubella virus. Thus, the strategy of gene expression of rubella virus appears to be similar to that of the alphaviruses.     

Mouse hepatitis virus A59: mRNA structure and genetic localization of the sequence divergence from hepatotropic strain MHV-3.

The composition and structure of the mouse hepatitis virus (MHV)-specific RNA in actinomycin D-treated, infected L-2 cells were studied. SEven virus-specific RNA species with molecular weights of 0.6 X 10(6), 0.9 X 10(6), 1.2 X 10(6), 1.5 X 10(6), 3.0 X 10(6), 4.0 X 10(6), and 5.4 X 10(6) (equivalent to the viral genome) were detected. T1 oligonucleotide fingerprinting studies suggested that the sequences of each RNA species were totally included within the next large RNa species. The oligonucleotides of each RNA species were mapped on the 60S RNA genome of the virus. Each RNA species contained the oligonucleotides starting from the 3' end of the genome and extending continuously for various lengths in the 3' leads to 5' direction. All of the viral RNA species contained a polyadenylate stretch of 100 to 130 nucleotides and probably identical sequences immediately next to the polyadenylate. These data suggested that the virus-specific RNAs are mRNA's and have a stairlike structure similar to that of infectious bronchitis virus, an avian coronavirus. A proposal is presented, based on the mRNA structure, for the designation of the genes on the MHV genome. Using this proposal, the sequence differences between A59, a weakly pathogenic strain, and MHV-3, a strongly hepatotropic strain, were localized primarily in mRNA's 1 and 3, corresponding t genes A and C.     

RNA synthesized in calicivirus-infected cells is atypical of picornaviruses.

RNA labeled with [3H]uridine from Vero cells infected with San Miguel sea lion virus in the presence of actinomycin D was analyzed by glycerol density gradient sedimentation and polyacrylamide gel electrophoresis. The predominant single-stranded RNA (36S, 2.6 x 10(6) molecular weight) was genome size. There was also a prominent 22S, 1.1 x 10(6)-molecular weight, single-stranded component and one or more double-stranded or partially double-stranded classes. Replicative forms, sedimenting at 18S, contained single-stranded RNA corresponding to the larger-molecular-weight class. All classes of intracellular RNA and virion RNA were polyadenylated. These findings and results with pig kidney cells infected with vesicular exanthema of swine virus and feline cells infected with feline calicivirus indicate that caliciviruses exhibit a strategy of replication different from typical picornaviruses and supports removal of the caliciviruses from the family Picornaviridae.     

Identification of virus-coded nonstructural polypeptides in bunyavirus-infected cells.

Analyses of bunyavirus-infected cell extracts identified at least two virus-induced nonstructural polypeptides. With snowshoe hare (SSH), La Crosse (LAC), and six SSH-LAC reassortant viruses, it was shown that one of these nonstructural polypeptides (NSs, approximate molecular weight, 7.4 X 10(3)) is coded by the SSH small (S)-size viral RNA species. This nonstructural polypeptide was not detected (at least in the same relative abundancies) in LAC virus-infected cells or in cells infected with reassortants having LAC S RNA. For SSH virus, tryptic peptide analyses of either [3H]leucine- or [3H]arginine-labeled NSs indicated that it contains unique sequences not present in the SSH nucleocapsid (N) polypeptide (also coded by the S RNA; J. R. Gentsch and D. H. L. Bishop, J. Virol. 28:417-419, 1978). Analyses of SSH virus-infected cell extracts and extracts of cells infected with SSH-LAC reassortants having SSH medium (M)-size RNA species indicated that a nonstructural polypeptide (NSM; approximate molecular weight, 12 X 10(3)) is coded by the SSH M RNA species. In extracts of LAC virus-infected cells (or cells infected with SSH-LAC reassortants having LAC M RNA), a polypeptide with an electrophoretic mobility slightly faster than that of the SSH NSM polypeptide was observed (approximate molecular weight, 11 X 10(3)); it has been designated LAC NSM. The relationships of the NSM polypeptides to the other M RNA-coded polypeptides (G1 and G2; J. R. Gentsch and D. H. L. Bishop, J. Virol. 30;767-770, 1979) have not been determined. Two additional polypeptides present in both LAC- and SSH-infected cell extracts also appear to be virus induced (one with an approximate molecular weight of 10 X 10(3), p10; the other with an approximate molecular weight of 18 X 10(3), p18). Whether these polypeptides are virus coded has not been determined.     

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.     

Replication of Sindbis Virus V. Polyribosomes and mRNA in Infected Cells

Cells infected with wild-type Sindbis virus contain at least two forms of mRNA, 26S and 49S RNA. Sindbis 26S RNA (molecular weight 1.6 x 10(6)) constitutes 90% by weight of the mRNA in infected cells, and is thought to specify the structural proteins of the virus. Sindbis 49S RNA, the viral genome (molecular weight 4.3 x 10(6)), constitutes approximately 10% of the mRNA in infected cells and is thought to supply the remaining viral functions. In cells infected with ts2, a temperature-sensitive mutant of Sindbis virus, the messenger forms also include a third species of RNA with a sedimentation coefficient of 33S and an apparent molecular weight of 2.3 x 10(6). Hybridization-competition experiments showed that 90% of the base sequences in 33S RNA from these cells are also present in 26S RNA. Sindbis 33S RNA was also isolated from cells infected with wild-type virus. After reaction with formaldehyde, this species of 33S RNA appeared to be completely converted to 26S RNA. These results indicate that 33S RNA isolated from cells infected with either wild-type Sindbis or ts2 is not a unique and separate form of Sindbis RNA.     

Structural analysis of the intracellular RNAs of murine mammary tumor virus.

We have characterized murine mammary tumor virus (MuMTV)-specific RNA in several types of cells in which viral DNA is transcribed into RNA: cultured GR mouse mammary tumor cells, S49 lymphoma cells from BALB/c mice, lactating mammary glands from C57BL/6 mice, and mink lung cells infected in vitro with MuMTV. In all cell types studied, there are three distinct species of intracellular viral RNA, with sedimentation coefficients of 35S, 24S, and 13S (or molecular weights of 3.1 X 10(6), 1.5 X 10(6), and 0.37 X 10(6), as determined by rate-zonal sedimentation in sucrose gradients and by electrophoresis in agarose gels under denaturing conditions. These three viral RNA species appear to be present regardless of viral RNA concentration, responsiveness to glucocorticoid hormones, production of extracellular virus, and use of either endogenous or acquired MuMTV proviral DNA as template. The three viral RNAs display characteristics of mRNAs in that they are polyadenylated, associated with polyribosomes, and released from polyribosomes by treatment with EDTA; hence all three species presumably direct the synthesis of virus-coded proteins. The two larger species of viral RNA are probably responsible for synthesis of the structural proteins of the virion, but the function of the 13S RNA is not known. Both of the subgenomic RNAs contain sequences found at the 3' terminus of 35S (or genomic) RNA. However, only the 24S RNA (not the 13S RNA) contains sequences which are located at the 5' terminus of 35S RNA and are apparently transposed during RNA synthesis of maturation, as described for subgenomic mRNA's of other retroviruses.     

Epstein-Barr virus RNA. VI. Viral RNA in restringently and abortively infected Raji cells.

Nuclear and polyadenylated RNA fractions of Raji cells are encoded by larger fractions of Epstein-Barr virus DNA (35 and 18%, respectively) than encode polyribosomal RNA (10%). Polyribosomal RNA is encoded by DNA mapping at 0.05 X 10(8) to 0.29 X 10(8), 0.63 X 10(8) to 0.66 X 10(8), and 1.10 X 10(8) to 0.03 X 10(8) daltons. An abundant, small (160-base), non-polyadenylated RNA encoded by EcoRI fragment J (0.05 X 10(8) to 0.07 X 10(8) daltons) is also present in the cytoplasm of Raji cells. After induction of early antigen in Raji cells, there was a substantial increase in the complexity of viral polyadenylated and polyribosomal RNAs. Thus, nuclear RNA was encoded by 40% of Epstein-Barr virus DNA, and polyadenylated and polyribosomal RNAs were encoded by at least 30% of Epstein-Barr virus DNA. Polyribosomal RNA from induced Raji cells was encoded by Epstein-Barr virus DNAs mapping at 0.05 X 10(8) to 0.29 X 10(8), 0.63 X 10(8) to 0.66 X 10(8), and 1.10 X 10(8) to 0.03 X 10(8) daltons and also by DNAs mapping within the long unique regions of Epstein-Barr virus DNA at 0.39 X 10(8) to 0.49 X 10(8), 0.51 X 10(8) to 0.59 X 10(8), 0.66 X 10(8) to 0.77 X 10(8), and 1.02 X 10(8) to 1.05 X 10(8) daltons.     

Replication of Sindbis virus. I. Relative size and genetic content of 26 s and 49 s RNA

The genome of Sindbis virus, 49 s RNA, is a single, intact polynucleotide chain having a molecular weight of 4.3±0.3 × 10⁶ daltons. This has been determined using a variety of methods including polyacrylamide gel electrophoresis, sedimentation after reaction with formaldehyde, determination of the molecular weights of the double-stranded forms of Sindbis-specific RNA and hybridization competition. The second major species of single-stranded RNA made after infection with Sindbis, 26 s RNA, has been found to have a molecular weight of 1.6 × 10⁶ daltons as determined by sedimentation in dimethylsulfoxide. Hybridizationcompetition experiments carried out between these two species of RNA, using double-stranded forms of Sindbis RNA isolated from infected cells, showed that 26 s RNA contains only one-third of the base sequences in 49 s RNA and thus represents a unique fraction of the viral genome.     

DNA-binding proteins induced by herpes simplex virus type 2 in HEp-2 cells.

Affinity chromatography on single-stranded and double-stranded DNA-cellulose indicates that 12 proteins previously identified from herpes simplex virus type 2-infected cells, ranging in molecular weight from 28 X 10(3) to 186 X 10(3), bind to DNA-cellulose. The DNA-binding proteins found in infected cells differed in relative binding strengths for denatured DNA-cellulose. The virus specificity of these DNA-binding proteins was further studied by comparison with DNA-binding proteins isolated from mock-infected cells, and by immunoprecipitation of infected-cell DNA-binding proteins with antisera specific for viral antigens. The promise this technique holds for the purification and study of polypeptides involved in virus DNA replication, recombination, or repair is discussed.     

Isolation and biochemical characterization of the microsomal fraction from the digestive gland of mussel Mytilus galloprovincialis Lam

A procedure to prepare microsomes from the mussel digestive gland is proposed. The data concerning the biochemical characterization of this subcellular fraction shows a typical RNA:protein ratio, but the presence of hydrolytic enzymes was also found; therefore a mixture of hydrolase inhibitors to study the different biochemical characteristics was used. The biochemical data demonstrate that glucose-6-phosphatase activity (G6Pase), a typical microsomal marker in mammalian cells, is not present in mussel digestive gland microsomes but a high non-specific phosphatase activity was detected. Benzo[a]pyrene hydroxylase activity was found to be present although in a minimal amount. The evaluation of the molecular weight of the rRNA demonstrates that the larger ribosomal subunit contains RNA of Mr 1.40 X 10(-6) (approximately 26S) and the smaller subunit is composed of RNA of Mr 0.65 X 10(-6) (18S). The data from mussel digestive gland microsomes was compared with that experimentally obtained from rat liver microsomes and discussed from a functional or an evolutionary point of view.     

Virus-specific RNA synthesis in the midgut of silkworm, Bombyx mori, infected with cytoplasmic-polyhedrosis virus

Virus-specific RNA synthesis in the midgut of silkworm infected with cytoplasmic-polyhedrosis virus was investigated under the condition inhibiting host RNA synthesis by actinomycin D injection. Two species of virus-induced RNA were formed; one was sensitive to ribonuclease (RNase) but the other was resistant. The resistant RNA had a sedimentation coefficient of 15 S and was considered as viral progeny with doublestranded RNA. The sensitive RNA, presumably single-stranded RNA, consisted of two classes with 15 S and 22 S sedimentation coefficients. Annealing the single-stranded RNA with heat-denatured CPV-RNA indicated that the single-stranded RNA was transcribed from viral genome RNA. The function of 22 S and 15 S single-stranded RNAs was discussed from the viewpoint of virus multiplication.     

Analysis of Aleutian disease virus infection in vitro and in vivo: demonstration of Aleutian disease virus DNA in tissues of infected mink.

Aleutian disease virus (ADV) infection was analyzed in vivo and in vitro to compare virus replication in cell culture and in mink. Initial experiments compared cultures of Crandell feline kidney (CRFK) cells infected with the avirulent ADV-G strain or the highly virulent Utah I ADV. The number of ADV-infected cells was estimated by calculating the percentage of cells displaying ADV antigen by immunofluorescence (IFA), and several parameters of infection were determined. Infected cells contained large quantities of viral DNA (more than 10(5) genomes per infected cell) as estimated by dot-blot DNA-DNA hybridization, and much of the viral DNA, when analyzed by Southern blot hybridization, was found to be of a 4.8-kilobase-pair duplex monomeric replicative form (DM DNA). Furthermore, the cultures contained 7 to 67 fluorescence-forming units (FFU) per infected cell, and the ADV genome per FFU ratio ranged between 2 X 10(3) and 164 X 10(3). Finally, the pattern of viral antigen detected by IFA was characteristically nuclear, although cytoplasmic fluorescence was often found in the same cells. Because no difference was noted between the two virus strains when cultures containing similar numbers of infected cells were compared, it seemed that both viruses behaved similarly in infected cell culture. These data were used as a basis for the analysis of infection of mink by virulent Utah I ADV. Ten days after infection, the highest levels of viral DNA were detected in spleen (373 genomes per cell), mesenteric lymph node (MLN; 750 genomes per cell), and liver (373 genomes per cell). In marked contrast to infected CRFK cells, the predominant species of ADV DNA in all tissues was single-stranded virion DNA; however, 4.8-kilobase-pair DM DNA was found in MLN and spleen. This observation suggested that MLN and spleen were sites of virus replication, but that the DNA found in liver reflected sequestration of virus produced elsewhere. A final set of experiments examined MLN taken from nine mink 10 days after Utah I ADV infection. All of the nodes contained ADV DNA (46 to 750 genomes per cell), and although single-stranded virion DNA was always the most abundant species, DM DNA was observed. All of the lymph nodes contained virus infectious for CRFK cells, but when the genome per FFU ratio was calculated, virus from the lymph nodes required almost 1,000 times more genomes to produce an FFU than did virus prepared from infected cell cultures.(ABSTRACT TRUNCATED AT 400 WORDS)     

Identification of Saint Louis encephalitis virus mRNA.

Saint Louis encephalitis (SLE) virus-specific RNA was recovered from infected HeLa cells by sodium dodecyl sulfate (SDS)-phenol-chloroform extraction, and the molecular species were resolved by SDS-sucrose gradient centrifugation and agarose gel electrophoresis. Sucrose gradient centrifugation revealed the presence of a 45S species, minor 20 to 30S heterogeneous species, and an 8 to 10 S RNA species in the cytoplasmic extract. Analysis of the same samples by electrophoresis on agarose gels, under both nondenaturing and denaturing conditions, revealed only two virus-specific RNA molecules, the 45S genome-sized RNA and an 8 to 10S species. Varying the gel concentration to facilitate analysis of nucleic acids with molecular weights ranging from 25,000 to 25 X 10(6) failed to reveal additional RNA species, although low levels of a putative double-stranded replicative form could conceivably have escaped detection. From our observations it appears that the heterogeneous RNA species and presumably the 20S RNase-resistant species reported in other investigations of flavivirus RNA are degradation products or conformers of the 45S molecule. Polysomes from SLE virus-infected cells were prepared and separated from contaminating nucleocapsid by centrifugation on discontinuous sucrose gradients. RNA extracted from these polysome preparations was analyzed by sucrose gradient centrifugation and agarose gel electrophoresis. The 45S SLE virus genome-size molecule was found to be the only RNA species associated with the polysomes. This molecule was sensitive to RNase digestion and was released from polysomes by EDTA and puromycin treatment. These findings provide direct evidence that the 45 S SLE virus RNA serves as the messenger during virus replication, in contrast to the 26S RNA species which functions as the predominant messenger during alphavirus replication.     

Characterization of Bluetongue Virus Ribonucleic Acid

An improved purification procedure yielded bluetongue virus free from any single-stranded ribonucleic acid (RNA) component. Double-stranded RNA obtained from purified virus or isolated from infected cells was fractionated into 5 components by means of sucrose gradient sedimentation analysis, and into 10 components by electrophoresis on polyacrylamide gels. The size of these components vary from 0.5 x 10(6) to 2.8 x 10(6) daltons, with a total molecular weight estimate of about 1.5 x 10(7) for the viral nucleic acid. The denaturation of the genome and separation of the resulting fragments are also discussed.