A.O. Jackson

Investigation of the complexity of barley stripe mosaic virus RNAs with recombinant dna clones

RNA isolated from the Type, ND18, and Norwich strains of barley stripe mosaic virus (BSMV) was electrophoresed in agarose gels, transferred to nitrocellulose, and hybridized with BSMV-specific complementary DNA (cDNA) or recombinant DNA clones derived from ND18 RNA. Genomic RNA components 1 (Mr = 1.43 x 10(6)) and 2 (Mr = 1.24 x 10(6)) were resolved in all three strains, but RNA 3 (Mr = 1.1 x 10(6)) was seen only in the ND18 and Norwich strains. A low-molecular-weight RNA (Mr = 0.27 x 10(6)), thought to be a subgenomic (SG) RNA, was also detected in RNA preparations from all three strains by staining with toluidine blue or ethidium bromide and by hybridizing with cDNA or selected recombinant DNA probes. Three classes of recombinant DNA clones, designated pBSM1, pBSM2, and pBSM3, were identified by hybridization of nick-translated recombinant DNA to electrophoretically separated viral RNAs. Clones in the pBSM1 class hybridized only to RNA 1 of all three strains and class pBSM2 clones hybridized only to RNA 2 of all three strains. Class pBSM3 clones hybridized to RNA 3 of the ND18 and Norwich strains and to RNA 2 of the Type strain, but not to RNA 2 of ND18 or Norwich. Based on the sizes of the BSMV-specified inserts in clones designated pBSM1a, pBSM2a, and pBSM3a, we estimate that a minimum of 44, 63, and 63% of the nucleotide sequences of ND18 and Norwich RNAs 1, 2, and 3, respectively, are unique. Furthermore, because the combined size of the inserts in pBSM2a and pBSM3a is approximately 15% greater than the estimated size of RNA 2, it is probable that the second RNA component of the Type strain actually consists of two RNA species which are similar in size but have different sequences. The SG RNA component is viral specific and contains sequences common to clones derived from RNA 3.

Comparative analysis of polypeptides synthesized in vivo and in vitro by two strains of barley stripe mosaic virus.

Proteins synthesized in a wheat germ system containing RNA from the Type or ND18 strain of barley stripe mosaic virus (BSMV), or polyribosomes from Type or ND18-infected barley, were compared with each other and with polypeptides from Type and ND18-infected plants. Polypeptides with apparent molecular weights (Mr) of 25, 67, and 120 x 10(3) that were induced in infected barley were synthesized in vitro by polyribosomes from infected plants or RNA from purified BSMV. The 25,000 Mr polypeptide was identified as BSMV coat protein by coelectrophoresis with coat protein from purified virions, by immunoprecipitation, and by absence of [35S]methionine incorporation into the protein. Strain-specific differences were observed in two additional translation products synthesized in both wheat germ and reticulocyte lysate systems containing Type of ND18 RNA. RNAs from both strains directed synthesis of 71 and 82 x 10(3) Mr polypeptides. However, ND18 RNA directed synthesis of a larger amount of the 71,000 Mr polypeptide, whereas with Type RNA the 82,000 Mr polypeptide predominated. These two proteins may be processed in vivo because reduced synthesis of both polypeptides in wheat germ extracts containing polyribosomes from infected plants was correlated with increased synthesis of the 67,000 Mr polypeptide. Several additional polypeptides synthesized in the wheat germ system were probably premature termination products because their synthesis in the reticulocyte lysate was greatly reduced.

Partial characterization of the structural proteins of sonchus yellow net virus

Abstract Sonchus yellow net virus (SYNV) proteins separate into four major and three minor proteins during polyacrylamide gel electrophoresis (PAGE). The major proteins consist of a glyoprotein (G), a nucleoprotein (N), and two proteins thought to be membrane proteins (M-1 and M-2). Minor proteins include two or three high molecular weight proteins (L) and a protein (possibly NS) with an electrophoretic mobility near the N protein. Proteins of potato yellow dwarf virus (PYDV) are similar in number and staining intensity to those of SYNV after PAGE, but the electrophoretic mobilities of proteins from the two viruses differ. SYNV dissociates with 2% Triton X-100 in 0.4 M NaCl at pH 7.5 to produce a 250 S nucleoprotein core plus solubilized L, G, and M proteins. A 200 S core is released at pH 7.5 in 0.8 M NaCl or at pH 8.4 in 0.4 or 0.8 M NaCl. The 250 S core contains all of the N protein and some undissociated L, G, and M proteins, but the 200 S core contains N protein and only traces of the L, G, and M proteins. After fixation with glutaraldehyde and negative staining, both 200 and 250 S cores are loosely coiled strands with a helical structure. The cores are infectious after being treated with detergent and separated on sucrose gradients, but the infectivity is considerably less than intact virus preparations. Iodination patterns of intact and detergent-disrupted virions suggest that L and G proteins are on the exterior of the virion, that M-2 is partially exposed on the virion surface, and that M-1 and N proteins are located beneath the surface of the virion.

Sequence complementarity of sonchus yellow net virus RNA with RNA isolated from the polysomes of infected tobacco.

Polyribosomal RNA from tobacco infected with sonchus yellow net virus (SYNV) contained sequences which hybridized to 125I-labeled SYNV RNA and which were complementary to 80 to 100% of the viral RNA genome. The poly(A)-containing RNA from polyribosomes was complementary to over 90% of the viral genome but the polyribosomal RNA lacking poly(A) hybridized to approximately 40-60% of the genome. The kinetics of hybridization of all three fractions are best explained by the presence of a single abundance class of viral-complementary RNA. However, titration hybridization of poly(A)+ RNA to an excess of SYNV RNA suggested that viral-complementary sequences which contain poly(A) may vary in concentration over a factor of about fivefold. About 1.5 to 4.6% of the fraction containing poly(A), 0.02 to 0.06% of the fraction lacking poly(A) and 0.04 to 0.18% of the total polyribosomal RNA was complementary to viral RNA as estimated from the kinetics of hybridization. The viral complementary RNA(vcRNA) was heterogeneous in size with a modal sedimentation coefficient of 12 S and a profile in sucrose density gradients similar to the polyadenylated polyribosomal RNA.

Complexity of the Argentina mild strain of barley stripe mosaic virus.

Barley stripe mosaic virus (BSMV) is known to have unusual strain-specific variations in the number of high molecular weight RNAs that can be resolved by gel electrophoresis. Analysis with recombinant DNA clones has revealed that all strains contain three distinct hybridizing species of RNA, which we now designate alpha, beta and y. The alpha- and beta-RNAs are about 4200 and 3650 nucleotides (NT), respectively, but the gamma-RNAs vary in size depending on the strain. The gamma-RNA of the Type strain is 3650 NT, and is difficult to resolve from beta-RNA by gel electrophoresis. However, the gamma-RNA of the North Dakota 18 (ND18) strain is 3250 NT and is clearly separated from beta-RNA. The results presented below show that the Argentina mild (AM) strain contains a mixture of gamma-RNAs of 3650, 3250, and 2900 NT. The 3650- and 3250-NT gamma-RNAs appear to be functional because AM subclones containing either 3650- or 3250-NT gamma-RNA can be isolated from plants inoculated with low concentrations of virus. The 2900-NT gamma-RNA is probably not capable of functioning as a genomic RNA because this RNA is not a component of any of the AM subclones.

Low-molecular-weight RNAs associated with tobacco ringspot virus are satellites.

The low-molecular-weight RNA associated with an isolate of tobacco ringspot virus [TRSV(F)] lacks polyadenylic acid [poly(A)] sequences. Complementary DNA (cDNA) specific to this RNA was made with an oligo(dT) primer after tailing the RNA with poly(A). However, a significant amount of cDNA was made only when the RNA was treated with alkaline phosphatase prior to tailing. Hybridization of RNA in solution and by a Northern blot technique showed that there was no detectable nucleotide sequence homology between TRSV(F) RNA and two of the low-molecular-weight RNAs associated with this virus. These associated RNAs are therefore, satellites of TRSV(F). The relationship of these satellites to a previously reported satellite of TRSV is discussed.

Subcellular distribution of RNA sequences complementary to sonchus yellow net virus RNA

RNA isolated from free and membrane-bound polyribosomes of sonchus yellow net virus (SYNV)-infected tobacco was hybridized to SYNV RNA. RNA from free polyribosomes was shown to be complementary to nearly 100% of the SYNV genome, whereas RNA from membrane-bound polyribosomes was complementary to only 40% of the SYNV RNA. When RNA derived from the two classes of polyribosomes was fractionated by chromatography on oligo(dT)-cellulose, sequences complementary to SYNV RNA were present in both bound and unbound fractions. Neither nuclear RNA nor poly(A)-containing nuclear RNA from SYNV-infected plants hybridized to SYNV RNA. The results suggest that SYNV messenger RNAs are selectively partitioned in the cytoplasm and that hybridizing sequences are not abundant in the nuclei of infected cells.

Characterization of Viral-complementary RNA Associated with Polyribosomes from Tobacco Infected with Sonchus Yellow Net Virus

SUMMARY Polyribosomal RNA from tobacco infected with sonchus yellow net virus was resolved by electrophoresis on agarose gels, transferred to nitrocellulose and hybridized to radioactive viral RNA. Polyadenylated RNA contained four major viral complementary species, molecular weights 2.2 x 106, 0.83 x 106, 0.62 x 106 and 0.46 × 106, plus a minor species, molecular weight approx. 3 × 106. No such species could be detected in the non-polyadenylated RNA fraction. The polyadenylated RNA from membrane-bound polyribosomes contained only the 0-83 × 106 and 0-46 x 106 molecular weight RNAs. The sizes of the RNAs were consistent with the expected sizes of messages for the virus proteins, calculated from the molecular weight of these proteins. Sonchus yellow net virus (SYNV) is a member of the rhabdovirus group (Jackson & Christie, 1977). Although at least 38 members of this group infect plants (Peters, 1981), the replicative mechanisms of these viruses, in contrast to some of the animal rhabdoviruses, have not been well characterized..The plant and animal rhabdoviruses share many morphological and biochemical properties. Vesicular stomatitis virus (VSV) has been extensively studied and its replicative mechanisms may share features with those of the plant rhabdoviruses. However, both plant and animal rhabdoviruses can be divided into subgroups on the basis of structural protein composition, serological relationships, intracellular site of virus accumulation, etc. (Toriyama & Peters, 1981; Tesh et al., 1983). The degree to which the mechanisms of replication of rhabdoviruses from different subgroups resemble each other and those of VSV remains unknown. In cells infected with VSV, five subgenomic polyadenylated RNAs are transcribed from the