Maize stripe tenuivirus RNA2 transcripts in plant and insect hosts and analysis of pvc2, a protein similar to the Phlebovirus virion membrane glycoproteins

The complete sequence of the maize stripe tenuivirus (MstV) RNA2 was determined (3337 nucleotides). RNA2 contains two large open reading frames (ORFs) arranged in an ambisense orientation and specific RNAs of ca. 700 and 2600 nucleotides corresponding to the ORFs were detected in MStV-infected plants and planthoppers. The deduced amino acid sequence of the 23,500 MW protein (pv2) encoded by viral RNA2 (vRNA2) was similar to proteins encoded by the rice stripe (RStV) and rice hoja blanca tenuiviruses vRNA2. Sequence analysis suggested that pv2 is membrane associated. The 93,900 MW protein (pvc2) encoded by viral complementary MStV RNA2 (vcRNA2) was similar to the 94,000 MW protein of RStV RNA2 and to the virion membrane glycoproteins for Phlebovirus members of the Bunyaviridae. The phlebovirus glycoprotein cleavage sitewas similar to a region in the MStV and RStV proteins suggesting that the tenuivirus pvc2 may be processed analogous to the phlebovirus glycoproteins.     

A comparison of the nucleotide sequence homologies of three isolates of bean yellow mosaic virus and their relationship to other potyviruses

3H-labelled complementary DNA (cDNA) was reverse transcribed from the RNA of three biologically distinct isolates of bean yellow mosaic virus (BYMV-G, Q, and S) by the random primer method of Taylor et al. [Biochim. Biophys. Acta 442, 324-330 (1976)]. Excess virus RNA was hybridized with each of the cDNAs and percentage hybrid formation was assayed using the single-strand-specific nuclease S1, The R0t (1/2) values obtained for the homologous hybridization reactions (1.2 x 10(-2) mol sec liter(-1)) showed that all three BYMV-RNAs were unique species, without detectable zones of reiteration. Reciprocal hybridizations between isolates G, Q, and S have shown that each was significantly different from the other. A comparison of these three isolates with a selection of other potyviruses showed that the relationship between them was closer than that observed between any of them and the other potyviruses tested.     

Electrophoresis of the complementary strands of the double-stranded Kemerovo virus RNAs in agarose-urea gel

Single-stranded (ss)RNAs derived from 10 double-stranded (ds)RNA segments of Kemerovo virus (KV) were separated into 13 RNA bands by agarose-urea gel electrophoresis. The complementary strands of the dsRNA segments 1, 9 and 10 displayed different electrophoretic mobility. An attempt was made to determine the origin of the ssRNA bands. The ssRNA bands originating from the dsRNA segments 1, 2, 3, 9 and 10 were identified unequivocally, while those originating from the dsRNA segments 4, 5, 6, 7 and 8 were characterized partially. The minus RNA strands of the dsRNA segments 9 and 10 exhibited higher electrophoretic mobilities as their complementary plus RNA strands.     

Replication of tobacco mosaic virus. VII. Further characterization of single- and double-stranded virus-related RNAs from TMV-infected plants

The single-stranded (ss) and double-stranded (ds) RNAs produced in tobacco tissue as a result of infection by tobacco mosaic virus (TMV) have been reinvestigated. 32P-labeled probes consisting of either cDNA or viral RNA, complementary to specific regions of either the viral RNA or its negative strand, respectively, were used in "Northern" hybridization experiments. Of the 10 ssRNA bands observed, all but four appeared to be artifacts of electrophoresis. These four RNAs were found on polyribosomes and are presumed to be true mRNAs; three were identified as the well-known genomic RNA, the I2-mRNA and the coat protein mRNA, or LMC. The fourth RNA species of MW approximately 1.2 x 10(6) had not previously been specifically identified as a subgenomic RNA of TMV. The viral RNA which gave rise to the six artifactual ssRNA bands was heterogeneous in size and was shown to be encapsidated in vivo. Upon electrophoresis, these heterogeneous RNA fragments comigrated approximately with plant rRNAs also present in the extracts, generating the observed artifactual bands. Four dsRNAs were also identified. From molecular weight and hybridization analyses, they appeared to be double-stranded forms of the above four polyribosome-associated ssRNAs. Attempts to translate proteins from the denatured dsRNAs in vitro were unsuccessful. A population of low-molecular-weight, TMV-specific ssRNAs, (+) and (-) in sequence, was generated during infection; however these RNAs were believed to be breakdown products.     

Analysis of the terminal sequences of the genome segments of four orbiviruses

The dsRNA genome segments of bluetongue virus (BTV) types 1 and 20 and Ibaraki virus (a member of the epizootic haemorrhagic disease (EHD) serogroup) have conserved sequences of six bases at both of their 3' termini. One strand of all the genome segments analysed ends in 3'CAUUCA ... 5' while the other strand ends in 3'CAAUUU ... 5'. These conserved sequences are identical to those previously reported for BTV types 10 and 11 (A. Kiuchi, C. D. Rao, and P. Roy (1983), "Double-Stranded RNA Viruses" (R. W. Compans and D. H. L. Bishop, eds.), pp. 55-64. Elsevier, New York; C. D. Rao, A. Kiuchi, and P. Roy (1983), J. Virol. 46, 378-383). The 3' terminal sequences of segments 3 and 10 of the BTV type 1 genome were confirmed by the detection of exactly complementary sequences at the 5' termini of the ssRNA strands of opposite polarity. This also confirmed for these dsRNA segments (and by analogy for all the genome segments of these viruses) that the dsRNA molecules are fully base paired end to end. Using in vitro synthesised mRNA of BTV type 1 in annealing experiments with the two ssRNAs separated from each of the individual genome segments, it was shown that in each case the strand ending in 3'CAUUCA ... 5' is of the same polarity as the mRNA (+ve), while the strand ending in 3'CAAUUU ... 5' is of the opposite (-ve) polarity. The fourth virus analysed (Tilligerry virus, a member of the Eubenangee serogroup) only had five conserved bases at the 3' termini of one strand of its genome segments (3'CAU-CA ... 5') and three conserved bases at the 3' termini of the other strand (3'CA--U ... 5'). Considerable sequence homology was found in the near-terminal nonconserved regions of comparable genome segments from the different viruses, particularly between the different BTV types. There was little evidence, however, for absolute conservation of "segment specific" sequences in these regions of the RNA.     

Nucleotide sequence homology of thirteen tobamovirus RNAs as determined by hybridization analysis with complementary DNA

The extent of RNA sequence homology between 13 isolates of tobamoviruses has been estimated by hybridization analysis using radioactive complementary DNA. Hybrid formation between complementary [3H]DNA prepared against 7 of the tobamovirus RNAs and the RNAs of the 13 isolates was assayed with the single-strand-specific S1 nuclease. The presence of mismatched regions in the DNA: RNA hybrids was shown by variation of the stringency of the conditions for the hybridization and S1 nuclease assay. The data obtained have allowed the allocation of the 13 isolates into five groups on the basis of significant sequence homology between members of each group. Members of two of these groups were further divided into subgroups on the basis of differences in the hybridization data. Although hybridization analysis using labeled complementary DNA is considered the method of choice at present for estimating sequence homology between viral RNAs, the limitations inherent in any hybridization approach are discussed.     

Calicivirus intracellular RNA: fractionation of 18-22 s RNA and lack of typical 5'-methylated caps on 36 S and 22 S San Miguel sea lion virus RNAs

Radiolabeled RNA from Vero cells infected with a calicivirus, San Miguel sea lion virus type 2, in the presence of actinomycin D, was fractionated by glycerol density gradient sedimentation to yield 36 S single-stranded RNA (ssRNA) and heterogenous RNA sedimenting in the 18–22 S region. Further fractionation of 18–22 S RNA by LiCl precipitation, CF-11 cellulose chromatography, and polyacrylamide gel electrophoresis yielded 22 S ssRNA, two doule-stranded RNAs )dsRNA), and a partially single-partially double-stranded RNA (ss/dsRNA). Sedimentation analysis indicated the major and minor dsRNAs were equivalent to two strands of 36 S and 22 S ssRNAs, respectively, and the dsRNA core of ss/dsRNA was equivalent to the major dsRNA. Neither 22 S ssRNA (presumed subgenomic mRNA) nor 36 S ssRNA (presummed full genomic RNA plus intracellular virion RNA) had the typical 5′-methylated cap.     

Fig mosaic virus mRNAs show generation by cap-snatching

Fig mosaic virus (FMV), a member of the newly described genus Emaravirus, has four negative-sense single-stranded genomic RNAs, and each codes for a single protein in the viral complementary RNA (vcRNA). In this study we show that FMV mRNAs for genome segments 2 and 3 contain short (12-18 nucleotides) heterogeneous nucleotide leader sequences at their 5′ termini. Furthermore, by using the high affinity cap binding protein eIF4E_K119A, we also determined that a 5′ cap is present on a population of the FMV positive-sense RNAs, presumably as a result of cap-snatching. Northern hybridization results showed that the 5′ capped RNA3 segments are slightly smaller than the homologous vcRNA3 and are not polyadenylated. These data suggest that FMV generates 5′ capped mRNAs via cap-snatching, similar to strategies used by other negative-sense multipartite ssRNA viruses.     

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.     

Transcription and replication of eight RNA segments of influenza virus

A novel quantitation system of both plus- and minus-strand RNAs for all eight genome segments of influenza virus was developed using single-strand cDNAs as the probes for hybridization, and employed for the measurement of various RNA species in influenza virus WSN-infected MDBK cells. The synthesis rate and accumulation level of plus-strand RNAs differed considerably among eight RNA segments and were under temporal control. In contrast, eight vRNA molecules of minus polarity were synthesized coordinately at similar rates. Newly synthesized plus-strand RNAs were rapidly transported into the cytoplasm, particularly during the early phase of virus infection, but vRNAs accumulated in the nuclei until the late infection phase. The present data supported the differential regulation of synthesis and the separate transport between plus- and minus-strand RNAs.     

Characterization of X-tropic endogenous retrovirus-specific RNA uninfected tissues and lymphosarcoma of BALB/c mice

Analysis of endogenous X-tropic BALB/c retrovirus-specific RNAs in a BALB/c mouse tissue containing mostly non-driving cells (liver), in two normal tissues having significant proportions of dividing cells (20-h regenerating liver and 12-day embryo) and in cells of a lymphosarcoma of BALB/c mice were carried out by determining the extent to which the RNAs from these tissues hybridized to the virus 3H-cDNA probe and by their relative sedimentation values in a sucrose gradient. RNAs from 20-h regenerating liver, 12-day embryo and lymphosarcoma, each containing a significant proportion of proliferating cells, showed 8 to 21 % higher hybridization values than normal liver RNA. Differences in the exact size classes of virus-specific RNAs and, in their relative proportions were found to exist in the four different tissue types examined and no correlation between a specific RNA size-profile and the proliferating activity of a tissue could be detected.