Shotgun sequencing of the negative-sense RNA genome of the rhabdovirus Maize mosaic virus

The maize-infecting nucleorhabdovirus, Maize mosaic virus (MMV), was sequenced to near completion using the random shotgun approach. Sequences of 102 clones from a cDNA library constructed from randomly-primed viral RNA were compiled into a 12,133 nucleotide (nt) contig containing six open reading frames. The contig consisted of 97 sequences averaging 660 bp in length. The average sequence coverage was six-fold, and 93% of the contig had sequence reads covering both strands. The remaining sequence was derived from single (5%) or multiple (2%) reads on the same strand. Three of the six ORFs showed significant similarities to the deduced protein sequences of the nucleocapsid, glycoprotein and polymerase sequences of other rhabdoviruses. The predicted gene order of the MMV genome was 3'-N-P-3-M-G-L-5'. Shotgun sequencing of the MMV genome took approximately 127 h and cost 0.38 dollars per nt (including labor), whereas the primer walking approach for sequencing the 13,782-nt MFSV genome [Tsai, C.-W., Redinbaugh, M.G., Willie, K.J., Reed, S., Goodin, M., Hogenhout, S. A., 2005. Complete genome sequence and in planta subcellular localization of maize fine streak virus proteins. J. Virol. 79, 5304-5314] took about 217 h and cost 0.50 dollars per nt. Thus, the shotgun approach gave good depth of coverage for the viral genome sequence while being significantly faster and less expensive than the primer walking method. This technique will facilitate the sequencing of multiple rhabdovirus genomes.     

Translation of turnip rosette virus RNA in rabbit reticulocyte lysates

Turnip rosette virus (TRosV) RNA was translated in nuclease-treated rabbit reticulocyte lysates, and four principal products (radioactive proteins always found under routine conditions) with molecular weights of 105,000, 67,000, 35,000, and 30,000 were synthesized. Optimum conditions for translation included 50 mM added K+, no added Mg2+, TRosV RNA at 25 to 50 mug/ml, incubation for 40 to 60 min; these conditions gave 20-fold stimulation in the incorporation of [35S]methionine over endogenous background levels. The 30,000 MW protein was identified as virus capsid protein by peptide mapping of specific immunoprecipitates. Most efficient synthesis of the capsid protein was observed for a virus-encapsidated subgenomic RNA (denatured MW 0.5 x 10(6)). The major genome products of 105,000, 67,000, and 35,000 MW were shown to be related by peptide analysis following partial proteolysis. Time course analyses suggested that the 67,000 product was processed from the 105,000 product by post-translational cleavage. The protease activity responsible for product cleavage was apparently viral coded and protease inhibitor studies further indicated the enzyme was of the thiol type. In an initiation assay using anisomycin, two ribosomes bound per TRosV RNA. The strategy of translation as determined for the virus here is discussed.     

Aminoacylation and messenger functions of eggplant mosaic virus RNA

The predominant RNA of eggplant mosaic virus (EMV) was found to have a molecular weight of 1.9 x 10(6) by formamide gel electrophoresis. Electrophoretic analysis of virion protein under dissociating conditions revealed two polypeptides, a major component of 21,000 daltons, and a minor component of 22,000 daltons. Both peptides were present in translation products coded by RNA isolated from virions, but the proportion of the 22,000-dalton peptide was higher in products synthesized using RNA isolated from EMV-infected Datura leaves as messenger. Since the viral RNA showed a marked tendency to aggregate, it is possible that these polypeptides were translated from trace amounts of small EMV RNAs present as contaminants of the 1.9 x 10(5)-dalton genomic RNA. Although the addition of tRNA from infected or healthy Datura leaves, or from wheat germ, stimulated amino acid incorporation, no changes were discerned in the profile of cell-free translation products after electrophoretic separation. Nonaminoacylated and valylated EMV RNA stimulated similar levels of amino acid incorporation, and the translation products appeared identical. Valine bound to genomic EMV RNA was not donated during protein synthesis.     

Infectivity of partially encapsidated tobacco mosaic virus RNA

Tobacco mosaic virus was reconstituted in vitro, and the infectivity of intermediate products at various stages of reconstitution was investigated in relation to their rod length. The infectivity of intermediates was exponentially related to their rod length throughout the reconstitution process, indicating that no particular region of TMV-RNA is preferentially susceptible to inactivation or more important for infection.     

Purification and characterization of brome mosaic virus RNA-dependent RNA polymerase

RNA-dependent RNA polymerase (RdRp) was solubilized from cellular membranes of brome mosaic virus (BMV)-infected barley. The solubilized enzyme was subsequently purified by glycerol gradient centrifugation and DEAE ion-exchange chromatography. The purified enzyme proved to be highly stable and both dependent on and specific for BMV RNAs. The enzyme is inhibited by high template RNA concentrations. This inhibition indicates feedback regulation of minus-strand synthesis. The nonstructural viral protein P1 was found to be a component of the RdRp complex (R. Quadt, H.J.M. Verbeek, and E.M.J. Jaspars, 1988, Virology 165, 256-261). Using antibodies directed against a C-terminal peptide of P1 a complex of seven 125I-labeled proteins was precipitated. This indicates that the P1 protein is associated with at least six proteins in the infected cell.     

Nucleotide sequence and translation of satellite tobacco mosaic virus RNA

Satellite tobacco mosaic virus (STMV) is a plant virus with a 17-nm icosahedral particle encapsidating a 0.3 X 10(6) Mr ssRNA genome that depends on tobamoviruses for its replication. The complete nucleotide sequence of STMV RNA deduced in the experiments described here was 1059 nucleotides in length. The efficiency of labeling viral RNA with [gamma-32P]ATP using T4 polynucleotide kinase was not affected by treatment with tobacco acid pyrophosphatase and/or bacterial alkaline phosphatase, indicating that the majority of the 5' termini of encapsidated STMV RNAs were not phosphorylated. The 240 3'-terminal nucleotides of STMV RNA and either tobacco mosaic virus (TMV) U1 RNA or TMV U2/U5 RNA had greater than 65% overall sequence similarity, with two nearly identical regions of 40 and 50 bases, respectively. There were no other regions of sequence relatedness to TMV RNA. The 19 5'-terminal nucleotides of STMV RNA had greater than 65% sequence similarity with the 16 5'-terminal nucleotides of brome mosaic virus (RNA 3 and 50% sequence similarity with the 12 5'-terminal nucleotides of the Q strain of cucumber mosaic virus RNA 3. The first open reading frame (ORF) beginning at base 53 encoded a 6800 Mr protein that corresponded in size to a major in vitro translation product directed by STMV RNA. A second ORF, beginning at nucleotide 163, had the capacity to code for a protein that corresponded in size (17,500 Mr) to the other major in vitro translation product. The first 12 codons of this ORF corresponded to the sequence of the N-terminal amino acids of the capsid protein. Western-blot analysis of the in vitro translation products revealed that the 17,500 Mr protein had the same electrophoretic mobility as the authentic capsid protein; it was also antigenically related to the capsid protein, but the 6800 Mr protein was not. Time course analysis of in vitro translation demonstrated that the 6800 Mr protein was synthesized at the same time as the capsid protein and did not arise by the proteolytic cleavage of a larger precursor polypeptide. These results suggest that the genome of STMV functioned as a polycistronic messenger RNA. It has not been determined if the 6800 Mr protein is synthesized in vivo. STMV RNA had untranslated regions of 52 and 418 nucleotides at its 5' and 3' termini, respectively.(ABSTRACT TRUNCATED AT 400 WORDS)     

Differential replication in zucchini squash of a cucumber mosaic virus satellite RNA maps to RNA 1 of the helper virus.

Cucumber mosaic virus (CMV) supports the replication and encapsidation of its satellite RNA, both in solanaceous and cucurbit host plants; however, different strains of CMV support the replication of satellite RNAs with different efficiency. In addition, replication of satellite RNA is very efficient in solanaceous host plants and generally poor in cucurbit host plants. The WL1-satellite (WL1-sat) RNA is an exception, and replicates to high levels in both solanaceous and curcubit host plants with most CMV strains as the helper virus. Two strains of CMV were used in this study: Fny-CMV, which replicates the WL1-sat RNA efficiently in all hosts tested; and Sny-CMV, which does not replicate the WL1-sat RNA to detectable levels in zucchini squash (Cucurbita pepo), but does replicate WL1-sat RNA efficiently in other hosts. Using pseudorecombinants constructed between Fny-CMV and Sny-CMV we have mapped to RNA 1 the ability to support the efficient replication of WL1-sat RNA in zucchini squash.     

Molecular epidemiology of Cucumber mosaic virus and its satellite RNA

Molecular analysis of viral isolates can yield information that facilitates an understanding of virus epidemiology and has been termed molecular epidemiology. This approach has only recently been applied to plant viruses. Results on the molecular epidemiology of Cucumber mosaic virus (CMV) and its satellite RNA (satRNA) in Spain, where CMV is endemic in vegetable crops are presented here. To characterise the genetic structure of CMV populations, c. 300 isolates, representing 17 outbreaks (i.e. sub-populations) in different crops, regions and years, were compared. Genetic analyses of CMV isolates were done by ribonuclease protection assay of cRNA probes representing RNA1, RNA2 and the two open reading frames in RNA3. All isolates belonged to one of three genetic types: Sub-group II and two types of Sub-group I. The genetic structure of the 17 sub-populations varied randomly, without correlation with location, year, or host plant species. Thus, CMV in Spain shows a metapopulation structure with local extinction and random recolonisation from local or distant virus reservoirs. The frequency of mixed infections and of new genetic types generated by reassortment of genomic segments or by recombination was also estimated. Results indicate that heterologous genetic combinations are not favoured. About 30% of CMV isolates were supporting a satRNA. The frequency of CMV isolates with a satRNA differed for each sub-population, being c. 1 in eastern Spain in 1990 and decreasing to c. 0 in distant regions and in subsequent years. Molecular analyses of CMV-satRNA isolates show high genetic diversity, due both to the accumulation of point mutations and to recombination. The CMV-satRNA population is a single, unstructured one. Thus, the CMV-satRNA population has a genetic structure and dynamics different from those of its helper virus. This indicates that CMV-satRNA has spread epidemically on the extant virus population from an original reservoir in eastern Spain. The relevance of these results for the control of CMV infections is discussed.     

Sequence and organization of southern bean mosaic virus genomic RNA

The genomic RNA sequence of the cowpea strain of southern bean mosaic virus (SBMV-C) has been determined. The genome is 4194 nucleotides in length and has four open reading frames. A 5' proximal open frame, from base 49 to base 603, corresponds to the length of the P4 proteins translated in cell-free extracts from full-length and smaller virion RNA. The largest open frame extends from base 570 to base 3437 and encodes the two largest proteins translated in cell-free extracts from full-length virion RNA. Segments of this open reading frame's predicted amino acid sequence resemble those of known viral RNA polymerases, ATP-binding proteins, and viral genome-linked proteins. A third open frame extends from base 1895 to base 2380 and has not been correlated with an in vitro translation product. The fourth open reading frame is located in the 3' terminal region of the genome extending from base 3217 to base 4053. This frame encodes the SBMV capsid protein which is translated from subgenomic, virion RNA.     

Does tobacco mosaic virus RNA contain cytokinins?

Tobacco mosaic virus was previously reported to contain between 5 and 17 modified nucleosides with cytokinin activity per RNA molecule. Using a new and highly sensitive method for gas chromatography of permethylated cytokinins, we were unable to detect cytokinins in enzymic or alkaline digests of TMV RNA. The method was sensitive enough to assay 1 cytokinin nucleoside per 10 TMV RNA molecules. Soybean callus bioassay also failed to detect activity in TMV RNA hydrolysates which corresponded to any of the cytokinin nucleosides known to occur in RNA. We therefore suggest that TMV RNA does not contain cytokinin nucleosides.     

Complete sequence of the Pepino mosaic virus RNA genome

We have determined the complete nucleotide sequence (Accession No. AF484251) of the Pepino mosaic virus (PepMV) RNA genome. PepMV is the etiological agent of a new disease which affects tomato crops in Europe and North America. The PepMV genome consists of one single stranded positive sense RNA 6410 nt long that contains five open reading frames (ORFs). ORF 1 is the putative RNA dependent RNA polymerase (RdRp), as it has the characteristic methyltransferase, NTP-binding and polymerase motifs. ORF 2 to 4 form the PepMV triple gene block. ORF 5 codes for the capsid protein. Two short untranslated regions flank the coding regions and there is a poly(A) tail at the 3'end of the genomic RNA. Thus, the genome organization of PepMV is that of a typical member of the genus Potexvirus. The nucleotide sequence obtained shares an overall 99% identity with the genomic RNA of a PepMV isolate from UK which has been partially sequenced. Protein coded by ORF4 is the least conserved between both isolates (95% amino acid identity), whereas proteins coded by ORF3 and ORF5 are identical.     

Investigation of RNA structure in satellite panicum mosaic virus

Three new crystal forms of satellite panicum mosaic virus (SPMV) were grown and their structures solved from X-ray diffraction data using molecular replacement techniques. The crystals were grown under conditions of pH and ionic strength that were appreciably different then those used for the original structure determination. In rhombohedral crystals grown at pH 8.5 and low ionic strength PEG 3350 solutions, Fourier syntheses revealed segments, ten amino acid residues long, of amino-terminal polypeptides not previously seen, as well as masses of electron density within concavities on the interior of the capsid, which appeared in the neighborhoods of icosahedral five- and threefold axes. The densities were compatible with secondary structural domains of RNA, and they included a segment of double helical RNA of about four to five base pairs oriented, at least approximately, along the fivefold axes. The distribution of RNA observed for SPMV appears to be distinctly different than the encapsidated nucleic acid conformation previously suggested for another satellite virus, satellite tobacco mosaic virus. This study further shows that analysis of viruses in crystals grown under different chemical conditions may reveal additional information regarding the structure of encapsidated RNA.     

The structure of cowpea mosaic virus replicative form RNA

The structure of Cowpea mosaic virus (CPMV) replicative form (RF) RNA has been investigated by in vitro labelling at the 5' and 3' termini. The results indicate that the 3' termini of the minus (-) strands of the RFs from both M and B RNA (RF-M and RF-B) are exact complements of the corresponding 5' sequences of the virion RNAs. The 5'-labelling studies revealed that both RF-M and RF-B have VPg linked to poly(U) at the 5' ends of the (-) strands and also show that there is heterogeneity in the lengths of the VPg-linked RNAse T1 products derived from the 5' ends of the plus (+) strands. In addition it appears that not all of the 5' ends in RF molecules are linked to the VPg. The results enable us to draw an overall structure common to both RF-M and RF-B.     

Cloning and sequencing of influenza C/Yamagata/1/88 virus NS gene

Summary It was previously shown that the shortest RNA of influenza C/California/78 virus contains 934 nucleotides and codes for two nonstructural proteins of 286 amino acids (NS 1) and 121 amino acids (NS 2). In this report, we determined the nucleotide sequence of the NS gene of the recently isolated influenza C/Yamagata/1/88 strain by using cloned cDNA derived from the viral RNA. Compared with the NS gene of C/California/78, one nucleotide insertion has occurred in the NS gene of C/Yamagata/1/88. This caused frame shifts of both the NS 1 and NS 2 reading frames, directing the synthesis of the NS 1 and NS 2 proteins consisting of 246 and 182 amino acids, respectively.     

Alfalfa mosaic virus coat protein bridges RNA and RNA-dependent RNA polymerase in vitro

Alfalfa mosaic virus (AMV) RNA replication requires the viral coat protein (CP). AMV CP is an integral component of the viral replicase; moreover, it binds to the viral RNA 3'-termini and induces the formation of multiple new base pairs that organize the RNA conformation. The results described here suggest that AMV coat protein binding defines template selection by organizing the 3'-terminal RNA conformation and by positioning the RNA-dependent RNA polymerase (RdRp) at the initiation site for minus strand synthesis. RNA-protein interactions were analyzed by using a modified Northwestern blotting protocol that included both viral coat protein and labeled RNA in the probe solution ("far-Northwestern blotting"). We observed that labeled RNA alone bound the replicase proteins poorly; however, complex formation was enhanced significantly in the presence of AMV CP. The RNA-replicase bridging function of the AMV CP may represent a mechanism for accurate de novo initiation in the absence of canonical 3' transfer RNA signals.