Nucleotide sequence of cucumber mosaic virus RNA. 1. Presence of a sequence complementary to part of the viral satellite RNA and homologies with other viral RNAs

The nucleotide sequence of the 3389 residues of RNA 1 (Mr 1.15 X 10(6) of the Q strain of cucumber mosaic virus (CMV) was determined, completing the primary structure of the CMV genome (8617 nucleotides). CMV RNA 1 was sequenced by the dideoxy-chain-termination method using M13 clones carrying RNA 1 sequences as well as synthetic oligonucleotide primers on RNA 1 as a template. At the 5' end of the RNA there are 97 noncoding residues between the cap structure and the first AUG (98-100), which is the start of a single long open-reading frame. This reading frame encodes a translation product of 991 amino acid residues (Mr 110791) and stops 319 nucleotide residues from the 3' end of RNA 1. In addition to the conserved 3' region present in all CMV RNAs (307 residues in RNA 1), RNAs 1 and 2 have highly homologous 5' leader sequences, a 12-nucleotide segment of which is also conserved in the corresponding RNAs of brome mosaic virus (BMV). CMV satellite RNA can form stable base pairs with a region of CMV RNAs 1 and 2 including this 12-nucleotide sequence, implying a regulatory function. This conserved sequence is part of a hairpin structure in RNAs 1 and 2 of CMV and BMV and in CMV satellite RNA. The entire translation products of RNA 1 of CMV and BMV could be aligned with significant homology. Less prominent homologies were found with alfalfa mosaic virus RNA 1 translation product and with tobacco mosaic virus Mr-126000 protein.     

Characterization of long guanosine-free RNA sequences from the Dahlemense and U2 strains of tobacco mosaic virus.

Four naturally occurring strains of tobacco mosaic virus, U2, Dahlemense, CV4, and the bean form of tobacco mosaic virus, were tested for the existence of long T1 RNAase oligonucleotides analogous to the oligonucleotide omega found in the common or U1 strain of tobacco mosaic virus and which makes up the 5' non-coding region of the RNA molecule. U2 and Dahlemense RNA were each found to contain this type of long T1 RNAase oligonucleotide with chain lengths of 54 and 74--77 residues, respectively. The sequence of the two oligonucleotides was determined mostly by using 5'-32P-labelled material in vitro and rapid polyacrylamide gel sequencing techniques.     

Complete nucleotide sequence of alfalfa mosaic virus RNA 1.

Double-stranded cDNA of alfalfa mosaic virus (AlMV) RNA 1 has been cloned and sequenced. From clones with overlapping inserts, and other sequence data, the complete primary sequence of the 3644 nucleotides of RNA 1 was deduced: a long open reading frame for a protein of Mr 125,685 is flanked by a 5'-terminal sequence of 100 nucleotides and a 3' noncoding region of 163 nucleotides, including the sequence of 145 nucleotides the three genomic RNAs of AlMV have in common. The two UGA-termination codons halfway RNA 1, that were postulated by Van Tol et al. (FEBS Lett. 118, 67-71, 1980) to account for partial translation of RNA 1 in vitro into Mr 58,000 and Mr 62,000 proteins, were not found in the reading frame of the Mr 125,685 protein.     

Sequence of 1000 nucleotides at the 3'' end of tobacco mosaic virus RNA.

The sequence of 1000 nucleotides at the 3' end of tobacco mosaic virus RNA has been determined. The sequence contains the entire coat protein cistron as well as regions to its left and right. Sequence characterization was by conventional methods for use with uniformly 32P labeled RNA complemented by newer methods for in vitro 5' and 3' 32P end-labeling of RNA and its subsequent rapid analysis. The noncoding region separating the coat protein cistron from the 3' terminus is 204 residues long and may be folded into a clover-leaf-type secondary structure. The distribution of termination codons to the left of the coat protein cistron suggests that the end of the adjacent cistron is separated from the beginning of the coat protein cistron by only two nucleotides. The subgenomic viral coat protein mRNA was isolated from infected tissue and shown to be capped. The nontranslated sequence separating the cap from the AUG initiation codon is 9 residues long and thus overlaps a portion of the adjacent cistron on the genome RNA.     

Complete nucleotide sequence of alfalfa mosaic virus RNA 4.

Alfalfa mosaic virus RNA 4, the subgenomic messenger for viral coat protein, was partially digested with RNase T1 or RNase A and the sequence of a number of fragments was deduced by in vitro labeling with polynucleotide kinase and application of RNA sequencing techniques. From overlapping fragments, the complete primary sequence of the 881 nucleotides of RNA 4 was constructed: the coding region of 660 nucleotides (not including the initiation and termination codon) is flanked by a 5' noncoding region of 39 nucleotides and a 3' noncoding region of 182 nucleotides. The RNA sequencing data completely confirm the amino acid sequence of the coat protein as deduced by Van Beynum et al. (Fur.J. Biochem. 72, 63-78, 1977).     

The organisation and interviral homologies of genes at the 3' end of tobacco rattle virus RNA1

The RNA1 of tobacco rattle virus (TRV) has been cloned as cDNA and the nucleotide sequence determined of 2 kb from the 3'-terminal region. The sequence contains three long open reading frames. One of these starts 5' of the cDNA and probably corresponds to the carboxy-terminal sequence of a 170-K protein encoded on RNA1. The deduced protein sequence from this reading frame shows homology with the putative replicases of tobacco mosaic virus (TMV) and tricornaviruses. The location of the second open reading frame, which encodes a 29-K polypeptide, was shown by Northern blot analysis to coincide with a 1.6-kb subgenomic RNA. The validity of this reading frame was confirmed by showing that the cDNA extending over this region could be transcribed and translated in vitro to produce a polypeptide of the predicted size which co-migrates in electrophoresis with a translation product of authentic viral RNA. The sequence of this 29-K polypeptide showed homology with two regions in the 30-K protein of TMV. This homology includes positions in the TMV 30-K protein where mutations have been identified which affect the transport of virus between cells. The third open reading frame encodes a potential 16-K protein and was shown by Northern blot hybridisation to be contained within the region of a 0.7-kb subgenomic RNA which is found in cellular RNA of infected cells but not virus particles. The many similarities between TRV and TMV in viral morphology, gene organisation and sequence suggest that these two viral groups may share a common viral ancestor.     

Studies on the sequence of the 3'-terminal region of turnip-yellow-mosaic-virus RNA.

A fragment representing the 3'-terminal 'tRNA-like' region of turnip yellow mosaic (TYM) virus RNA has been purified following incubation of intact TYM virus RNA with Escherichia coli 'RNase P'. This fragment, which is 112+3-nucleotides long has been completely digested with T1 RNase and pancreatic RNase and all the oligonucleotides present in such digests have been sequenced using 32P-end labelling techniques in vitro. The TYM virus RNA fragment is free of modified nucleosides and does not contain a G-U-U-C-R sequence. Using nuclease P1 from Penicillium citrinum, the sequence of 26 nucleotides from the 5' end and 16 nucleotides from the 3' end of this fragment has been deduced. The nucleotide sequence at the 5' end of the TYM virus RNA fragment indicates that this fragment includes the end of the TYM virus coat protein gene.     

Complete nucleotide sequences of the coat protein messenger RNAs of brome mosaic virus and cowpea chlorotic mottle virus

The nucleotide sequences of the subgenomic coat protein messengers (RNA4's) of two related bromoviruses, brome mosaic virus (BMV) and cowpea chlorotic mottle virus (CCMV), have been determined by direct RNA and CDNA sequencing without cloning. BMV RNA4 is 876 b long including a 5' noncoding region of nine nucleotides and a 3' noncoding region of 300 nucleotides. CCMV RNA 4 is 824 b long, including a 5' noncoding region of 10 nucleotides and a 3' noncoding region of 244 nucleotides. The encoded coat proteins are similar in length (188 amino acids for BMV and 189 amino acids for CCMV) and display about 70% homology in their amino acid sequences. Length difference between the two RNAs is due mostly to a single deletion, in CCMV with respect to BMV, of about 57 b immediately following the coding region. Allowing for this deletion the RNAs are indicate that mutations leading to divergence were constrained in the coding region primarily by the requirement of maintaining a favorable coat protein structure and in the 3' noncoding region primarily by the requirement of maintaining a favorable RNA spatial configuration.     

Intercistronic as well as terminal sequences are required for efficient amplification of brome mosaic virus RNA3.

The genome of brome mosaic virus (BMV) is divided among messenger polarity RNA1, RNA2, and RNA3 (3.2, 2.9, and 2.1 kilobases, respectively). cis-Acting sequences required for BMV RNA amplification were investigated with RNA3. By using expressible cDNA clones, deletions were constructed throughout RNA3 and tested in barley protoplasts coinoculated with RNA1 and RNA2. In contrast to requirements for 5'- and 3'-terminal noncoding sequences, either of the two RNA3 coding regions can be deleted individually and both can be simultaneously inactivated by N-terminal frameshift mutations without significantly interfering with amplification of RNA3 or production of its subgenomic mRNA. However, simultaneous major deletions in both coding regions greatly attenuate RNA3 accumulation. RNA3 levels can be largely restored by insertion of a heterologous, nonviral sequence in such mutants, suggesting that RNA3 requires physical separation of its terminal domains or a minimum overall size for normal replication or stability. Unexpectedly, deletions in a 150-base segment of the intercistronic noncoding region drastically reduce RNA3 accumulation. This segment contains a sequence element homologous to sequences found near the 5' ends of BMV RNA1 and RNA2 and in analogous positions in the three genomic RNAs of the related cucumber mosaic virus, suggesting a possible role in plus-strand synthesis.     

Circular dichroism and sedimentation studies on the reconstitution of tobacco mosaic virus

Reconstitution of tobacco mosaic virus from its constituents, the coat protein and RNA, was investigated by means of ultracentrifugation and circular dichroism measurement. Tobacco mosaic virus protein forms a 20S double-layer disc under conditions favorable for tobacco mosaic virus reconstitution. Dibromination of the tyrosine 139 residue of tobacco mosaic virus protein prevents formation of the 20S disc.Acidification of the tobacco mosaic virus protein solution causes 20S discs to polymerize into long helical rods. Changes in the CD spectra of tobacco mosaic virus protein in the near-ultraviolet region suggest that stacking of the aromatic sidechains of amino acid residues stabilizes the helical rod. The dibrominated tobacco mosaic virus protein also has the ability of rod elongation under acidic condition. CD studies reveal that assembly of tobacco mosaic virus particles from its constituents is stabilized by the stacking effect between the base residues of RNA and the aromatic residues of tobacco mosaic virus protein.Cucumber green mottle mosaic virus protein, which acts as a substituent for tobacco mosaic virus protein in tobacco mosaic virus reconstitution, was also investigated.     

Complete nucleotide sequence of alfalfa mosaic virus RNA3.

A full-length cDNA clone of alfalfa mosaic virus (AMV) RNA3 was prepared and sequenced. The 2,037 base sequence contains two open reading frames of 903 and 666 nucleotides that code for a 32,400 dalton protein (32.4K protein) and the 24,380 dalton coat protein, respectively. A 5'-noncoding sequence of 240 bases preceeding the 32.4K protein contains homologous regions that may have a function in its translation. The intercistronic junction is 49 bases long, the last 36 bases representing the 5'-end of the subgenomic RNA4. The remaining 179 bases comprise the 3'-terminal noncoding sequence.     

The Complete Nucleotide Sequence of Odontoglossum Ringspot Virus (Cy-1 Strain) Genomic RNA

The complete nucleotide sequence of the genomic RNA of odontoglossum ringspot virus Cy-1 strain (ORSV Cy-1) was determined using cloned cDNA. This sequence is 6611 nucleotides long containing four open reading frames, which correspond to 126 K, 183 K, 31 K, and 18 K proteins. Its genomic organization is similar to other tobamoviruses, TMV-V(vulgare), TMV-L (tomato strain), tobacco mild green mosaic virus (TMGMV) and cucumber green mottle mosaic virus (CGMMV). The 5′ non-coding regions of ORSV Cy-1 is 62 nucleotides. The ORFs encoded a 126 K polypeptide and a 183 K read-through product in which helicase-sequence and polymerase-sequence motifs are found. The ORFs encoding the 126 K and 183 K proteins have 61% and 63% identities with those of TMV-V. The third ORF encoded a 31 K protein homologous to TMV cell-to-cell movement protein. It has 63% identities with that of TMV-V. The fourth ORF encoded an 18 K coat protein. The 5′ non-coding region, which extends from base 1 to 62 has 2 G residues and a ribosome binding site (AUU). The 3′ non-coding region, 414 nucleotides in length, is entirely different from that of other tobamoviruses.     

Calanthe Mild Mosaic Virus, a New Potyvirus Causing a Mild Mosaic Disease of Calanthe Orchid in Japan

Calanthe mild mosaic potyvirus (CalMMV), a previously undescribed virus found in several locations in Japan, causes mild leaf mosaic and flower colour breaking of Calanthe plants. CalMMV was mechanically transmitted only to Calanthe sp., Phalaenopsis sp. and Tetragonia expansa of 50 plant species tested and was transmitted by the aphid Myzus persicae in a nonpersistent manner. The virus has flexuotis particles about 764 nm long and induced the formation of intracellular cytoplasmic cylindrical inclusions. The virus particles contain a single poly-peptide of 32.0 kDa and a single RNA of mol. weight 3.1 × 10⁶. As determined by immuno-electron microscopy, CalMMV is distantly related to the Japanese isolate of dendrobium mosaic potyvirus (DeMV-J), but it showed no serological relationship to any of seven other potyviruses. The sequence of the 3'-terminal 1306 nucleo-tides of the viral genome was determined. The coat protein (CP) coding sequence is predicted to be 804 nucleotides in length, encoding a protein of 268 amino acids with a calculated mol. weight of 30 389. The 3' noncoding region is 169 nucleotides long and is followed by a polyadenylate tract. The amino acid sequence of the CP of CalMMV was 73% homologous to that of DeMV-J, but less than 66% to other potyviruses.     

Biological activities of hybrid RNAs generated by 3''-end exchanges between tobacco mosaic and brome mosaic viruses.

Sequences within the conserved, aminoacylatable 3' noncoding regions of brome mosaic virus (BMV) genomic RNAs 1, 2, and 3 direct initiation of negative-strand synthesis by BMV polymerase extracts and, like sequences at the structurally divergent but aminoacylatable 3' end of tobacco mosaic virus (TMV) RNA, are required in cis for RNA replication in vivo. A series of chimeric RNAs in which selected 3' segments were exchanged between the tyrosine-accepting BMV and histidine-accepting TMV RNAs were constructed and their amplification was examined in protoplasts inoculated with or without other BMV and TMV RNAs. TMV derivatives whose 3' noncoding region was replaced by sequences from BMV RNA3 were independently replication competent when the genes for the TMV 130,000-M(r) and 180,000-M(r) replication factors remained intact. TMV replicase can thus utilize the BMV-derived 3' end, though at lower efficiency than the wild-type (wt) TMV 3' end. Providing functional BMV RNA replicase by coinoculation with BMV genomic RNAs 1 and 2 did not improve the amplification of these hybrid genomic RNAs. By contrast, BMV RNA3 derivatives carrying the 3' noncoding region of TMV were not amplified when coinoculated with wt BMV RNA1 and RNA2, wt TMV RNA, or all three. Thus, BMV replicase appeared to be unable to utilize the TMV 3' end, and there was no evidence of intervirus complementation in the replication of any of the hybrid RNAs. In protoplasts coinoculated with BMV RNA1 and RNA2, the nonamplifiable RNA3 derivatives bearing TMV 3' sequences gave rise to diverse new rearranged or recombined RNA species that were amplifiable.     

Probable reassortment of genomic elements among elongated RNA-containing plant viruses

Summary The relationships of genome organization among elongated (rod-shaped and filamentous) plant viruses have been analyzed. Sequences in coding and noncoding regions of barley stripe mosaic virus (BSMV) RNAs 1, 2, and 3 were compared with those of the monopartite RNA genomes of potato virus X (PVX), white clover mosaic virus (WClMV), and tobacco mosaic virus, the bipartite genome of tobacco rattle virus (TRV), the quadripartite genome of beet necrotic yellow vein virus (BNYVV), and icosahedral tricornaviruses. These plant viruses belong to a supergroup having 5-capped genomic RNAs. The results suggest that the genomic elements in each BSMV RNA are phylogenetically related to those of different plant RNA viruses. RNA 1 resembles the corresponding RNA 1 of tricornaviruses. The putative proteins encoded in BSMV RNA 2 are related to the products of BNYVV RNA 2, PVX RNA, and WClMV RNA. Amino acid sequence comparisons suggest that BSMV RNA 3 resembles TRV RNA 1. Also, it can be proposed that in the case of monopartite genomes, as a rule, every gene or block of genes retains phylogenetic relationships that are independent of adjacent genomic elements of the same RNA. Such differential evolution of individual elements of one and the same viral genome implies a prominent role for gene reassortment in the formation of viral genetic systems.     

Anti-sense regions in satellite RNA of cucumber mosaic virus form stable complexes with the viral coat protein gene.

The interaction in vitro of the RNA of the Q-strain of cucumber mosaic virus (CMV) with its satellite RNA (sat-RNA) has been studied. In hybridisation reactions containing 30% formamide at 45 degrees, sat-RNA binds to CMV RNA 3 and 4 but not to CMV RNA 1 and 2 or RNA from tobacco mosaic virus and alfalfa mosaic virus. The viral coat protein gene present in RNA 3 and 4 contains the site of binding but this region does not contain complementary sequences of any significant length to the sat-RNA sequence. However, the optimum alignment of short complementary sequences present in these regions revealed a stable structure in which it is proposed that sat-RNA twists around the coat protein gene so that two separate blocks of nucleotides in sat-RNA base pair in opposite directions with two adjacent blocks in the coat protein gene to form a knot-like structure. The binding site is a region of 33 nucleotides within the coding region of the coat protein gene which base pairs with residues 98-113 and 134-152 of sat-RNA. The possibility of the binding region of sat-RNA functioning as an "anti-sense" sequence in regulation of the viral coat protein synthesis is discussed.     

Near identity of 3′ RNA secondary structure in bromoviruses and cucumber mosaic virus

The 3′ terminal sequences of RNAs 1, 2, 3 and 4 from each of the three bromoviruses (brome mosaic, cowpea chlorotic mottle and broad bean mottle viruses) and also from cucumber mosaic virus display interviral sequence similarity in addition to strong intraviral homology. Interviral similarity is much more evident when RNA secondary, rather than primary, structures are compared. The last 190 bases of the various RNAs can fold into strikingly similar, extensively base-paired secondary structures whose common features are supported by RNA structure mapping. The extreme 3′ end of each viral RNA can base-pair in two distinct configurations. Bromovirus RNA 3s each contain an unusually accessible internal oligo(A) sequence which, in brome mosaic virus at least, is located in the intercistronic noncoding region. Functional implications of these structural features are discussed.     

La autoantigen enhances and corrects aberrant translation of poliovirus RNA in reticulocyte lysate.

Translation initiation on poliovirus RNA occurs by internal binding of ribosomes to a sequence within the 5' untranslated region. We have previously characterized a HeLa cell protein, p52, that binds to a fragment of the poliovirus 5' untranslated region (K. Meerovitch, J. Pelletier, and N. Sonenberg, Genes Dev. 3:1026-1034, 1989). Here we report the purification of the HeLa p52. Protein microsequencing identified p52 as La autoantigen. The La protein is a human antigen that is recognized by antibodies from patients with autoimmune disorders such as systemic lupus erythematosus and Sjögren's syndrome. We show that the La protein stimulates translation of poliovirus RNA, but not brome mosaic virus, tobacco mosaic virus, and alfalfa mosaic virus 4 RNA, translation in a reticulocyte lysate. In addition, La corrects aberrant translation of poliovirus RNA in a reticulocyte lysate. Subcellular immunolocalization showed that La protein is mainly nuclear, but after poliovirus infection, La is redistributed to the cytoplasm. Our results suggest that La protein is involved in poliovirus internal initiation of translation and might function through a similar mechanism in the translation of cellular mRNAs.