Characterization and engineering of sequences controlling in vivo synthesis of brome mosaic virus subgenomic RNA.

Expression of brome mosaic virus (BMV) coat protein and internal genes of many other positive-strand RNA viruses requires initiation of subgenomic mRNA synthesis from specific internal sites on minus-strand genomic RNA templates. Biologically active viral cDNA clones were used to investigate the sequences controlling production of BMV subgenomic RNA in vivo. Suitable duplications directed production of specifically initiated, capped subgenomic RNAs from new sites in the BMV genome. Previously implicated promoter sequences extending 20 bases upstream (-20) and 16 bases downstream (+16) of the subgenomic RNA initiation site directed only low-level synthesis. Subgenomic RNA production at normal levels required sequences extending to at least -74 but not beyond -95. Loss of an (rA)18 tract immediately upstream of the -20 to +16 "core promoter" particularly inhibited subgenomic RNA synthesis. The -38 to -95 region required for normal initiation levels contains repeats of sequence elements in the core promoter, and duplications creating additional upstream copies of these repeats stimulated subgenomic RNA synthesis above wild-type levels. At least four different subgenomic RNAs can be produced from a single BMV RNA3 derivative. For all derivatives producing more than one subgenomic RNA, a gradient of accumulation progressively favoring smaller subgenomic RNAs was seen.     

Occurrence of chloroplast ribosome recognition sites within conserved elements of the RNA genomes of carlaviruses

The nucleotide sequences upstream from the carlavirus open reading frames were examined for direct sequence homology. Blocks of homology were evident upstream from the 25 K ORFs of potato virus S (PVS), potato virus M (PVM) and lily symptomless virus (LSV), and upstream from the coat protein initiation codons of PVS, PVM, LSV, carnation latent virus and Helenium virus S. These blocks, which correspond to the 5′-terminal regions of the subgenomic RNAs, were shown to contain potential ribosome recognition sequences. The distances between the binding sites and initiation codons ranged from 20 to 40 nucleotides on the viral RNAs. Whilst the majority of chloroplasts mRNAs have a distance of 8 nucleotides between binding site and initiation codon, the remaining have a distance of 23 nucleotides which is similar to that reported here for the carlaviruses.     

The complete nucleotide sequence of PEBV RNA2 reveals the presence of a novel open reading frame and provides insights into the structure of tobraviral subgenomic promoters.

The 3374 nucleotide sequence of RNA2 from the British PEBV strain SP5 has been determined. The RNA includes three open reading frames flanked by 5' and 3' noncoding regions of 509 and 480 nucleotides. The open reading frames specify coat protein, a 29.6K product homologous to the 29.1K product of TRV(TCM) RNA2 and a 23K product not homologous to any previously described protein. The homology demonstrated between the coat proteins of PRV, TRV and PEBV indicates a common evolutionary origin for these proteins. Upstream of each ORF are located sequences homologous to those with which subgenomic RNAs of other tobraviruses start. Subgenomic RNAs for the expression of the three ORFs may start at these points. On all five tobraviral RNA2 molecules sequenced to date, these sequences were found upstream of the coat protein ORF in association with a strongly-conserved potential secondary structural element. Similar potential structures were identified upstream of other tobraviral ORFs. These structures may contribute to the activity of the tobraviral subgenomic promoter.     

Potato virus X-related single- and double-stranded RNAs: Characterization and identification of terminal structures

Six species of 3′-coterminal poly(A) -containing RNAs of subgenomic (sg) size have been found in plants infected with potato virus X (PVX): two major (0.9 kb — the coat protein mRNA, and 2.1 kb) and four minor (1.4, 1.8, 3.0 and 3.6 kb). The 5′-end of the shortest sgRNA is located 26 nucleotides upstream of the initiating codon of the coat protein gene (812 nucleotides from the 3′-terminal poly(A) tract of the PVX genomic RNA). Double-stranded analogues have been found for most sgRNAs. The genomic-size double-stranded RNA (the replicative form) is shown to carry a poly(A)-poly(U) hybrid of a predominant length of 150–250 bp on one end, and an unpaired G residue on the other (the 3′-end of the negative chain). In contrast to this(—) the chains of double-stranded 0.9 and 2.1 kbp sgRNAs lack the unpaired G and both end in C.     

Analysis of the genome structure of tobacco rattle virus strain PSG.

The sequence of the 3'-terminal 2077 nucleotides of genomic RNA 1 and the complete sequence of genomic RNA 2 of tobacco rattle virus (TRV, strain PSG) has been deduced. RNA 2 (1905 nucleotides) contains a single open reading frame for the viral coat protein (209 amino acids), flanked by 5'- and 3'-noncoding regions of 570 and 708 nucleotides, respectively. A subgenomic RNA (RNA 4) was found to lack the 5'-terminal 474 nucleotides of RNA 2 and is the putative messenger for coat protein. The deduced RNA 1 sequence contains the 3'-terminal part of a reading frame that probably corresponds to the TRV 170K protein and reading frames for a 29K protein and a 16K protein. Proteins encoded by the first two reading frames show significant amino acid sequence homology with corresponding proteins encoded by tobacco mosaic virus. Subgenomic RNAs 3 (1.6 kb) and 5 (0.7 kb) were identified as the putative messengers for the 29K and 16K proteins, respectively. At their 3'-termini all PSG-RNAs have an identical sequence of 497 nucleotides; at the 5'-termini homology is limited to 5 to 10 bases.     

Structure-function relationship of Rous sarcoma virus leader RNA.

Cells infected by RSV synthesize viral 35S RNA as well as subgenomic 28S and 22S RNAs coding for the Env and Src genes respectively. In addition, at least the 5' 101 nucleotides of the leader are also conserved and we have shown previously that this sequence contains a strong ribosome binding site (J.-L. Darlix et al., J. Virol. 29, 597). We now report the RNA sequence of Rous Sarcoma virus (RSV) leader RNA and propose a folding of this 5' untranslated region which brings the Cap, the initiation codon for Gag and the strong ribosome binding site close to each other. We also show that ribosomes protect a sequence just upstream from initiator Aug of Gag in vitro, and believed to interact with part of the strong ribosome binding site according to the folding proposed for the leader RNA.     

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.     

Complete genome sequence and analyses of the subgenomic RNAs of sweet potato chlorotic stunt virus reveal several new features for the genus Crinivirus

The complete nucleotide sequences of genomic RNA1 (9,407 nucleotides [nt]) and RNA2 (8,223 nt) of Sweet potato chlorotic stunt virus (SPCSV; genus Crinivirus, family Closteroviridae) were determined, revealing that SPCSV possesses the second largest identified positive-strand single-stranded RNA genome among plant viruses after Citrus tristeza virus. RNA1 contains two overlapping open reading frames (ORFs) that encode the replication module, consisting of the putative papain-like cysteine proteinase, methyltransferase, helicase, and polymerase domains. RNA2 contains the Closteroviridae hallmark gene array represented by a heat shock protein homologue (Hsp70h), a protein of 50 to 60 kDa depending on the virus, the major coat protein, and a divergent copy of the coat protein. This grouping resembles the genome organization of Lettuce infectious yellows virus (LIYV), the only other crinivirus for which the whole genomic sequence is available. However, in striking contrast to LIYV, the two genomic RNAs of SPCSV contained nearly identical 208-nt-long 3' terminal sequences, and the ORF for a putative small hydrophobic protein present in LIYV RNA2 was found at a novel position in SPCSV RNA1. Furthermore, unlike any other plant or animal virus, SPCSV carried an ORF for a putative RNase III-like protein (ORF2 on RNA1). Several subgenomic RNAs (sgRNAs) were detected in SPCSV-infected plants, indicating that the sgRNAs formed from RNA1 accumulated earlier in infection than those of RNA2. The 5' ends of seven sgRNAs were cloned and sequenced by an approach that provided compelling evidence that the sgRNAs are capped in infected plants, a novel finding for members of the Closteroviridae.     

The simian immunodeficiency virus 5' untranslated leader sequence plays a role in intracellular viral protein accumulation and in RNA packaging

We investigated the role of 5' untranslated leader sequences of simian immunodeficiency virus (SIV(mac239)) in RNA encapsidation and protein expression. A series of progressively longer deletion mutants was constructed with a common endpoint six nucleotides upstream of the gag initiation codon and another endpoint at the 3' end of the primer binding site (PBS). We found that efficient intracellular Gag-Pol protein accumulation required the region between the PBS and splice donor (SD) site. Marked reduction of genomic RNA packaging was observed with all the deletion mutants that involved sequences at both the 5' and at the 3' ends of the major SD site, and increased nonspecific RNA incorporation could be detected in these mutants. RNA encapsidation was affected only modestly by a deletion of 54 nucleotides at the 3' end of the SD site when the mutant construct pDelta54 was transfected alone. In contrast, the amount of pDelta54 genomic RNA incorporated into particles was reduced more than 10-fold when this mutant was cotransfected with a construct specifying an RNA molecule with a wild-type packaging signal. Therefore, we conclude that the 175 nucleotides located 5' of the gag initiation codon are critical for efficient and selective incorporation of genomic RNA into virions. This location of the SIV Psi element provides the means for efficient discrimination between viral genomic and spliced RNAs.