Subgenomic RNA as a riboregulator: negative regulation of RNA replication by Barley yellow dwarf virus subgenomic RNA 2

Barley yellow dwarf virus (BYDV) generates three 3'-coterminal subgenomic RNAs (sgRNAs) in infected cells. Translation of BYDV genomic RNA (gRNA) and sgRNA1 is mediated by the BYDV cap-independent translation element (BTE) in the 3' untranslated region. sgRNAs 2 and 3 are unlikely to be mRNAs. We proposed that accumulation of sgRNA2, which contains the BTE in its 5' UTR, regulates BYDV replication by trans-inhibiting translation of the viral polymerase from genomic RNA (gRNA). Here, we tested this hypothesis and found that: (i) co-inoculation of the BTE or sgRNA2 with BYDV RNA inhibits BYDV RNA accumulation in protoplasts; (ii) Brome mosaic virus (BMV), engineered to contain the BTE, trans-inhibits BYDV replication; and (iii) sgRNA2 generated during BYDV infection trans-inhibits both GFP expression from BMV RNA and translation of a non-viral reporter mRNA. We conclude that sgRNA2, via its BTE, functions as a riboregulator to inhibit translation of gRNA. This may make gRNA available as a replicase template and for encapsidation. Thus, BYDV sgRNA2 joins a growing list of trans-acting regulatory RNAs.     

Synthesis of genomic and subgenomic RNAs by a membrane-bound RNA-dependent RNA polymerase isolated from oat plants infected with cereal yellow dwarf virus

Summary. A membrane-bound RNA-dependent RNA polymerase (RdRp) complex was isolated by differential sedimentation from oat plants infected with cereal yellow dwarf virus (CYDV). When incubated with ³²P-labelled UTP, unlabelled ATP, CTP and GTP, and Mg²⁺ ions, the RdRp preparation catalysed the synthesis of double-stranded (ds) RNAs corresponding in size to the virus genomic RNA (5.7 kbp) and two putative subgenomic RNAs (2.8 and 0.7 kbp). Hybridisation using strand-specific hybridization targets showed that the 5.7-kbp dsRNA was labelled mainly in the plus strand, whereas the 2.8- and 0.7-kbp dsRNAs were labelled only in the minus strand. Genomic-length single-stranded, plus-strand RNA of 5.7 kb and single-stranded, plus-strand subgenomic RNAs of 2.8 and 0.7 kbp were detected in RNA isolated from oat plants infected with CYDV. Mapping experiments were consistent with the genomic and subgenomic RNAs having common 3′ ends, but different 5′ ends, whether produced in vitro or in vivo. The RdRp-encoding region of the CYDV genome was cloned and expressed in Escherichia coli, and the purified protein was used to raise antibodies in a rabbit. In immunoblots, the antibodies detected a protein of about 68 kDa in RdRp preparations from CYDV-infected oat plants, but not from equivalent preparations from healthy oats. As far as we are aware, this is the first report of an in vitro RNA synthesis system for a phloem-limited virus.     

Sequence comparison of the 3' ends of a subgenomic RNA and the genomic RNAs of barley stripe mosaic virus

All strains of barley stripe mosaic virus examined encapsidate small amounts of an 800-nucleotide (NT) gamma-subgenomic (sg) RNA. This sgRNA has been isolated from genomic (g) RNAs of the Type and North Dakota 18 (ND18) strains and the sequence of these RNAs has been compared near the 3' end. The immediate 3' termini of the gRNAs terminate in the icosomer-GGUCCCCCAAGGGAAGACCAOH-3' and differ from the sgRNAs, which are polyadenylated. The poly(A) tracts of the sgRNAs are heterogeneous with lengths ranging from 10 to greater than 150 NT. Polyacrylamide gel electrophoresis of complementary (c) DNAs transcribed in the presence of dideoxynucleotides reveals that the sgRNAs from Type and ND18 have almost identical sequences for at least 160 NT adjacent to the 5' side of the poly(A) region. This region of the sgRNA from the ND18 strain is nearly identical to a 95-NT sequence adjacent to a poly(A) tract located at the 3' end of a 2050-base pair cDNA cloned from the gamma-genomic RNA of ND18. These results suggest that the sequences encoding the sgRNA are located upstream of an internal poly(A) region situated more than 200 NT from the 3' end of the gamma-genomic RNA.     

Identification of the 5' end of the rubella virus subgenomic RNA

The 5' end of the subgenomic RNA of rubella virus was determined by primer extension. The Maxam-Gilbert sequence ladder of the primer extension product contained a determinable sequence which was colinear with the complement of the sequence of the genomic RNA through nucleotide 3325 from the 3' end of the genomic RNA and a pair of bands in every lane above the determinable sequence. These results indicated that synthesis of the subgenomic RNA is initiated internally on the minus-polarity genome-length RNA template and that the length of subgenomic RNA is 3327 nucleotides excluding the poly(A) tail. There are thus 77 nucleotides between the 5' end of the subgenomic RNA and the first AUG, which is the initiation codon for the structural protein open reading frame. The initiation site of rubella virus subgenomic RNA synthesis is 20 nucleotides downstream from a block of 28 nucleotides which shares homology with the nucleotide sequence which is conserved at the subgenomic RNA initiation site in the Alphaviruses, the other genus of Togaviruses.     

Encapsidation of genomic but not subgenomic Turnip yellow mosaic virus RNA by coat protein provided in trans

We have studied the encapsidation requirements of Turnip yellow mosaic virus (TYMV) genomic and subgenomic RNA using an "agroinfiltration" procedure involving transient expression of RNAs and coat protein (CP) in Nicotiana benthamiana leaves. Although N. benthamiana is a nonhost, expression of TYMV RNA in its leaves by agroinfiltration resulted in efficient local infection and production of the expected virions containing genomic and subgenomic RNAs together with empty capsids. A nonreplicating genomic RNA with a mutation in the polymerase domain was efficiently encapsidated by CP provided in trans, even though RNA levels were a thousand-fold lower than in normal infections. In contrast, encapsidation of CP mRNA was not observed under these conditions, even when the CP mRNA had authentic 5'- and 3'-termini. Deletion of the 3'-tRNA-like structure from the genomic RNA did not alter the encapsidation behavior, suggesting that this feature does not play a role in the encapsidation of TYMV RNA. Our results indicate differences in the encapsidation process between genomic and subgenomic RNAs, and suggest an interaction between RNA replication and the packaging of subgenomic RNA.     

Rose spring dwarf-associated virus has RNA structural and gene-expression features like those of Barley yellow dwarf virus

We determined the complete nucleotide sequence of the Rose spring dwarf-associated virus (RSDaV) genomic RNA (GenBank accession no. EU024678) and compared its predicted RNA structural characteristics affecting gene expression. A cDNA library was derived from RSDaV double-stranded RNAs (dsRNAs) purified from infected tissue. Nucleotide sequence analysis of the cloned cDNAs, plus for clones generated by 5'- and 3'-RACE showed the RSDaV genomic RNA to be 5808 nucleotides. The genomic RNA contains five major open reading frames (ORFs), and three small ORFs in the 3'-terminal 800 nucleotides, typical for viruses of genus Luteovirus in the family Luteoviridae. Northern blot hybridization analysis revealed the genomic RNA and two prominent subgenomic RNAs of approximately 3 kb and 1 kb. Putative 5' ends of the sgRNAs were predicted by identification of conserved sequences and secondary structures which resembled the Barley yellow dwarf virus (BYDV) genomic RNA 5' end and subgenomic RNA promoter sequences. Secondary structures of the BYDV-like ribosomal frameshift elements and cap-independent translation elements, including long-distance base pairing spanning four kb were identified. These contain similarities but also informative differences with the BYDV structures, including a strikingly different structure predicted for the 3' cap-independent translation element. These analyses of the RSDaV genomic RNA show more complexity for the RNA structural elements for members of the Luteoviridae.     

Comparison of the 5' and 3' termini of tomato ringspot virus RNA1 and RNA2: evidence for RNA recombination

The sequences of the 5' terminal 1140 and 3' terminal 1546 nt of tomato ringspot virus (TomRSV) RNA1 have been determined. These sequences share a high degree of nucleotide sequence similarity with the previously determined TomRSV RNA2 sequence. Eighty-eight percent of the 5' terminal 907 nt of TomRSV RNA1 and RNA2 contain identical nucleotide residues; the first 459 nt are identical at all positions, whereas the next 447 nt are identical at only 75.8% of the nucleotide positions. The region of similarity includes not only the 5' nontranslated leader but also sequence probably encoding polyproteins. The 3' terminal 1533 nt of TomRSV RNA1 and RNA2 are identical and are noncoding. The sequences common to RNA1 and RNA2 account for almost 35% of the total genomic sequence. It is possible that the similar sequences at both ends of TomRSV RNA1 and RNA2 are a result of recombination between these two genomic RNA components.     

The Taiwanese hepatitis C virus genome: sequence determination and mapping the 5' termini of viral genomic and antigenomic RNA.

The complete nucleotide sequence of hepatitis C virus (HCV) cloned from the liver tissue of a Taiwanese patient with post-transfusion type C hepatitis was determined. The 5' end of HCV genomic RNA was located 341 nucleotides upstream from the initiation codon for the viral polyprotein open reading frame. The 5' end of the viral antigenomic RNA was shown to have 13 consecutive As. Thus the 3' terminus of the viral genome is a stretch of U which ends about 50 nucleotides downstream from the stop codon of the large open reading frame. The nucleotide sequence homology between this HCV strain and two Japanese isolates was 90.5 and 90.7%, respectively. Homology with the United States strain, however, was only 77.8%. Accordingly, the indigenous Taiwanese HCV strain is of the same subtype as the Japanese isolates. Novel features of the viral genome termini are possibly relevant to HCV genome replication.     

Recombination of 5' subgenomic RNA3a with genomic RNA3 of Brome mosaic bromovirus in vitro and in vivo

RNA-RNA recombination salvages viral RNAs and contributes to their genomic variability. A recombinationally-active subgenomic promoter (sgp) has been mapped in Brome mosaic bromovirus (BMV) RNA3 (Wierzchoslawski et al., 2004. J. Virol.78, 8552-8864) and mRNA-like 5′ sgRNA3a was characterized (Wierzchoslawski et al., 2006. J. Virol. 80, 12357-12366). In this paper we describe sgRNA3a-mediated recombination in both in vitro and in vivo experiments. BMV replicase-directed co-copying of (−) RNA3 with wt sgRNA3a generated RNA3 recombinants in vitro, but it failed to when 3′-truncated sgRNA3a was substituted, demonstrating a role for the 3′ polyA tail. Barley protoplast co-transfections revealed that (i) wt sgRNA3a recombines at the 3′ and the internal sites; (ii) 3′-truncated sgRNA3as recombine more upstream; and (iii) 5′-truncated sgRNA3 recombine at a low rate. In planta co-inoculations confirmed the RNA3-sgRNA3a crossovers. In summary, the non-replicating sgRNA3a recombines with replicating RNA3, most likely via primer extension and/or internal template switching.     

Characterization of the cell-free translation products of carnation mottle virus genomic and subgenomic RNAs

The in vitro translation products of carnation mottle virus (CarMV) genomic and subgenomic RNAs were analysed using a rabbit reticulocyte lysate system. Viral RNAs directed synthesis of three main polypeptides, p80, p40, and p34. p40, which was the predominant product using unfractionated virion RNA as template, was identified as coat protein based on electrophoretic mobility through SDS-polyacrylamide gels and immunoprecipitation with anti-CarMV serum. Upon size-fractionation of virion RNAs by sucrose gradient centrifugation and subsequent translational analysis, p40 was found to be encoded by subgenomic RNA, whereas p80 and p34 were synthesized from templates of genome length. p80 and p34 were found to contain common or overlapping amino acid sequences by analysis of cleavage peptides formed during proteolysis with alpha-chymotrypsin. These data support CarMV translational mechanisms other than those proposed in a previous study (R. Saloman, M. Bar-Joseph, H. Soreq, I. Gozes, and U. Z. Littauer, 1978,Virology 90, 288-298).     

Isolation of replicative forms of 3' terminal subgenomic RNAs of tobacco necrosis virus

Three double-stranded RNAs of molecular weights 2.6 x 10(6), 1.05 x 10(6), and 0.94 x 10(6) are found to be synthesized during TNV infection of tobacco leaves. These double-stranded RNAs, isolated by LiCl fractionation and polyacrylamide gel electrophoresis, have been studied using RNA-RNA hybridization and RNase T(1)-resistant oligonucleotide finger-printing techniques. By these methods all three double-stranded RNAs are shown to be viral in origin and the smaller double-stranded RNA subsets of the larger RNAs. RNase T(1) oligonucleotide mapping of the viral RNA shows that both smaller double-stranded RNAs are derived from the 3' end of the viral genome. The possible role of these small double-stranded RNAs as replicative forms for mRNA synthesis and the organization and expression of the TNV genome is discussed.     

Rubella virus capsid protein modulation of viral genomic and subgenomic RNA synthesis

The ratio of the subgenomic (SG) to genome RNA synthesized by rubella virus (RUB) replicons expressing the green fluorescent protein reporter gene (RUBrep/GFP) is substantially higher than the ratio of these species synthesized by RUB (4.3 for RUBrep/GFP vs. 1.3-1.4 for RUB). It was hypothesized that this modulation of the viral RNA synthesis was by one of the virus structural protein genes and it was found that introduction of the capsid (C) protein gene into the replicons as an in-frame fusion with GFP resulted in an increase of genomic RNA production (reducing the SG/genome RNA ratio), confirming the hypothesis and showing that the C gene was the moiety responsible for the modulation effect. The N-terminal one-third of the C gene was required for the effect of be exhibited. A similar phenomenon was not observed with the replicons of Sindbis virus, a related Alphavirus. Interestingly, modulation was not observed when RUBrep/GFP was co-transfected with either other RUBrep or plasmid constructs expressing the C gene, demonstrating that modulation could occur only when the C gene was provided in cis. Mutations that prevented translation of the C protein failed to modulate RNA synthesis, indicating that the C protein was the moiety responsible for modulation; consistent with this conclusion, modulation of RNA synthesis was maintained when synonymous codon mutations were introduced at the 5' end of the C gene that changed the C gene sequence without altering the amino acid sequence of the C protein. These results indicate that C protein translated in proximity of viral replication complexes, possibly from newly synthesized SG RNA, participate in regulating the replication of viral RNA.     

Analysis of the in vitro coding properties of the 3' region of turnip yellow mosaic virus genomic RNA

The genomic RNA of turnip yellow mosaic virus (TYMV) codes in vitro for two proteins of 195,000 (195K) and 150,000 (150K) daltons initiated at the same site of the RNA. In the presence of yeast amber suppressor tRNA, synthesis of the 195K protein is reduced in favor of a 210,000-dalton (210K) protein. Using TYMV RNA sequence data and comparison of tryptic peptides of the 150K, 195K, 210K, and coat proteins, we postulate that the silent region between the 195K gene and the coat protein gene located in the 3' region of TYMV genomic RNA is only 14 nucleotides long.     

Structural plasticity of Barley yellow dwarf virus-like cap-independent translation elements in four genera of plant viral RNAs

The 3′ untranslated regions (UTRs) of many plant viral RNAs contain cap-independent translation elements (3′ CITEs). Among the 3′ CITEs, the Barley yellow dwarf virus (BYDV)-like translation elements (BTEs) form a structurally variable and widely distributed group. Viruses in three genera were known to harbor 3′ BTEs, defined by the presence of a 17-nt consensus sequence. To understand BTE function, knowledge of phylogenetically conserved structure is essential, yet the secondary structure has been determined only for the BYDV BTE. Here we show that Rose spring dwarf-associated luteovirus, and two viruses in a fourth genus, Umbravirus, contain functional BTEs, despite deviating in the 17 nt consensus sequence. Structure probing by selective 2′-hydroxyl acylation and primer extension (SHAPE) revealed conserved and highly variable structures in BTEs in all four genera. We conclude that BTEs tolerate striking evolutionary plasticity in structure, while retaining the ability to stimulate cap-independent translation.     

Mechanism of synthesis of turnip yellow mosaic virus coat protein subgenomic RNA in vivo

Turnip yellow mosaic virus (TYMV) possesses a monopartite single-stranded (+) sense RNA genome in which the coat protein (cp) gene is 3' proximal and is expressed in vivo via a subgenomic RNA. Evidence is presented here that this subgenomic RNA is synthesized in vivo by internal initiation of replication on (-) RNA strands of genomic length. The double-stranded RNAs (dsRNAs) from TYMV-infected plants have been isolated, purified, and characterized. Under native conditions, no dsRNAs (replicative intermediates and/or replicative forms) of subgenomic length corresponding to subgenomic cp RNA can be detected by ethidium bromide staining of RNA-sizing gels or by Northern blot hybridization using RNA probes. The presence of nascent subgenomic cp (+) RNA strands on the dsRNA of genomic length has been demonstrated using two different approaches: (1) Northern blot hybridization using (-) RNA probes under denaturing conditions and (2) characterization of the 5' ends of nascent (+) RNA strands upon labeling by vaccinia virus nucleoside-2'-methyltransferase.     

Core protein-mediated 5'-3' annealing of the West Nile virus genomic RNA in vitro

Genome cyclization through conserved RNA sequences located in the 5' and 3' terminal regions of flavivirus genomic RNA is essential for virus replication. Although the role of various cis-acting RNA elements in panhandle formation is well characterized, almost nothing is known about the potential contribution of protein cofactors to viral RNA cyclization. Proteins with nucleic acid chaperone activities are encoded by many viruses (e.g., retroviruses, coronaviruses) to facilitate RNA structural rearrangements and RNA-RNA interactions during the viral replicative cycle. Since the core protein of flaviviruses is also endowed with potent RNA chaperone activities, we decided to examine the effect of West Nile virus (WNV) core on 5'-3' genomic RNA annealing in vitro. Core protein binding resulted in a dramatic, dose-dependent increase in 5'-3' complex formation. Mutations introduced in either the UAR (upstream AUG region) or CS (conserved sequence) elements of the viral RNA diminished core protein-dependent annealing, while compensatory mutations restored the 5'-3' RNA interaction. The activity responsible for stimulating RNA annealing was mapped to the C-terminal RNA-binding region of WNV core protein. These results indicate that core protein - besides its function in viral particle formation - might be involved in the regulation of flavivirus genomic RNA cyclization, and thus virus replication.