Replication of tobacco mosaic virus. VIII. Characterization of a third subgenomic TMV RNA

In an earlier study we concluded that tobacco mosaic virus (TMV) infections engender a third subgenomic RNA in infected tissue (P. Palukaitis, F. Garcia-Arenal, M. A. Sulzinski, and M. Zaitlin (1983), Virology 131, 533-545). This RNA of approximate MW of 1.1 x 10(6), termed I1-RNA, was shown to be polyribosome-associated and thus was presumed to serve as a messenger RNA in vivo. Upon in vitro translation of I1-RNA in a rabbit reticulocyte lysate system, a major product of MW approximately 50K was generated. When RNA isolated from polyribosomes of infected tissues was analyzed with clones representing distinct regions of the TMV genome, the I1-RNA was shown to be a subset of the TMV genome, representing the 3'-half of the molecule. A TMV-specific DNA fragment (from a phage M13 clone) containing sequences overlapping the 5' end of I1-RNA was used in nuclease S1-mapping experiments with TMV-RNAs isolated from polyribosomes. I1-RNA was thus shown to be a distinct RNA species and not a class of heterogeneous molecules of approximately the same size. The I1-RNA 5' terminus is residue 3405 in the genome. Based on these findings and on consideration of the TMV-RNA sequence, we propose a model for the translation of I1-RNA: after an untranslated sequence of 90 bases, an AUG codon at residues 3495-3497 initiates a protein of MW 54K, terminating at residue 4915. Thus, the amino acid sequence of the 54K protein is coincident with those residues of the carboxy terminus of the well-known 183K TMV protein.     

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.     

Molecular weights of the RNA genome segments of a cytoplasmic polyhedrosis virus determined by a new comparative approach.

A cytoplasmic polyhedrosis virus (CPV) from Heliothis armigera contains 10 double-stranded RNA genome segments which are all well resolved on polyacrylamide gel electrophoresis. The relationship between log molecular weight and electrophoretic mobility in polyacrylamide gels has been studied using ³²P-labeled CPV RNA, and has been shown to deviate markedly from linearity at the higher molecular weight values. A new relationship between molecular weight and electrophoretic mobility for doubles-tranded RNA has therefore been defined and used to determine the absolute molecular weight of the CPV RNA by comparison with reovirus type 3 RNA.     

Togavirus RNA: Reversible effect of urea on genomes and absence of subgenomic viral RNA in Kunjin virus-infected cells

Electrophoretic analyses showed that no RNase-sensitive RNA smaller than the genome was specified by the flavivirus Kunjin in infected Vero cells during the period of maximum RNA and protein synthesis. In contrast, RNA extracted from Sindbis virus-infected cells under similar conditions included the expected 42S RNA (equivalent to the genome) and the smaller 26S (interjacent) RNA. Treatment of the genome of both togaviruses with 12 M urea produced a reversible (possibly conformational) change; measurement of the molecular weights of the treated RNAs by co-electrophoresis with fully denatured ribosomal RNA markers in SDS-polyacrylamide gels yielded a value of 2.1 X 10(6) if 8 M urea was incorporated in the gels and 4.2 X 10(6) if urea was omitted from the gels. These results indicate that flavivirus messenger RNA is represented solely by the intact genome of m.wt. 4.2 X 10(6).     

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.     

Replication timing of the human genome

We apologize for errors in the magnitude of reagent concentrations     

Electrophoretic separation of double-stranded RNA genome segments from Warrego and Mitchell River viruses

Summary Warrego and Mitchell River viruses form the Warrego serological group of orbiviruses. Their double-stranded RNA genomes differ in molecular weight and in thermal denaturation profiles.With 2 Figures     

Replication of an incomplete alfalfa mosaic virus genome in plants transformed with viral replicase genes

RNAs 1 and 2 of alfalfa mosaic virus (AIMV) encode proteins P1 and P2, respectively, both of which have a putative role in viral RNA replication. Tobacco plants were transformed with DNA copies of RNA1 (P1-plants), RNA2 (P2-plants) or a combination of these two cDNAs (P12-plants). All transgenic plants were susceptible to infection with the complete AIMV genome (RNAs 1, 2, and 3). Inoculation with incomplete mixtures of AIMV RNAs showed that the P1-plants were able to replicate RNAs 2 and 3, that the P2-plants were able to replicate RNAs 1 and 3, and that the P12-plants were able to replicate RNA3. Initiation of infection of nontransgenic plants, P1-plants, or P2-plants requires the presence of AIMV coat protein in the inoculum, but no coat protein was required to initiate infection of P12-plants with RNA3. Results obtained with P12-protoplasts supported the conclusion that coat protein plays an essential role in the replication cycle of AIMV RNAs 1 and 2.     

The nucleotide sequence and genome organization of the RNA2 and RNA3 segments in broad bean mottle virus.

Complete nucleotide sequences of broad bean mottle virus (BBMV) genomic RNAs 2 and 3 were determined. They consist of 2811 and 2293 nucleotides, respectively. Both RNAs are caped and, unlike in other tricornaviruses, both initiate with an A residue. BBMV RNA2 is monocistronic and encodes an 815 amino acid 2a protein, whereas RNA3 is dicistronic, encoding for a 295 amino acid 3a protein and for the 190 amino acid coat protein. A central, 423 amino acid 2a protein core region is highly homologous among the three bromoviruses, whereas both N- and C-termini are more heterologous. Most of the homologies among 3a proteins are concentrated within the N-termini two-thirds of the molecule that is predominantly hydrophobic, whereas the C-terminal one-third contains a large number of charged amino acids. The homologies among coat proteins are clustered within several mostly hydrophobic, or neutral, domains. The 5' noncoding region of the RNA2 has 110 nucleotides, whereas that of RNA3 contains 330 nucleotides. As in cowpea chlorotic mottle virus, but unlike in Brome mosaic virus, the 5' noncoding region includes subgenomic promoter-like sequences. The BBMV RNA3 intercistronic region also has subgenomic promoter sequences and contains a long poly(A) stretch. At the 3' end, BBMV RNAs 2 and 3 have 257 and 236 noncoding nucleotides, respectively.     

Down-regulation of Groundnut Rosette Virus Replication by a Variant Satellite RNA

Symptom production in groundnut plants infected with groundnut rosette virus (GRV) depends on the presence of satellite RNA (sat-RNA) in the GRV culture, and sat-RNA variants that induce only mild symptoms are known. One such variant drastically diminished the replication of GRV genomic RNA in infectedNicotiana benthamianaplants. This down-regulating ability did not involve either of the two open reading frames in the sat-RNA but was controlled by a region near its 5′ end, which is required for sat-RNA replication. WhenN. benthamianaplants were inoculated with GRV and the mild satellite and challenged by inoculation with a GRV isolate (YB) containing a sat-RNA that induces yellow blotch symptoms, no symptoms appeared and little GRV genomic RNA or sat-RNA was detected in the plants, provided the two inoculations were no more than 2 days apart. A GRV isolate containing a sat-RNA that neither induces symptoms inN. benthamiananor affects genomic RNA accumulation also provided protection against yellow blotch symptom production if inoculated before or up to 2 days after isolate YB. However, in this case protection was incomplete and both GRV RNA and sat-RNA accumulated to normal levels. It is suggested that sequences from the mild sat-RNA may provide a novel source of resistance against rosette disease.     

Cross-inhibition to heterologous foot-and-mouth disease virus infection induced by RNA interference targeting the conserved regions of viral genome

RNA interference (RNAi) is the process by which double-stranded RNA (dsRNA) directs sequence-specific degradation of messenger RNA in animal and plant cells. In mammalian cells, RNAi can be triggered by 21-23 nucleotide duplexes of small interfering RNA (siRNA). Strategies to inhibit RNA virus multiplication based on the use of siRNAs have to consider the high genetic polymorphism exhibited by this group of virus. Here we described a significant cross-inhibition of foot-and-mouth disease (FMD) virus (FMDV) replication in BHK-21 cells by siRNAs targeted to various conserved regions (5'NCR, VP4, VPg, POL, and 3'NCR) of the viral genome. The results showed that siRNAs generated in vitro by human recombinant dicer enzyme gave an inhibition of 10- to 1000-fold in virus yield of both homologous (HKN/2002) and heterologous (CHA/99) isolates of FMDV serotype O at 48 h post-infection (hpi). The inhibition extended to at least 6 days post-infection. For serotype Asia1, the virus yield in YNBS/58-infected cells examined at 12, 24, and 48 hpi decreased by approximately 10-fold in cells pretreated with HKN/2002-specific siRNAs, but there was no significant decrease at 60 hpi. The inhibition was specific to FMDV replication, as no reduction was observed in virus yield of pseudorabies virus, an unrelated virus. Moreover, we also demonstrated an enhanced viral suppression could be achieved in BHK-21 cells with siRNA transfection after an infection had been established. These results suggested that siRNAs directed to several conserved regions of the FMDV genome could inhibit FMDV replication in a cross-resistance manner, providing a strategy candidate to treat high genetic variability of FMDV.     

The left part of the viral genome is sufficient for interferon induction by adenovirus type 12

Summary Adenovirus type 12 is a potent interferon inducer on chick embryio cells. Incomplete particles induce similar levels of interferon as complete virions, even if they contain only the left 20 per cent of the genome. Empty capsids lacking DNA are not able to induce interferon, suggesting that part of the viral genome is required to trigger the cells to produce interferon.With 5 Figures     

Cucumber necrosis virus p20 is a viral suppressor of RNA silencing

The p20 protein encoded by the tombusvirus, Cucumber necrosis virus has previously been shown to be involved in host pathogenicity and shares sequence similarity with the Tomato bushy stunt virus p19 suppressor of silencing. Using a virus-induced gene silencing (VIGS) assay, we show that p20 is a viral suppressor of RNA silencing (VSR) in infected plants. In addition, a CNV p20-knockout mutant showed a decline in viral RNA accumulation in infected plants, consistent with the role of p20 in suppression of RNA silencing. However, unexpectedly, all GFP transgenic plants co-infiltrated with p20 and GFP displayed RNA silencing using an Agrobacterium-mediated silencing assay. Detailed RNA analysis of GFP mRNA levels in p20 agro-infiltrated plants revealed that p20 did initially display suppressor activity but this was rapidly overcome by RNA silencing. p20 expression levels in agro-infiltrated plants were shown to be approximately 50-fold lower than that of the TBSV p19 silencing suppressor, consistent with the notion that p20 dosage levels are not sufficient to suppress RNA silencing in the Agrobacterium-mediated system. Our results suggest that a viral-based VIGS assay may be required for identifying VSRs encoded by some plant viruses. Based on bioinformatics studies the mechanism of suppression of silencing by p20 is predicted to be similar to that of the TBSV p19 suppressor.