Replication of cucumber mosaic virus satellite RNA in vitro by an RNA-dependent RNA polymerase from virus-infected tobacco.

An RNA-dependent RNA polymerase purified from tobacco infected with cucumber mosaic virus catalyzes the synthesis of (-) and (+) strands of the viral satellite RNA, CARNA 5, but fails to replicate the satellite RNA of peanut stunt virus (PSV). The enzyme replicates the genomic RNAs of the three principal cucumoviruses CMV, PSV and tomato aspermy virus (TAV) with varying efficiencies. The specificity with which CMV RdRp replicates different sequence-unrelated RNA templates suggests that the site of their recognition requires secondary or higher level structural organization.     

Perspective on CARNA 5, cucumber mosaic virus-dependent replicating RNAs capable of modifying disease expression

CARNA 5, the disease regulatory RNA associated with cucumber mosaic virus, consists of a family of sequence-related variant molecules. This type of multiplicity is the result of the selectivity with which specific combinations of virus strain and host species during infection allow certain CARNA 5 variants to emerge and replicate faster than others.     

Rapid synthesis of double-stranded cucumber mosaic virus-associated RNA 5: Mechanism controlling viral pathogenesis?

In cucumber mosaic virus infections of tobacco where disease attenuation is observed, viral RNA synthesis is quickly overtaken by the synthesis of cucumber mosaic virus-associated RNA 5, a satellite-like RNA dependent upon the virus for its replication, and that of its double-stranded form. A disease regulatory mechanism is proposed in which the sequestration of rapidly synthesized cucumber mosaic virus-associated RNA 5 molecules of complementary nucleotide sequence enables their successful competition with and suppression of, viral RNA synthesis.     

Replication footprint analysis of cucumber mosaic virus electroporated into tomato protoplasts

Total RNA extracted from cucumber mosaic virus (CMV) strains WT, with its associated satellite CARNA 5 (CMV-associated RNA 5), was successfully electroporated into isolated tomato protoplasts. At various time intervals samples were extracted for total nucleic acids and analyzed by semidenaturing polyacrylamide gel electrophoresis (PAGE). Sequence-specific hybridization probes were used for the detection of viral and satellite RNAs following Northern transfer. The resulting PAGE patterns and/or autoradiographs depict the proportional presence of viral and satellite RNAs in the extracts over time and have been referred to as "replication footprint profiles" (RFPs) of specific CMV/CARNA 5 combinations. The effective isolation and infection of tomato protoplasts, combined with the ability to follow virus/satellite titers during the infection by RFP analysis, yield results similar to those of infected plants and reduces experiments of 21 or more days in whole plants to less than 72 h in protoplasts.     

Double-stranded cucumovirus associated RNA 5: experimental analysis of necrogenic and non-necrogenic variants by temperature-gradient gel electrophoresis.

Cucumber mosaic virus (CMV) and peanut stunt virus (PSV) each contain a fifth major RNA in the size range of 334 to 393 nucleotides. This fifth RNA is a satellite capable of modulating the expression of viral disease symptoms. It is present in infected tissue in single-stranded and double-stranded form. Nucleotide sequence variants of the double-stranded CMV-associated RNA 5 (dsCARNA 5) and PSV-associated RNA 5 (dsPARNA 5) were analysed by temperature-gradient gel electrophoresis. Gels were 5% polyacrylamide, containing 8 M urea in 8.9 mM Tris-borate buffer, with temperature differences of 25-40 degrees C establishing gradients either perpendicular or parallel to the direction of the electric field. For dsCARNA 5 two characteristic transitions were detected with increasing temperature: at temperatures between 40 degrees C and 46 degrees C a drastic retardation in electrophoretic mobility induced by partial dissociation of the duplex structure from the ends and at temperatures above 52 degrees C an abrupt increase in mobility due to complete strand dissociation. dsPARNA 5 exhibited both transitions at up to 10 degrees C higher temperatures and an additional retardation between the transitions mentioned. Seven different variants of dsCARNA 5, 4 necrogenic and 3 non-necrogenic, were analysed. Some showed only one single band, others gave rise to up to six well separated bands corresponding to six molecular species. From all experimental results a correlation between the temperature of the retardation transition and the necrogenicity of CARNA 5 was derived. The diagnostic application of the temperature-gradient gel analysis in agriculture, particularly for the use of non-necrogenic variants as biological control agents to impede CMV-infections, is discussed.     

Suppression of RNA interference by adenovirus virus-associated RNA

We show that human adenovirus inhibits RNA interference (RNAi) at late times of infection by suppressing the activity of two key enzyme systems involved, Dicer and RNA-induced silencing complex (RISC). To define the mechanisms by which adenovirus blocks RNAi, we used a panel of mutant adenoviruses defective in virus-associated (VA) RNA expression. The results show that the virus-associated RNAs, VA RNAI and VA RNAII, function as suppressors of RNAi by interfering with the activity of Dicer. The VA RNAs bind Dicer and function as competitive substrates squelching Dicer. Further, we show that VA RNAI and VA RNAII are processed by Dicer, both in vitro and during a lytic infection, and that the resulting short interfering RNAs (siRNAs) are incorporated into active RISC. Dicer cleaves the terminal stem of both VA RNAI and VA RNAII. However, whereas both strands of the VA RNAI-specific siRNA are incorporated into RISC, the 3' strand of the VA RNAII-specific siRNA is selectively incorporated during a lytic infection. In summary, our work shows that adenovirus suppresses RNAi during a lytic infection and gives insight into the mechanisms of RNAi suppression by VA RNA.     

Nature of the single-stranded DNA in replicating adenovirus type 5 DNA.

Single-stranded regions in replicating adenovirus type 5 DNA were isolated and hybridized in solution to the separated strands of adenovirus 2 or 5 DNA. The results showed that the two strands of adenovirus 5 DNA are exposed to almost the same extent during replication, suggesting that displacement synthesis may start from either end of the viral DNA.     

Isolation and partial characterization of single-stranded adenoviral DNA produced during synthesis of adenovirus type 2 DNA.

Single-stranded fragments of adenovirus type 2 DNA were isolated from infected KB cells under conditions which retarded reassociation of complementary sequences but did not denature native viral DNA. Of the total intracellular, virus-specific DNA labeled during a 1-h pulse with tritiated thymidine begining 15 h after infection, about 20% was single stranded when fractionated on hydroxylapatite. This DNA shifted predominantly to the double-stranded fraction on hydroxylapatite during an extended chase incubation, suggesting that it may represent single-stranded DNA in replicating intermediates. Furthermore, the single-stranded DNA annealed nearly equally to both strands of the adenovirus genome. These findings indicate that at least portions of both complementary strands of adenovirus type 2 DNA are exposed as single strands during the period of viral DNA synthesis.     

Crystal structure of RIG-I C-terminal domain bound to blunt-ended double-strand RNA without 5' triphosphate

RIG-I recognizes molecular patterns in viral RNA to regulate the induction of type I interferons. The C-terminal domain (CTD) of RIG-I exhibits high affinity for 5' triphosphate (ppp) dsRNA as well as blunt-ended dsRNA. Structures of RIG-I CTD bound to 5'-ppp dsRNA showed that RIG-I recognizes the termini of dsRNA and interacts with the ppp through electrostatic interactions. However, the structural basis for the recognition of non-phosphorylated dsRNA by RIG-I is not fully understood. Here, we show that RIG-I CTD binds blunt-ended dsRNA in a different orientation compared to 5' ppp dsRNA and interacts with both strands of the dsRNA. Overlapping sets of residues are involved in the recognition of blunt-ended dsRNA and 5' ppp dsRNA. Mutations at the RNA-binding surface affect RNA binding and signaling by RIG-I. These results provide the mechanistic basis for how RIG-I recognizes different RNA ligands.     

Control of Replication in RNA Bacteriophages

The rates of viral RNA and protein syntheses for wild-type RNA bacteriophages and their nonpolar, coat protein amber mutants were determined in amber suppressor (S26R1E, Su-1 and H12R8a, Su-3) and nonsuppressor (AB259, S26, and Q13) strains of Escherichia coli in the presence of rifamycin. It was demonstrated that the rates of synthesis of phage-specific replicase and RNA minus strands drop off concurrently in both wild-type and coat protein mutant-infected Su(-) and Su(+) cells after 10 and 15 min postinfection, respectively. The rate of synthesis of RNA plus strands started to decline 5 to 10 min later in both cases. Excessive synthesis of replicase in the coat protein mutant-infected cells was accompanied by a similar overproduction of RNA minus strands, but not of plus strands. Partial suppression of protein synthesis in wild-type phage-infected cells abolishing coat protein control over replicase accumulation led to prolongation of replicase synthesis. Such an effect was observed also in coat protein mutant-infected cells, indicating that the excess of replicase itself may be capable of suppression of replicase synthesis in the absence of coat protein. The prolongation of replicase synthesis was followed by the prolonged synthesis of RNA minus strands in both cases. Moreover, replicase and minus strands were formed in nearly equal amounts when protein synthesis was partially inhibited. Assuming functional instability of phage RNAs, the observed coupling of replicase and minus-strand RNA synthesis offers a possibility for control of viral RNA replication by means of control of replicase synthesis on the translational level. A hypothesis is put forward to explain the molecular mechanism of such coupling between the syntheses of replicase and RNA minus strands.     

RNA complementary to the genome of RNA tumor viruses in virions and virus-producing cells.

Cells producing type C (avain sarcoma virus) or type B (mouse mammary tumor virus) RNA tumor viruses contain small amounts of RNA complementary to the viral genomes. The negative strands are complementary to at least 30 to 45% of the viral genomes and are found as RNA-RNA duplexes in the nucleus and cytoplasm of infected cells and in mature virions.     

Launching of the yeast 20 s RNA narnavirus by expressing the genomic or antigenomic viral RNA in vivo

20 S RNA virus is a persistent positive strand RNA virus found in Saccharomyces cerevisiae. The viral genome encodes only its RNA polymerase, p91, and resides in the cytoplasm in the form of a ribonucleoprotein complex with p91. We succeeded in generating 20 S RNA virus in vivo by expressing, from a vector, genomic strands fused at the 3'-ends to the hepatitis delta virus antigenomic ribozyme. Using this launching system, we analyzed 3'-cis-signals present in the genomic strand for replication. The viral genome has five-nucleotide inverted repeats at both termini (5'-GGGGC... GCCCC-OH). The fifth G from the 3'-end was dispensable for replication, whereas the third and fourth Cs were essential. The 3'-terminal and penultimate Cs could be eliminated or modified to other nucleotides; however, the generated viruses recovered these terminal Cs. Furthermore, extra nucleotides added at the viral 3'-end were eliminated in the launched viruses. Therefore, 20 S RNA virus has a mechanism(s) to maintain the correct size and sequence of the viral 3'-end. This may contribute to its persistent infection in yeast. We also succeeded in generating 20 S RNA virus similarly from antigenomic strands provided active p91 was supplied from a second vector in trans. Again, a cluster of four Cs at the 3'-end in the antigenomic strand was essential for replication. In this work, we also present the first conclusive evidence that 20 S and 23 S RNA viruses are independent replicons.     

Structure and function of the non-coding regions of hepatitis C viral RNA

At the 5' and 3' end of genomic HCV RNA there are two highly conserved, untranslated regions, 5'UTR and 3'UTR. These regions are organized into spatially ordered structures and they play key functions in regulation of processes of the viral life cycle. Most nucleotides of the region located at the 5' side of the coding sequence serve as an internal ribosomal entry site, IRES, which directs cap-independent translation. The RNA fragment present at the 3' end of the genome is required for virus replication and probably contributes to translation of viral proteins. During virus replication its genomic strand is transcribed into a strand of minus polarity, the replicative strand. Its 3' terminus is responsible for initiation of synthesis of descendant genomic strands. This article summarizes our current knowledge on the structure and function of the non-coding regions of hepatitis C genomic RNA, 5'UTR and 3'UTR, and the complementary sequences of the replicative viral strand.     

The existence of DNA sequences homologous to adenovirus 5 DNA in the genome of normal rat cells.

Three discrete bands specifically hybridizing to adenovirus 5 DNA were found in the rat liver DNA restricted BY Bam HI endonuclease and fractionated electrophoretically. The hybridization with different regions of the viral genome takes place. Similar bands are present in the DNA from different lines of adenovirus 5 transformed cells, but in these cases high molecular weight DNA fragments containing the integrated viral genomes can also be found.