Atypical RNA Elements Modulate Translational Readthrough in Tobacco Necrosis Virus D.

Tobacco necrosis virus, strain D (TNV-D), is a positive-strand RNA virus in the genus Betanecrovirus and family Tombusviridae The production of its RNA-dependent RNA polymerase, p82, is achieved by translational readthrough. This process is stimulated by an RNA structure that is positioned immediately downstream of the recoding site, termed the readthrough stem-loop (RTSL), and a sequence in the 3' untranslated region of the TNV-D genome, called the distal readthrough element (DRTE). Notably, a base pairing interaction between the RTSL and the DRTE, spanning ∼3,000 nucleotides, is required for enhancement of readthrough. Here, some of the structural features of the RTSL, as well as RNA sequences and structures that flank either the RTSL or DRTE, were investigated for their involvement in translational readthrough and virus infectivity. The results revealed that (i) the RTSL-DRTE interaction cannot be functionally replaced by stabilizing the RTSL structure, (ii) a novel tertiary RNA structure positioned just 3' to the RTSL is required for optimal translational readthrough and virus infectivity, and (iii) these same activities also rely on an RNA stem-loop located immediately upstream of the DRTE. Functional counterparts for the RTSL-proximal structure may also be present in other tombusvirids. The identification of additional distinct RNA structures that modulate readthrough suggests that regulation of this process by genomic features may be more complex than previously appreciated. Possible roles for these novel RNA elements are discussed.IMPORTANCE The analysis of factors that affect recoding events in viruses is leading to an ever more complex picture of this important process. In this study, two new atypical RNA elements were shown to contribute to efficient translational readthrough of the TNV-D polymerase and to mediate robust viral genome accumulation in infections. One of the structures, located close to the recoding site, could have functional equivalents in related genera, while the other structure, positioned 3' proximally in the viral genome, is likely limited to betanecroviruses. Irrespective of their prevalence, the identification of these novel RNA elements adds to the current repertoire of viral genome-based modulators of translational readthrough and provides a notable example of the complexity of regulation of this process.

Identification and characterization of the 480-kilodalton template-specific RNA-dependent RNA polymerase complex of red clover necrotic mosaic virus.

Replication of positive-strand RNA viruses occurs through the assembly of membrane-associated viral RNA replication complexes that include viral replicase proteins, viral RNA templates, and host proteins. Red clover necrotic mosaic virus (RCNMV) is a positive-strand RNA plant virus with a genome consisting of RNA1 and RNA2. The two proteins encoded by RNA1, a 27-kDa protein (p27) and an 88-kDa protein containing an RNA-dependent RNA polymerase (RdRP) motif (p88), are essential for RCNMV RNA replication. To analyze RCNMV RNA replication complexes, we used blue-native polyacrylamide gel electrophoresis (BN/PAGE), which enabled us to analyze detergent-solubilized large membrane protein complexes. p27 and p88 formed a complex of 480 kDa in RCNMV-infected plants. As a result of sucrose gradient sedimentation, the 480-kDa complex cofractionated with both endogenous template-bound and exogenous template-dependent RdRP activities. The amount of the 480-kDa complex corresponded to the activity of exogenous template-dependent RdRP, which produced RNA fragments by specifically recognizing the 3'-terminal core promoter sequences of RCNMV RNAs, but did not correspond to the activity of endogenous template-bound RdRP, which produced genome-sized RNAs without the addition of RNA templates. These results suggest that the 480-kDa complex contributes to template-dependent RdRP activities. We subjected those RdRP complexes to affinity purification and analyzed their components using two-dimensional BN/sodium dodecyl sulfate-PAGE (BN/SDS-PAGE) and mass spectrometry. The 480-kDa complex contained p27, p88, and possible host proteins, and the original affinity-purified RdRP preparation contained HSP70, HSP90, and several ribosomal proteins that were not detected in the 480-kDa complex. A model for the formation of RCNMV RNA replication complexes is proposed.

The Cucumber leaf spot virus p25 auxiliary replicase protein binds and modifies the endoplasmic reticulum via N-terminal transmembrane domains.

Cucumber leaf spot virus (CLSV) is a member of the Aureusvirus genus, family Tombusviridae. The auxiliary replicase of Tombusvirids has been found to localize to endoplasmic reticulum (ER), peroxisomes or mitochondria; however, localization of the auxiliary replicase of aureusviruses has not been determined. We have found that the auxiliary replicase of CLSV (p25) fused to GFP colocalizes with ER and that three predicted transmembrane domains (TMDs) at the N-terminus of p25 are sufficient for targeting, although the second and third TMDs play the most prominent roles. Confocal analysis of CLSV infected 16C plants shows that the ER becomes modified including the formation of punctae at connections between ER tubules and in association with the nucleus. Ultrastructural analysis shows that the cytoplasm contains numerous vesicles which are also found between the perinuclear ER and nuclear membrane. It is proposed that these vesicles correspond to modified ER used as sites for CLSV replication.

Identification of amino acids in auxiliary replicase protein p27 critical for its RNA-binding activity and the assembly of the replicase complex in Red clover necrotic mosaic virus.

The specific recognition of genomic RNAs by viral replicase proteins is a key regulatory step during the early replication process in positive-strand RNA viruses. In this study, we characterized the RNA-binding activity of the auxiliary replicase protein p27 of Red clover necrotic mosaic virus (RCNMV), which has a bipartite genome consisting of RNA1 and RNA2. Aptamer pull-down assays identified the amino acid residues of p27 involved in its specific interaction with RNA2. The RNA-binding activity of p27 correlated with its activity in recruiting RNA2 to membranes. We also identified the amino acids required for the formation of the 480-kDa replicase complex, a key player of RCNMV RNA replication. These amino acids are not involved in the functions of p27 that bind viral RNA or replicase proteins, suggesting an additional role for p27 in the assembly of the replicase complex. Our results demonstrate that p27 has multiple functions in RCNMV replication.

Mitochondrial targeting and membrane anchoring of a viral replicase in plant and yeast cells.

Replication of the Carnation Italian ringspot virus genomic RNA in plant cells occurs in multivesicular bodies which develop from the mitochondrial outer membrane during infection. ORF1 in the viral genome encodes a 36-kDa protein, while ORF2 codes for the 95-kDa replicase by readthrough of the ORF1 stop codon. We have shown previously that the N-terminal part of ORF1 contains the information leading to vesiculation of mitochondria and that the 36-kDa protein localizes to mitochondria. Using infection, in vivo expression of green fluorescent protein fusions in plant and yeast cells, and in vitro mitochondrial integration assays, we demonstrate here that both the 36-kDa protein and the complete replicase are targeted to mitochondria and anchor to the outer membrane with the N terminus and C terminus on the cytosolic side. Analysis of deletion mutants indicated that the anchor sequence is likely to correspond approximately to amino acids 84 to 196, containing two transmembrane domains. No evidence for a matrix-targeting presequence was found, and the data suggest that membrane insertion of the viral proteins is mediated by an import receptor-independent signal-anchor mechanism relying on the two transmembrane segments and multiple recognition signals present in the N-terminal part of ORF1.

Nucleotide sequence and infectivity of a full-length cDNA clone of panicum mosaic virus.

The sequence of an infectious cDNA clone of panicum mosaic virus (PMV) showed that the single-stranded RNA genome is 4326 nucleotides (nt) and a single highly abundant subgenomic (sg) RNA of 1475 nt was synthesized during PMV infection of pearl millet plants and protoplasts. Computer comparisons revealed strong similarities between the predicted amino acid sequences of the p48 and p112 open reading frames (ORFs) and replicase proteins of members of the Tombusviridae. The sgRNA has the potential to encode five proteins. Three small ORFs, p8, p8-FS, and/or p6.6 have similarity to ORFs of carmo-, necro-, and machlomoviruses thought to be involved in virus spread in plants. The sgRNA also has the potential to encode a 26-kDa capsid protein and a 15-kDa nested gene (p15) of unknown function. PMV transcripts also supported replication and movement of SPMV, the satellite virus. Genome organization, physicochemical properties, and biological features indicate that PMV is a member of the Tombusviridae family. However, PMV differs sufficiently from previously described members to warrant its placement in a new genus provisionally designated Panicovirus.