Yeast as a model host to study replication and recombination of defective interfering RNA of Tomato bushy stunt virus

Defective interfering (DI) RNA associated with Tomato bushy stunt virus (TBSV), which is a plus-strand RNA virus, requires p33 and p92 proteins of TBSV or the related Cucumber necrosis virus (CNV), for replication in plants. To test if DI RNA can replicate in a model host, we coexpressed TBSV DI RNA and p33/p92 of CNV in yeast. We show evidence for replication of DI RNA in yeast, including (i) dependence on p33 and p92 for DI replication; (ii) presence of active CNV RNA-dependent RNA polymerase in isolated membrane-containing preparations; (iii) increasing amount of DI RNA(+) over time; (iv) accumulation of (-)stranded DI RNA; (v) presence of correct 5' and 3' ends in DI RNA; (vi) inhibition of replication by mutations in the replication enhancer; and (vii) evolution of DI RNA over time, as shown by sequence heterogeneity. We also produced evidence supporting the occurrence of DI RNA recombinants in yeast. In summary, development of yeast as a host for replication of TBSV DI RNA will facilitate studies on the roles of viral and host proteins in replication/recombination.     

The majority of the nucleotides in the top loop of the genomic 3' terminal stem loop structure are cis-acting in a West Nile virus infectious clone

The flavivirus genome RNA terminates with a conserved 3' stem loop (SL) structure that was shown to be essential for virus replication. A stretch of conserved nts is located in the top loop (TL) of this structure. Mutation of the TL nts (5' ACAGUGC 3') in a WNV infectious clone indicated that 3 of the 7 TL nts (5' ACAGUGC 3') are critical for virus replication. Mutation of 3 of the other nts reduced the efficiency of virus replication. The four 5' TL nts are conserved in both mosquito- and tick-borne flavivirus genomes, while the TL 3' C is conserved in mosquito-borne viruses. The conservation of two or three G-C base pairs in the TL flanking sequences suggests that a stable stem is necessary for precise presentation of the TL sequence. The TL may participate in RNA as well as protein interactions.     

New Satellite RNAs,but No DI RNAs,Are Found in Natural Populations of Tomato Bushy Stunt Tombusvirus

A collection of 57 field isolates of the tombusvirus tomato bushy stunt virus was obtained from eggplant and tomato during 1994–1997 and was examined for the presence of defective interfering (DI) RNA species by Northern blot hybridization and RT-PCR. No DI RNA species were detected associated with any of the field TBSV isolates. However, serial passaging of two field isolates inNicotiana clevelandiiat high multiplicity of infection resulted in the rapid generation of DI-like RNA species, indicating that the absence of DI RNAs in natural populations of the virus was not due to the inability of the TBSV field isolates to generate them in a suitable host. The results indicate that DI RNAs may not play a role in modulating natural TBSV infections in the hosts examined. In 4 of 57 isolates analyzed we have detected less than full-length RNAs and we show here that they are true satellite RNAs. Two different satellite RNA species were detected, named TBSV sat RNAs B1 (822 nt) and B10 (612 nt). TBSV sat RNAs lack significant open reading frames and do not present sequence homology except in a central box that is also conserved in TBSV-Ch genomic RNA and in all the DI RNAs derived from it. TBSV sat RNA B10 attenuated the symptoms induced by the helper virus inN. clevelandiiwhile sat RNA B1 did not modify the symptoms. This is the first report of sat RNAs associated with TBSV and the first time that sat RNAs are associated with natural tombusvirus infections.     

Modular arrangement of viral cis-acting RNA domains in a tombusvirus satellite RNA

Satellite (sat) RNAs are parasitic sub-viral RNA replicons found associated with certain positive-strand RNA viruses. Typical sat RNAs, such as those associated with members of the genus Tombusvirus, share little or no sequence identity with their helper virus genomes. Here, we have investigated a tombusvirus sat RNA and determined that it contains two functionally-relevant higher-order RNA domains, a T-shaped domain and a downstream domain, that are similar to elements shown previously to be present in the 5' untranslated regions (UTRs) of tombusvirus genomes. Although the two sat RNA domains showed only limited sequence identity with their viral counterparts, they were able to adopt comparably-folded RNA secondary structures. Interestingly, the relative spacing between the domains in the viral and satellite contexts was notably different. In the viral 5' UTR, the two domains are adjacent and separated by a small hairpin, however, in the sat RNA they are separated by a 137-nt long segment. Despite this distal modular arrangement, the two domains were found to be united spatially in the sat RNA through the formation of an RNA-RNA bridge. This co-localization facilitated an important inter-domain interaction and was essential for efficient helper-mediated sat RNA accumulation in protoplasts. These results indicate that the tombusvirus sat RNA and helper genome contain structurally and functionally equivalent RNA domains. It is proposed that the limited sequence identity observed between these corresponding higher-order RNA structures is related to a strategy that reduces the induction of gene silencing, which presumably would be detrimental to both viral and sat RNA replicons.     

Structure and prevalence of replication silencer-3' terminus RNA interactions in Tombusviridae

Tombusviridae is a large positive-strand RNA virus family. Tomato bushy stunt virus (TBSV), the type virus of this family, has a genome ending with AGCCC(-OH), termed the 3'-complementary silencer sequence (3'CSS). The 3'CSS is able to base pair with a complementary internally-located sequence, 5'GGGCU, called the replication silencer element (RSE). In TBSV, previous compensatory mutational analysis of the RSE-3'CSS interaction showed it to be functionally important for viral RNA synthesis both in vitro and in vivo. However, these investigations also revealed that the RSE and 3'CSS are very sensitive to nucleotide changes, even when base pairing potential between the two elements is maintained. Consequently, an alternative investigative approach was used in this study where the wild-type sequences of these elements were preserved and their surrounding contexts were modified. Results from these analyses, using a TBSV DI RNA, revealed important new structural requirements necessary for the RSE and 3'CSS to operate in vivo. Collectively, the data suggest that accessibility of the elements and their proximity to adjoining stem structures are important functional parameters. Based on these findings, a working structural model for the TBSV RSE-3'CSS interaction is proposed that involves coaxial stacking of adjacent helices at either end of the RSE-3'CSS interaction. Components of this structural model are extendable to potential RSE-3'CSS interactions that were identified throughout Tombusviridae by comparative sequence analysis. This survey also revealed a significant level of diversity and modularity with respect to RSEs, 3'CSSs and their structural contexts and, moreover, suggests that RSE-3'CSS interactions are prevalent in Tombusviridae and related viruses.     

Similar roles for yeast Dbp2 and Arabidopsis RH20 DEAD-box RNA helicases to Ded1 helicase in tombusvirus plus-strand synthesis

Recruited host factors aid replication of plus-strand RNA viruses. In this paper, we show that Dbp2 DEAD-box helicase of yeast, which is a homolog of human p68 DEAD-box helicase, directly affects replication of Tomato bushy stunt virus (TBSV). We demonstrate that Dbp2 binds to the 3′-end of the viral minus-stranded RNA and enhances plus-strand synthesis by the viral replicase in a yeast-based cell-free TBSV replication assay. In vitro data with wt and an ATPase-deficient Dbp2 mutant indicate that Dbp2 unwinds local secondary structures at the 3′-end of the TBSV (−)RNA. We also show that Dbp2 complements the replication deficiency of TBSV in yeast containing reduced amount of Ded1 DEAD-box helicase, another host factor involved in TBSV replication, suggesting that Dbp2 and Ded1 helicases play redundant roles in TBSV replication. We also show that the orthologous AtRH20 DEAD-box helicase from Arabidopsis can increase tombusvirus replication in vitro and in yeast.     

Genome sequence and molecular characterization of Homalodisca coagulata virus-1, a novel virus discovered in the glassy-winged sharpshooter (Hemiptera: Cicadellidae)

The complete nucleotide sequence of a novel single-stranded RNA virus infecting the glassy-winged sharpshooter, Homalodisca coagulata, has been determined. In silico analysis of H. coagulata virus-1 (HoCV-1) revealed a 9321-nt polyadenylated genome encoding two large open reading frames (ORF1 and ORF2) separated by a 182-nt intergenic region (IGR). The deduced amino acid sequence of the 5'-proximal ORF (ORF1, nt 420-5807) exhibited conserved core motifs characteristic of the helicases, cysteine proteases, and RNA-dependent RNA polymerases of other insect-infecting picorna-like viruses. A structural model created using Mfold exposed a series of stem loop (SL) structures immediately preceding the second ORF which are analogous to an internal ribosome entry site (IRES), suggesting that ORF2 begins with a noncognate GCA triplet rather than the canonical AUG. This 3' ORF2 (5990-8740) showed significant similarity to the structural proteins of members of the family Dicistroviridae, particularly those belonging to the genus Cripavirus. Evidence demonstrating relatedness of these viruses regarding genome organization, amino acid sequence similarity, and putative replication strategy substantiate inclusion of HoCV-1 into this taxonomic position.     

Expression of the Arabidopsis Xrn4p 5'-3' exoribonuclease facilitates degradation of tombusvirus RNA and promotes rapid emergence of viral variants in plants

Rapid RNA virus evolution is a major problem due to the devastating diseases caused by human, animal and plant-pathogenic RNA viruses. A previous genome-wide screen for host factors affecting recombination in Tomato bushy stunt tombusvirus (TBSV), a small monopartite plant virus, identified Xrn1p 5'-3' exoribonuclease of yeast, a model host, whose absence led to increased appearance of recombinants [Serviene, E., Shapka, N., Cheng, C.P., Panavas, T., Phuangrat, B., Baker, J., Nagy, P.D., (2005). Genome-wide screen identifies host genes affecting viral RNA recombination. Proc. Natl. Acad. Sci. U. S. A. 102 (30), 10545-10550]. In this paper, we tested if over-expression of Xrn1p in yeast or expression of the analogous Xrn4p cytoplasmic 5'-3' exoribonuclease, which has similar function in RNA degradation in Arabidopsis as Xrn1p in yeast, in Nicotiana benthamiana could affect the accumulation of tombusvirus RNA. We show that over-expression of Xrn1p led to almost complete degradation of TBSV RNA replicons in yeast, suggesting that Xrn1p is involved in TBSV degradation. Infection of N. benthamiana expressing AtXrn4p with Cucumber necrosis tombusvirus (CNV) led to enhanced viral RNA degradation, suggesting that the yeast and the plant cytoplasmic 5'-3' exoribonuclease play similar roles. We also observed rapid emergence of novel CNV genomic RNA variants formed via deletions of 5' terminal sequences in N. benthamiana expressing AtXrn4p. Three of the newly emerging 5' truncated CNV variants were infectious in N. benthamiana protoplasts, whereas one CNV variant caused novel symptoms and moved systemically in N. benthamiana plants. Altogether, this paper establishes that a single plant gene can contribute to the emergence of novel viral variants.     

Exploiting alternative subcellular location for replication: tombusvirus replication switches to the endoplasmic reticulum in the absence of peroxisomes

Plus-strand RNA virus replication takes place on distinct membranous surfaces in infected cells via the assembly of viral replicase complexes involving multiple viral and host proteins. One group of tombusviruses, such as Tomato bushy stunt virus (TBSV), replicate on the surfaces of peroxisomal membranes in plant and yeast cells. Surprisingly, previous genome-wide screen performed in yeast demonstrated that a TBSV replicon RNA replicated as efficiently in yeast defective in peroxisome biogenesis as in the wt yeast (Panavas et al., Proc Natl Acad Sci U S A, 2005). To further test how the lack of peroxisomes could affect tombusvirus replication, we used yeast cells missing either PEX3 or PEX19 genes, which are absolutely essential for peroxisome biogenesis. Confocal microscopy-based approach revealed that the wild-type tombusvirus p33 replication protein accumulated in the endoplasmic reticulum (ER) in pex3Delta or pex19Delta yeast, suggesting that tombusvirus replication could take place on the surface of ER membrane. The activities of the isolated tombusvirus replicase preparations from wt, pex3Delta or pex19Delta yeasts were comparable, demonstrating that the assembly of the replicase was as efficient in the ER as in the authentic subcellular environments. The generation/accumulation of tombusvirus recombinants was similar in wt, pex3Delta and pex19Delta yeasts, suggesting that the rate of mistakes occurring during tombusvirus replication is comparable in the presence or absence of peroxisomes. Overall, this work demonstrates that a tombusvirus, relying on the wt replication proteins, can efficiently replicate on an alternative intracellular membrane. This suggests that RNA viruses might have remarkable flexibility for using various host membranes for their replication.     

De novo generation of defective interfering RNAs of tomato bushy stunt virus by high multiplicity passage

Defective interfering (DI) RNAs were generated de novo in each of 12 independent isolates of tomato bushy stunt virus (TBSV) upon serial passage at high multiplicities of infection (m.o.i.) in plants, but not in any of 4 additional isolates after 11 serial passages at low m.o.i. The DI RNAs were detected in RNA isolated from virus particles and in 2.3 M LiCl-soluble RNA fractions isolated from inoculated leaves. Symptom attenuation leading to persistent infections was closely correlated with the passage in which DIs first developed. Comparisons of nucleotide sequences of 10 cDNA clones from 2 DI RNA populations and with a previously characterized TBSV DI RNA revealed the same four regions of sequence from the TBSV genome were strictly conserved in each of the DI RNAs: the virus 5' leader sequence of 168 bases; a region of approximately 200-250 bases from the viral polymerase gene; approximately 70 bases from the 3' terminus of the viral p19 and p22 genes; and approximately 130 bases from the 3' terminal noncoding region. Conservation of the sequence motif present in all of the DIs suggests that there might be a common mechanism of DI formation as well as selection pressure to maintain sequences essential for replication and encapsidation.     

Host transcription factor Rpb11p affects tombusvirus replication and recombination via regulating the accumulation of viral replication proteins

Previous genome-wide screens identified over 100 host genes whose deletion/down-regulation affected tombusvirus replication and 32 host genes that affected tombusvirus RNA recombination in yeast, a model host for replication of Tomato bushy stunt virus (TBSV). Down-regulation of several of the identified host genes affected the accumulation levels of p33 and p92(pol) replication proteins, raising the possibility that these host factors could be involved in the regulation of the amount of viral replication proteins and, thus, they are indirectly involved in TBSV replication and recombination. To test this model, we developed a tightly regulated expression system for recombinant p33 and p92(pol) replication proteins in yeast. We demonstrate that high accumulation level of p33 facilitated efficient viral RNA replication, while the effect of p33 level on RNA recombination was less pronounced. On the other hand, high level of p92(pol) accumulation promoted TBSV RNA recombination more efficiently than RNA replication. As predicted, Rpb11p, which is part of the polII complex, affected the accumulation levels of p33 and p92(pol) as well as altered RNA replication and recombination. An in vitro assay with the tombusvirus replicase further supported that Rpb11p affects TBSV replication and recombination only indirectly, via regulating p33 and p92(pol) levels. In contrast, the mechanism by which Rpt4p endopeptidase/ATPase and Mps1p threonine/tyrosine kinase affect TBSV recombination is different from that proposed for Rpb11p. We propose a model that the concentration (molecular crowding) of replication proteins within the viral replicase is a factor affecting viral replication and recombination.     

Silencing of Nicotiana benthamiana Xrn4p exoribonuclease promotes tombusvirus RNA accumulation and recombination

The cytosolic 5′-to-3′ exoribonuclease Xrn1p plays a major role in recombination and degradation of Tomato bushy stunt tombusvirus (TBSV) replicon (rep)RNA in yeast, a model host (Serviene, E., Shapka, N., Cheng, C.P., Panavas, T., Phuangrat, B., Baker, J., and Nagy, P.D., 2005. Genome-wide screen identifies host genes affecting viral RNA recombination. Proc. Natl. Acad. Sci. U. S. A. 102(30), 10545-10550.). To test if the plant cytosolic 5′-to-3′ exoribonuclease Xrn4p, similar to the yeast Xrn1p, could also affect TBSV recombination, in this paper, we silenced XRN4 in Nicotiana benthamiana, an experimental host. The accumulation of tombusvirus genomic RNA and repRNA increased by 50% and 220%, respectively, in XRN4-silenced N. benthamiana. We also observed up to 125-fold increase in the emergence of new recombinants and partly degraded viral RNAs in the silenced plants. Using a cell-free assay based on a yeast extract, which supports authentic replication and recombination of TBSV, we demonstrate that the purified recombinant Xrn1p efficiently inhibited the accumulation of recombinants and partly degraded viral RNAs. Altogether, the data from a plant host and cell-free system confirm a central role for the plant cytosolic 5′-to-3′ exoribonuclease in TBSV replication, recombination and viral RNA degradation.     

Ubiquitination of tombusvirus p33 replication protein plays a role in virus replication and binding to the host Vps23p ESCRT protein

Post-translational modifications of viral replication proteins could be widespread phenomena during the replication of plus-stranded RNA viruses. In this article, we identify two lysines in the tombusvirus p33 replication co-factor involved in ubiquitination and show that the same lysines are also important for the p33 to interact with the host Vps23p ESCRT-I factor. We find that the interaction of p33 with Vps23p is also affected by a “late-domain”-like sequence in p33. The combined mutations of the two lysines and the late-domain-like sequences in p33 reduced replication of a replicon RNA of Tomato bushy stunt virus in yeast model host, in plant protoplasts, and plant leaves, suggesting that p33-Vps23p ESCRT protein interaction affects tombusvirus replication. Using ubiquitin-mimicking p33 chimeras, we demonstrate that high level of p33 ubiquitination is inhibitory for TBSV replication. These findings argue that optimal level of p33 ubiquitination plays a regulatory role during tombusvirus infections.     

Structural and mutational analyses of cis-acting sequences in the 5'-untranslated region of satellite RNA of bamboo mosaic potexvirus

The satellite RNA of Bamboo mosaic virus (satBaMV) contains on open reading frame for a 20-kDa protein that is flanked by a 5'-untranslated region (UTR) of 159 nucleotides (nt) and a 3'-UTR of 129 nt. A secondary structure was predicted for the 5'-UTR of satBaMV RNA, which folds into a large stem-loop (LSL) and a small stem-loop. Enzymatic probing confirmed the existence of LSL (nt 8-138) in the 5'-UTR. The essential cis-acting sequences in the 5'-UTR required for satBaMV RNA replication were determined by deletion and substitution mutagenesis. Their replication efficiencies were analyzed in Nicotiana benthamiana protoplasts and Chenopodium quinoa plants coinoculated with helper BaMV RNA. All deletion mutants abolished the replication of satBaMV RNA, whereas mutations introduced in most of the loop regions and stems showed either no replication or a decreased replication efficiency. Mutations that affected the positive-strand satBaMV RNA accumulation also affected the accumulation of negative-strand RNA; however, the accumulation of genomic and subgenomic RNAs of BaMV were not affected. Moreover, covariation analyses of natural satBaMV variants provide substantial evidence that the secondary structure in the 5'-UTR of satBaMV is necessary for efficient replication.     

The host Pex19p plays a role in peroxisomal localization of tombusvirus replication proteins

Replication of Tomato bushy stunt virus (TBSV) RNA takes place on the cytosolic membrane surface of peroxisomes in plants and in yeast, a model host. To identify the host proteins involved in assisting the peroxisomal localization of the tombusvirus p33 replication protein, we tested if p33 could bind directly to yeast proteins involved in peroxisomal transport in vitro. This work has led to the demonstration of Pex19p-p33 interaction via pull-down and co-purification experiments. Pex19p was also detected in the tombusvirus replicase after protein cross-linking, suggesting that Pex19p transiently binds to the replicase as could be expected from a transporter. To validate the importance of Pex19p-p33 interaction in TBSV replication in yeast, we re-targeted Pex19p to the mitochondria, which resulted in the re-distribution of a large fraction of p33 to the mitochondria. The expression of the mitochondrial-targeted Pex19p inhibited TBSV RNA accumulation by 2-4-fold in vivo and reduced the in vitro activity of the tombusvirus replicase by 80%. These data support the model that Pex19p is a cellular transporter for localization of p33 replication protein to the host peroxisomal membranes.     

Mutations in the RNA-binding domains of tombusvirus replicase proteins affect RNA recombination in vivo

RNA recombination, which is thought to occur due to replicase errors during viral replication, is one of the major driving forces of virus evolution. In this article, we show evidence that the replicase proteins of Cucumber necrosis virus, a tombusvirus, are directly involved in RNA recombination in vivo. Mutations within the RNA-binding domains of the replicase proteins affected the frequency of recombination observed with a prototypical defective-interfering (DI) RNA, a model template for recombination studies. Five of the 17 replicase mutants tested showed delay in the formation of recombinants when compared to the wild-type helper virus. Interestingly, two replicase mutants accelerated recombinant formation and, in addition, these mutants also increased the level of subgenomic RNA synthesis (Virology 308 (2003), 191-205). A trans-complementation system was used to demonstrate that mutation in the p33 replicase protein resulted in altered recombination rate. Isolated recombinants were mostly imprecise (nonhomologous), with the recombination sites clustered around a replication enhancer region and a putative cis-acting element, respectively. These RNA elements might facilitate the proposed template switching events by the tombusvirus replicase. Together with data in the article cited above, results presented here firmly establish that the conserved RNA-binding motif of the replicase proteins is involved in RNA replication, subgenomic RNA synthesis, and RNA recombination.     

A new positive-strand RNA virus with unique genome characteristics from the red imported fire ant, Solenopsis invicta

We report the discovery of a new virus with unique genome characteristics from the red imported fire ant, Solenopsis invicta. This virus represents the second identified from this ant species. It is provisionally named Solenopsis invicta virus 2 (SINV-2). The SINV-2 genome was constructed by compiling sequences from successive 5' RACE reactions, a 3' RACE reaction, and expressed sequence tag, c246 (accession number EH413675), from a fire ant expression library. The SINV-2 genome structure was monopartite, polycistronic and RNA-based. The genome consensus sequence (EF428566) was 11,303 nucleotides in length, excluding the poly(A) tail present on the 3' end. Analysis of the genome revealed 4 major open reading frames (ORFs; comprised of > or =100 codons) and 5 minor ORFs (comprised of 50-99 codons) in the sense orientation. No large ORFs were found in the inverse orientation suggesting that the SINV-2 genome was from a positive-strand RNA virus. Further evidence for this conclusion includes abolished RT-PCR amplification by RNase treatment of SINV-2 nucleic acid template, and failure to amplify without first conducting cDNA synthesis. Blastp analysis indicated that ORF 4 contained conserved domains of an RNA-dependent RNA polymerase, helicase, and protease, characteristic of positive-strand RNA viruses. However, the protease domain and putative structural proteins (ORFs 1, 2, and 3) were less well conserved. Phylogenetic analysis of the RdRp, helicase, and ORF 1 indicate unique placement of SINV-2 exclusive from the Dicistroviridae, iflaviruses, Picornaviridae, and plant small RNA viruses.     

Mutational analysis of three predicted 5'-proximal stem-loop structures in the genome of tick-borne encephalitis virus indicates different roles in RNA replication and translation

Flavivirus gene expression is modulated by RNA secondary structure elements at the terminal ends of the viral RNA molecule. For tick-borne encephalitis virus (TBEV), four stem-loop (SL) elements have been predicted in the first 180 nucleotides of the viral genome: 5′-SL1, 5′-SL2, 5′-SL3 and 5′-SL4. The last three of these appear to be unique to tick-borne flaviviruses. Here, we report their characterization by mutagenesis in a TBEV luciferase reporter system. By manipulating their thermodynamic properties, we found that an optimal stability of the 5′-SL2 is required for efficient RNA replication. 5′-SL3 formation is also important for viral RNA replication, but although it contains the viral start codon, its formation is dispensable for RNA translation. 5′-SL4 appears to facilitate both RNA translation and replication. Our data suggest that maintenance of the balanced thermodynamic stability of these SL elements is important for temporal regulation of its different functions.