A Discontinuous RNA Platform Mediates RNA Virus Replication: Building an Integrated Model for RNA–based Regulation of Viral Processes

104

The capsid protein (CP) of Turnip crinkle virus (TCV) is a multifunctional protein needed for virus assembly, suppression of RNA silencing-based antiviral defense, and long-distance movement in infected plants. In this report, we have examined genetic requirements for the different functions of TCV CP and evaluated the interdependence of these functions. A series of TCV mutants containing alterations in the CP coding region were generated. These alterations range from single-amino-acid substitutions and domain truncations to knockouts of CP translation. The latter category also contained two constructs in which the CP coding region was replaced by either the cDNA of a silencing suppressor of a different virus or that of green fluorescent protein. These mutants were used to infect Arabidopsis plants with diminished antiviral silencing capability (dcl2 dcl3 dcl4 plants). There was a strong correlation between the ability of mutants to reach systemic leaves and the silencing suppressor activity of mutant CP. Virus particles were not essential for entry of the viral genome into vascular bundles in the inoculated leaves in the absence of antiviral silencing. However, virus particles were necessary for egress of the viral genome from the vasculature of systemic leaves. Our experiments demonstrate that TCV CP not only allows the viral genome to access the systemic movement channel through silencing suppression but also ensures its smooth egress by way of assembled virus particles. These results illustrate that efficient long-distance movement of TCV requires both functions afforded by the CP.

mentioning

confidence: 99%

The Capsid Protein of Turnip Crinkle Virus Overcomes Two Separate Defense Barriers To Facilitate Systemic Movement of the Virus in Arabidopsis

Cao¹,

Willie³

et al. 2010

104

“…Some, but not all, plus-strand RNA viruses contain a substantial number of genome-wide secondary structure elements (genome-scale ordered RNA structure [GORS]) and adopt a compact spheroid shape that correlates with viral persistence (5,6). While RNA viruses likely make extensive use of local and long-distance RNA interactions between such secondary structure elements to control basic processes, only a limited number of such interactions have been reported, with identified connections forming more readily discernible canonical base pairings (3,4,9,10,12,13,14,15,23,26,28,40,41).…”

Section: Discussionmentioning

confidence: 99%

“…For dengue virus (DENV), several sets of overlapping 5=-and 3=-interacting sequences have been identified that control the balance between linear and circular forms of the genome, which is critical for replication but not translation (13,38). Tomato bushy stunt virus (TBSV) requires a complex network of long-distance RNA-RNA interactions to promote replication, subgenomic RNA (sgRNA) synthesis, translation initiation, and translational recoding (4,14,20,40). For both examples, however, only canonical WatsonCrick interactions have been elucidated.…”

mentioning

confidence: 99%

A Local, Interactive Network of 3′ RNA Elements Supports Translation and Replication of Turnip Crinkle Virus

Yuan

Shi

Simon

2012

The majority of the 3= untranslated region (UTR) of Turnip crinkle virus (TCV) was previously identified as forming a highly interactive structure with a ribosome-binding tRNA-shaped structure (TSS) acting as a scaffold and undergoing a widespread conformational shift upon binding to RNA-dependent RNA polymerase (RdRp). Tertiary interactions in the region were explored by identifying two highly detrimental mutations within and adjacent to a hairpin H4 upstream of the TSS that reduce translation in vivo and cause identical structural changes in the loop of the 3= terminal hairpin Pr. Second-site changes that compensate for defects in translation/accumulation and reverse the structural differences in the Pr loop were found in the Pr stem, as well as in a specific stem within the TSS and within the capsid protein (CP) coding region, suggesting that the second-site changes were correcting a conformational defect and not restoring specific base pairing. The RdRp-mediated conformational shift extended upstream through this CP open reading frame (ORF) region after bypassing much of an intervening, largely unstructured region, supporting a connection between 3= elements and coding region elements. These data suggest that the Pr loop, TSS, and H4 are central elements in the regulation of translation and replication in TCV and allow for development of an RNA interactome that maps the higher-order structure of a postulated RNA domain within the 3= region of a plus-strand RNA virus.T he genomes of positive-strand RNA viruses control fundamental processes such as translation and replication through multiple RNA elements that interact dynamically with each other and with viral and host proteins. Upon entry into host cells, the viral genomic RNA (gRNA) is recognized as a template by the host translational apparatus for production of replication-associated proteins, which combine with an increasingly diverse variety of host factors to synthesize negative-strand RNAs that then serve as the templates for synthesis of progeny positive-strand RNAs (1,7,8,19). The widespread positioning of cis-elements that function in translation and/or replication requires short-range or long-range bridges within the RNA to deliver bound elements to locations where the specific processes initiate.Understanding how functional, dynamic RNA structures regulate viral processes requires a detailed knowledge of the topology of important regions of the RNA genome and the canonical and noncanonical tertiary interactions that connect various elements. Attempts to decipher networks of short-and long-distance RNA-RNA interactions have been limited to a few viruses. For dengue virus (DENV), several sets of overlapping 5=-and 3=-interacting sequences have been identified that control the balance between linear and circular forms of the genome, which is critical for replication but not translation (13,38). Tomato bushy stunt virus (TBSV) requires a complex network of long-distance RNA-RNA interactions to promote replication, subgenomic RNA (sgRNA) synthesis, tr...

“…These studies led to the identification of ϳ150 host genes that either stimulated or inhibited virus replication and RNA recombination. Additional global proteomics approaches, such as protein arrays, a cDNA library screen and mass spectrometry of the purified tombusvirus replicase, have led to the identification of an additional ϳ150 host proteins that interact with either p33 and p92 or the TBSV RNA (25,27,32,67).Functions of several of the identified host proteins in TBSV replication have been dissected in yeast and in vitro as well as validated in a native plant host (3,13,16,17,28,38,40,65,84,88).In spite of the intensive genome-wide and global proteomics screens for TBSV host factors, it seems that previous screens have not reached saturation level, since the overlap among the identified set of host genes from various screens is somewhat low, albeit significant (37,40). Therefore, we are continuing systematic screening for TBSV host factors in yeast.…”

mentioning

confidence: 99%

Proteome-Wide Overexpression of Host Proteins for Identification of Factors Affecting Tombusvirus RNA Replication: an Inhibitory Role of Protein Kinase C

Nawaz-ul-Rehman

Martinez-Ochoa

Pascal

et al. 2012

Self Cite

To identify host genes affecting replication of Tomato bushy stunt virus (TBSV), a small model positive-stranded RNA virus, we overexpressed 5,500 yeast proteins individually in Saccharomyces cerevisiae , which supports TBSV replication. In total, we identified 141 host proteins, and overexpression of 40 of those increased and the remainder decreased the accumulation of a TBSV replicon RNA. Interestingly, 36 yeast proteins were identified previously by various screens, greatly strengthening the relevance of these host proteins in TBSV replication. To validate the results from the screen, we studied the effect of protein kinase C1 (Pkc1), a conserved host kinase involved in many cellular processes, which inhibited TBSV replication when overexpressed. Using a temperature-sensitive mutant of Pkc1p revealed a high level of TBSV replication at a semipermissive temperature, further supporting the idea that Pkc1p is an inhibitor of TBSV RNA replication. A direct inhibitory effect of Pkc1p was shown in a cell-free yeast extract-based TBSV replication assay, in which Pkc1p likely phosphorylates viral replication proteins, decreasing their abilities to bind to the viral RNA. We also show that cercosporamide, a specific inhibitor of Pkc-like kinases, leads to increased TBSV replication in yeast, in plant single cells, and in whole plants, suggesting that Pkc-related pathways are potent inhibitors of TBSV in several hosts.