Defining the Roles of cis -Acting RNA Elements in Tombusvirus Replicase Assembly In Vitro

Abstract

In addition to its central role as a template for replication and translation, the viral plus-strand RNA genome also has nontemplate functions, such as recruitment to the site of replication and assembly of the viral replicase, activities that are mediated by cis-acting RNA elements within viral genomes. Two noncontiguous RNA elements, RII(؉)-SL (located internally in the tombusvirus genome) and RIV (located at the 3=-terminus), are involved in template recruitment into replication and replicase assembly; however, the importance of each of these RNA elements for these two distinct functions is not fully elucidated. We used an in vitro replicase assembly assay based on yeast cell extract and purified recombinant tombusvirus replication proteins to show that RII(؉)-SL, in addition to its known requirement for recruitment of the plus-strand RNA into replication, is also necessary for assembly of an active viral replicase complex. Additional studies using a novel two-component RNA system revealed that the recruitment function of RII(؉)-SL can be provided in trans by a separate RNA and that the replication silencer element, located within RIV, defines the template that is used for initiation of minus-strand synthesis. Collectively, this work has revealed new functions for tombusvirus cis-acting RNA elements and provided insights into the pioneering round of minus-strand synthesis. P lus-strand RNA viruses exhibit many similarities during genome replication, including the formation of membranebound viral replicase complexes, the production of minus-strand and abundant plus-strand RNAs, and the use of co-opted host factors (1,15,16,18). One of the best-studied plus-strand RNA viruses is Tomato bushy stunt virus (TBSV), which has a single 4,800-nucleotide (nt) RNA genome (43). The virus-encoded replication proteins p33 RNA chaperone and p92 pol RNA-dependent RNA polymerase (RdRp) participate in the formation of the membrane-bound viral replicase complex, which also contains several host proteins (3,12,16,38,40).Replication of TBSV and other plus-strand RNA viruses involve several sequential steps, including selection of the viral plusstrand RNA template for replication, recruitment of the viral plusstrand RNA and the viral replication proteins from the cytosol to the subcellular membrane surfaces where replication takes place, assembly and activation of the viral replicase, minus-strand and then plus-strand RNA synthesis, and the release of progeny plusstrand RNAs from the replicase complex (5,15,17). This complex process helps to ensure that authentic viral templates are replicated and that the replication process is rapid and efficient.The TBSV plus-strand RNA plays multiple roles during viral replication. In addition to its main function as a store of genetic information, the viral plus-strand RNA also regulates its own intracellular localization and recruitment to the site of RNA replication (20,26,32,33,44). Moreover, the TBSV plus-strand RNA serves as an assembly platform for the viral replicase, consisting of vir...

Template Role of Double-Stranded RNA in Tombusvirus Replication

“…For example, cis-acting RNA elements located on the plus strand, such as the internal p33 recognition element (p33RE), the 3=-proximal silencer element, and the 3=-terminal minus-strand initiation promoter (gPR), consist of secondary structures needed for function, but these hairpin structures are unlikely to exist and are not functional when they are part of dsRNA structures (32,33,(36)(37)(38). Thus, masking of cis-acting elements located on the plus strand through the formation of dsRNA structures prevents competition between (ϩ)-and (Ϫ)RNAs for the replicase, and it allows the tombusvirus replicase become committed to plus-strand synthesis at later time points.…”

The Resistance Protein Tm-1 Inhibits Formation of a Tomato Mosaic Virus Replication Protein-Host Membrane Protein Complex

“…Such cell-free viral RNA replication systems enable the biochemical analysis of replication processes and have been used to analyze how the replication complex is formed (33)(34)(35)(36)(37)(38)(39)(40). Using this in vitro system, we previously demonstrated that the Tm-1 protein inhibits ToMV RNA replication when added before, but not after, the ToMV replication complex is formed on membranes (28).…”

The Hop-Like Stress-Induced Protein 1 Cochaperone Is a Novel Cell-Intrinsic Restriction Factor for Mitochondrial Tombusvirus Replication

“…and activation of the polymerase function of the replication protein (102,121). The specific recognition of p33RE is performed by arginines within the RPR motif in p33/p92 pol (122,123).…”

“…4D, construct p36⌬RPR). The RPR motif in replication proteins is required for specific viral (ϩ)RNA recruitment and replicase assembly, and it also binds to Cpr7p Cyp40-like cyclophilin (41,(102)(103)(104). Interestingly, the same RPR-containing region in p36 replication protein could bind to both the TPR1 and TPR2 sequences in Sti1p (Fig.…”