Positive-sense RNA ((+)RNA) viruses such as hepatitis C virus exploit host cells by subverting host proteins, remodelling subcellular membranes, co-opting and modulating protein and ribonucleoprotein complexes, and altering cellular metabolic pathways during infection. To facilitate RNA replication, (+)RNA viruses interact with numerous host molecules through protein-protein, RNA-protein and protein-lipid interactions. These interactions lead to the formation of viral replication complexes, which produce new viral RNA progeny in host cells. This Review presents the recent progress that has been made in understanding the role of co-opted host proteins and membranes during (+)RNA virus replication, and discusses common themes employed by different viruses.

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To gain insights into the functions of a viral RNA replicase, we have assembled in vitro and entirely from nonplant sources, a fully functional replicase complex of Tomato bushy stunt virus (TBSV). The formation of the TBSV replicase required two purified recombinant TBSV replication proteins, which were obtained from E. coli, the viral RNA replicon, rATP, rGTP, and a yeast cell-free extract. The in vitro assembly of the replicase took place in the membraneous fraction of the yeast extract, in which the viral replicase-RNA complex became RNase-and proteinase-resistant. The assembly of the replicase complex required the heat shock protein 70 (Hsp70 ‫؍‬ yeast Ssa1/2p) present in the soluble fraction of the yeast cell-free extract. The assembled TBSV replicase performed a complete replication cycle, synthesizing RNA complementary to the provided RNA replicon and using the complementary RNA as template to synthesize new TBSV replicon RNA.hsp70 ͉ RNA-dependent RNA polymerase ͉ viral replicase ͉ virus replication ͉ yeast cell-free extract R eplication of plus-strand (ϩ)RNA viruses is driven by the viral replicase complex in the infected cells. The viral replicase complex assembles on intracellular membrane surfaces from viral replication proteins, the recruited viral RNA and a set of coopted host proteins. This is followed by complementary (Ϫ)-strand synthesis and the production of excess amounts of new (ϩ)RNA progeny, which is released from the site of replication to the cytosol (1, 2). Despite its key significance in all RNA virus infections, the composition of the viral replicase complex is not yet fully revealed and the mechanism of assembly is poorly understood (3, 4). Dissection of the functions of the viral and host proteins in the replicase complex would be greatly facilitated by an in vitro approach that leads to the assembly of functional viral replicase complex composed at least partially of defined components.The major difficulties in achieving in vitro assembly are related to the complex composition of the viral replicase and the requirement for activation of the viral RNA-dependent RNA polymerase (RdRp) during the assembly of the viral replicase in membraneous structures, as evidenced by results with p92 pol of Tomato bushy stunt virus (TBSV), 2a pol of Brome mosaic virus (BMV), P2 of Alfalfa mosaic virus (AMV), 180K of Tomato mosaic virus (ToMV), and the hepatitis C virus (HCV) NS5B. Most of our knowledge on viral RdRps is based on detergent-solubilized, ribonuclease treated (to remove the endogenous viral RNA), template-dependent viral replicase preparations, which are mostly capable of initiation, elongation, and termination of complementary RNA synthesis or RNA recombination (5-7). These replicase preparations are incapable of performing full replication cycle, show less stringent template specificity than in vivo viral replicases, and use exogenously added RNA templates inefficiently. In addition, cell-free replication assays using extracts obtained from noninfected cells, which are based on coupled...

Purified recombinant viral replicases are useful for studying the mechanism of viral RNA replication in vitro. In this work, we obtained a highly active template-dependent replicase complex for Cucumber necrosis tombusvirus (CNV), which is a plus-stranded RNA virus, from Saccharomyces cerevisiae. The recombinant CNV replicase showed properties similar to those of the plant-derived CNV replicase (P. D. Nagy and J. Pogany, Virology 276:279-288, 2000), including the ability (i) to initiate cRNA synthesis de novo on both plus-and minus-stranded templates, (ii) to generate replicase products that are shorter than full length by internal initiation, and (iii) to perform primer extension from the 3 end of the template. We also found that isolation of functional replicase required the coexpression of the CNV p92 RNA-dependent RNA polymerase and the auxiliary p33 protein in yeast. Moreover, coexpression of a viral RNA template with the replicase proteins in yeast increased the activity of the purified CNV replicase by 40-fold, suggesting that the viral RNA might promote the assembly of the replicase complex and/or that the RNA increases the stability of the replicase. In summary, this paper reports the first purified recombinant tombusvirus replicase showing high activity and template dependence, a finding that will greatly facilitate future studies on RNA replication in vitro.Plus-stranded RNA viruses, which constitute the largest group among plant and animal viruses, replicate in infected cells by using the viral replicase complex. The replicase complex consists of virus-coded proteins, such as the RNA-dependent RNA polymerase (RdRp), auxiliary proteins, and possibly host-derived proteins, and the RNA template (1,4,5,20,27). To study the mechanism of viral RNA replication, functional replicases are purified from virus-infected hosts (3,10,12,16,23,26,38,41,42,53,55) or from heterologous systems, including Escherichia coli (17,19,21,24,44,45), yeast (40), insect (22,24,58), Xenopus (13), and mammalian cells (14,24). The advantage of the heterologous systems is that expression of the replicase proteins can be achieved without dependence on virus replication, thus facilitating mutational analysis of the replicase genes. These studies have established that the RdRp of several viruses, including Turnip crinkle virus, Tobacco etch virus, Bamboo mosaic virus, Hepatitis C virus, Bovine viral diarrhea virus (17,[19][20][21][22]44,45), etc., are active when expressed without other virus-coded auxiliary proteins. On the contrary, RdRps for several other viruses, such as Brome mosaic virus (BMV) and Alfalfa mosaic virus (AMV), required the presence of several factors, such as the RdRp, a viral auxiliary protein, and the viral RNA, in order to be functional in vitro (40, 54). In summary, viral replicase systems, which are very useful to dissect the protein (trans-acting) and RNA (cis-acting) factors that control virus replication, have been developed only for a limited number of plus-stranded RNA viruses.Tombusviruses are small pl...