Purification of the Cucumber Necrosis Virus Replicase from Yeast Cells: Role of Coexpressed Viral RNA in Stimulation of Replicase Activity

Abstract

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...

Yeast genome-wide screen reveals dissimilar sets of host genes affecting replication of RNA viruses

“…1). We found that the autonomously replicating DI-72 RNA accumulated to ribosomal RNA levels in yeast cells, comparable with the virus replication level in plant cells (15,19). We also confirmed that, similar to plant infections, replication of DI-72 RNA replicon in yeast depends on cis-acting regulatory sequences in the RNA template and the transacting p33 and p92 proteins, which are part of the tombusvirus replicase (13,15).…”

“…We found that the autonomously replicating DI-72 RNA accumulated to ribosomal RNA levels in yeast cells, comparable with the virus replication level in plant cells (15,19). We also confirmed that, similar to plant infections, replication of DI-72 RNA replicon in yeast depends on cis-acting regulatory sequences in the RNA template and the transacting p33 and p92 proteins, which are part of the tombusvirus replicase (13,15). The DI-72 RNA replicon, which is derived naturally from TBSV infections of plants, does not encode proteins, and high levels of DI-72 RNA were achieved without artificial selection pressure to maintain this RNA in yeast cells.…”

“…Each strain in the YKO library was cotransformed with plasmids pGBK-His 33 and pGAD-His 92, which express p33 and p92 replicase proteins separately from the constitutive ADH1 promoter (15), and pYC͞defective interfering (DI)-72, which expresses the TBSV-derived DI-72 RNA replicon from an inducible GAL1 promoter (13).…”

“…13 and 15. The primary antibodies were anti-6ϫ His (Invitrogen), and the secondary antibodies were alkaline-phosphatase-conjugated antimouse IgG (Sigma) (15).…”

“…We and others have developed systems for inducing yeast cells to support the replication of certain positive-strand RNA viruses or their surrogates (11)(12)(13)(14). Here, we apply our previously developed system for robust replication of a small RNA replicon of the tomato bushy stunt virus (TBSV) in yeast (13,15) to screen the entire YKO library for genes influencing the efficiency of viral replication. A total of 96 YKO strains were identified.…”