Isolation and characterization of an RNA-dependent RNA polymerase from Nicotiana clevelandii plants infected with red clover necrotic mosaic dianthovirus

Replication of positive-strand RNA viruses occurs through the assembly of membrane-associated viral RNA replication complexes that include viral replicase proteins, viral RNA templates, and host proteins. Red clover necrotic mosaic virus (RCNMV) is a positive-strand RNA plant virus with a genome consisting of RNA1 and RNA2. The two proteins encoded by RNA1, a 27-kDa protein (p27) and an 88-kDa protein containing an RNA-dependent RNA polymerase (RdRP) motif (p88), are essential for RCNMV RNA replication. To analyze RCNMV RNA replication complexes, we used blue-native polyacrylamide gel electrophoresis (BN/PAGE), which enabled us to analyze detergent-solubilized large membrane protein complexes. p27 and p88 formed a complex of 480 kDa in RCNMV-infected plants. As a result of sucrose gradient sedimentation, the 480-kDa complex cofractionated with both endogenous template-bound and exogenous template-dependent RdRP activities. The amount of the 480-kDa complex corresponded to the activity of exogenous template-dependent RdRP, which produced RNA fragments by specifically recognizing the 3-terminal core promoter sequences of RCNMV RNAs, but did not correspond to the activity of endogenous template-bound RdRP, which produced genome-sized RNAs without the addition of RNA templates. These results suggest that the 480-kDa complex contributes to template-dependent RdRP activities. We subjected those RdRP complexes to affinity purification and analyzed their components using two-dimensional BN/sodium dodecyl sulfate-PAGE (BN/SDS-PAGE) and mass spectrometry. The 480-kDa complex contained p27, p88, and possible host proteins, and the original affinity-purified RdRP preparation contained HSP70, HSP90, and several ribosomal proteins that were not detected in the 480-kDa complex. A model for the formation of RCNMV RNA replication complexes is proposed.Positive-strand RNA viruses replicate their RNA on intracellular membranes (5). One or more viral nonstructural proteins, including RNA-dependent RNA polymerase (RdRP) and accessory proteins, play essential roles in RNA replication as integral membrane proteins (13,25,28) or as peripheral membrane proteins (4, 6). These viral nonstructural proteins interact with each other and with host proteins and viral RNA templates to form the membrane-associated viral RNA replication complexes. The membrane-associated RNA replication complexes of positive-strand RNA viruses have been isolated from the membrane fraction of virally infected tissues by the use of detergents (2,11,29,32,40,49,50). The purified fractions contain viral and host proteins and retain two types of RdRP activities. One is an endogenous template-bound RdRP activity that synthesizes virus-related RNAs without adding RNA templates. The other is an exogenous template-dependent RdRP activity capable of de novo initiation of cRNA synthesis from selected RNA templates. Although there has been intensive work performed on viral RdRP complexes, their organizations, properties, and functions have not been fully characterized.Red cl...

Section: Resultsmentioning

confidence: 99%

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confidence: 99%

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confidence: 99%

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Identification and Characterization of the 480-Kilodalton Template-Specific RNA-Dependent RNA Polymerase Complex of Red Clover Necrotic Mosaic Virus

Mine

Takeda

Taniguchi

et al. 2010

“…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.…”

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confidence: 99%

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

Panaviene

Panavas

Serva

et al. 2004

123

278

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...

“…RNA1 encodes a functionally unknown 27-kDa protein (p27) and an 88-kDa protein (p88) with an RNA-dependent RNA polymerase (RdRp) motif (23) translated by the extension of p27 because of a ribosome frameshift (22,52). Proteins p27 and p88 are components of the partially purified template-dependent RNA polymerase extracted from RCNMV-infected tissue (1). RNA1 also encodes a 37-kDa coat protein (CP) expressed from subgenomic RNA (53).…”

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Cap-Independent Translational Enhancement by the 3′ Untranslated Region of Red Clover Necrotic Mosaic Virus RNA1

Mizumoto

Tatsuta

Kaido

et al. 2003

117

Red clover necrotic mosaic virus (RCNMV) is a member of the genus Dianthovirus and has a bipartite positive-sense genomic RNA with 3 ends that are not polyadenylated. In this study, we show that both genomic RNA1 and RNA2 lack a 5 cap structure and that uncapped in vitro transcripts of RCNMV RNA1 replicated to a level comparable to that for capped transcripts in cowpea protoplasts. Because the 5 cap and 3 poly(A) tail play important roles in the translation of many eukaryotic mRNAs, genomic RNAs of RCNMV should contain an element(s) responsible for 5 cap-and poly(A) tail-independent translation of viral protein. By using a luciferase reporter assay system in vivo, we showed that the 3 untranslated region (UTR) of RNA1 alone significantly enhanced translation of the luciferase reporter gene in the absence of the 5 cap structure. Deletion studies revealed that the middle region (between nucleotides 3596 and 3732) in the 3 UTR, designated the 3 translation element of Dianthovirus RNA1 (3TE-DR1), plays an important role in cap-independent translation. This region contained a stem-loop structure conserved among members of the genera Dianthovirus and Luteovirus. A five-base substitution in the loop abolished cap-independent translational activity, as reported for a luteovirus, indicating that this stem-loop is one of the functional structures in the 3TE-DR1 involved in cap-independent translation. Finally, we suggest that cap-independent translational activity is required for RCNMV RNA1 replication in protoplasts.The genomic RNA of a positive-sense single-stranded RNA virus serves two essential functions at the start of the viral replication cycle in infected cells: as a template for negative strands and as mRNA for viral proteins. Virtually all eukaryotic cellular mRNAs are capped and polyadenylated. The 5Ј cap structure (m 7 GpppN) serves as the binding site for eukaryotic initiation factor 4F (eIF4F), composed of eIF4G, eIF4E, and eIF4A, which assists the binding of 40S ribosomes to mRNAs (15). The poly(A) tail serves as the binding site for the poly(A)-binding protein, which stabilizes eIF4F binding to the 5Ј cap (43). Consequently, the 5Ј cap and 3Ј poly(A) tail are critical in recruiting translational machinery for the efficient translation of encoded proteins. However, a variety of eukaryotic viral mRNAs lack the 5Ј cap and/or poly(A) tail and have therefore developed alternative strategies for translation regulation.For Tobacco mosaic virus, which has a capped genome and which lacks the poly(A) tail, the pseudoknot domain in the 3Ј untranslated region (UTR) appears to substitute functionally for the poly(A) tail and enhances translation (27). In addition, the poly(CAA) region in the 5Ј UTR (termed ⍀) is responsible for translation enhancement (9). The genomic RNA of picornaviruses has a poly(A) tail but lacks the 5Ј cap. In this case, the internal ribosome entry site (IRES) in the 5Ј UTR recruits ribosomes directly and enables cap-independent translation initiation to occur (21). In contrast to the IRESs of pi...