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...

Viral replicases are the key enzymes in the replication of plus-stranded RNA viruses (1, 7). The viral replicase complexes, which are assembled on intracellular membranes, consist of virus-encoded proteins, such as the RNA-dependent RNA polymerase (RdRp) and auxiliary viral protein(s), as well as host-derived proteins and the viral RNA template (2, 19). Studies with a small number of plant RNA viruses identified several host factors which are likely involved in the targeting of the viral replication proteins to intracellular compartments and replicase assembly and/or function. For example, subunit 45 of eukaryotic initiation factor 3 was found in association with brome mosaic virus (BMV) RdRp (35), while subunit 56 of eukaryotic initiation factor 3 was detected in a tobacco mosaic virus replicase preparation (25). Moreover, a molecular chaperone (Ydj1p) is known to be essential for the activation of the BMV replicase (45). TOM1 and TOM3 integral membrane proteins of Arabidopsis were found to interact with the tobacco mosaic virus replicase proteins and cofractionated with the RdRp activity (46, 53).Host factors interacting with and/or affecting viral replicase activities have also been identified in animal plus-stranded RNA viruses. For example, hepatitis C virus (HCV) RdRp is known to bind to nucleolin (an RNA binding protein) and initiation factor 4A (11,15,20). The HCV NS5A and NS5B replication proteins interacted with hVAP-33, a SNARE-like protein bound to cellular membranes (47). hVAP-33 has been suggested to serve as a docking site to anchor the HCV RdRp to the membrane during assembly of the replicase complex (12, 47). Also, host chaperones are involved in proteolytic processing of the HCV nonstructural proteins (44). The Hsp90 molecular chaperone has been shown to enhance Flock House virus replication (18). Poly(C) and poly(A) binding proteins were found to interact with both the poliovirus RNA and the viral polymerase precursor 3CD (14, 49). Nucleolin and Sam68, which are involved in virus replication, are relocalized in the cell during poliovirus infection (21, 48). In addition, the cellular COPII proteins are involved in the assembly of the poliovirus replication complex (39). hnRNP A1 was found to be part of the mouse hepatitis virus transcription/replication complex, and it is involved in subgenomic RNA synthesis and genome replication (42,50). Overall, the above examples illustrate that host factors play complex and significant roles in the replication of many plus-stranded RNA viruses.Tomato bushy stunt virus (TBSV) and the closely related cucumber necrosis virus (CNV) are nonsegmented plusstranded viruses of plants. Among the five TBSV-encoded proteins, only p33 and p92 are required for replication (26,30,40,52), whereas the other proteins are involved in cell-to-cell movement, encapsidation, and suppression of gene silencing (52). The p92 replication protein has the RdRp signature motifs in its unique C terminus, whereas the auxiliary p33, which overlaps with the N-terminal sequence of p92, play...

Knowledge of viral diversity is expanding greatly, but many lineages remain underexplored. We surveyed RNA viruses in 52 cultured monoxenous relatives of the human parasite ( and ), as well as plant-infecting was a hotbed for viral discovery, carrying a virus (Leptomonas pyrrhocoris ostravirus 1) with a highly divergent RNA-dependent RNA polymerase missed by conventional BLAST searches, an emergent clade of tombus-like viruses, and an example of viral endogenization. A deep-branching clade of trypanosomatid narnaviruses was found, notable as bearing Narna-like virus 1 (LepseyNLV1) have been reported in cultures recovered from patients with visceral leishmaniasis. A deep-branching trypanosomatid viral lineage showing strong affinities to bunyaviruses was termed "" (LBV) and judged sufficiently distinct to warrant assignment within a proposed family termed "" Numerous relatives of trypanosomatid viruses were found in insect metatranscriptomic surveys, which likely arise from trypanosomatid microbiota. Despite extensive sampling we found no relatives of the totivirus (LRV1/2), implying that it was acquired at about the same time the became able to parasitize vertebrates. As viruses were found in over a quarter of isolates tested, many more are likely to be found in the >600 unsurveyed trypanosomatid species. Viral loss was occasionally observed in culture, providing potentially isogenic virus-free lines enabling studies probing the biological role of trypanosomatid viruses. These data shed important insights on the emergence of viruses within an important trypanosomatid clade relevant to human disease.

Kinetic and binding studies involving a model DNA cytosine-5-methyltransferase, M.HhaI, and a 37-mer DNA duplex containing a single hemimethylated target site were applied to characterize intermediates on the reaction pathway. Stopped-flow fluorescence studies reveal that cofactor S-adenosyl-L-methionine (AdoMet) and product S-adenosyl-L-homocysteine (AdoHcy) form similar rapidly reversible binary complexes with the enzyme in solution. ), and the Thr-250 mutations confer further dramatic decrease of the rate of the covalent methylation k chem . We suggest that activation of the pyrimidine ring via covalent addition at C-6 is a major contributor to the rate of the chemistry step (k chem ) in the case of cytosine but not 5-fluorocytosine. In contrast to previous reports, our results imply a random substrate binding order mechanism for M.HhaI.Methylation of cytosine residues in DNA occurs in diverse organisms from bacteria to humans. Cytosine methylation in DNA is catalyzed by DNA methyltransferases (MTases) 1 that transfer methyl groups from the ubiquitous donor S-adenosyl-L-methionine (AdoMet) producing modified cytosines with a methyl group at either C-5 or N-4 (1). In higher organisms, where only 5-methylcytosine is found, DNA methylation is essential for controlling a number of cellular processes including transcription, genomic imprinting, developmental regulation, mutagenesis, DNA repair, and chromatin organization (2). Aberrations in cytosine-5 methylation correlate with human genetic disease, and therefore, the MTases are potent candidate targets for developing new therapies (3). In prokaryotes, MTases are usually but not exclusively found as components of restriction modification systems (1).Besides their important physiological role, the MTases are attractive models for the study of protein-DNA interactions, a central event in many biological processes. The major advantages of bacterial C5-MTases as model systems are as follows: (a) wide diversity of targets recognized (over 200 specificities known); (b) ability to promote covalent reactions within the DNA; (c) their relatively simple molecular organization; and (d) high level of sequence and structural homology with eukaryotic enzymes. It is not surprising that most evidence of the catalytic mechanism of cytosine-5 methylation has been obtained from the studies of prokaryotic MTases. A particular example is HhaI MTase, a component of a type II restriction-modification system from Haemophilus haemolyticus. M.HhaI recognizes the tetranucleotide sequence GCGC and methylates the inner cytosine residue (boldface) and is one of the smallest in the C5-MTase family. This enzyme has been extensively examined by employing a variety of methods. Interaction with the substrates was shown to lead to dramatic conformational changes in both the bound DNA and the enzyme itself. MTase-mediated rotation of the target nucleotide out of the DNA helix (baseflipping) serves to deliver the base into a concave catalytic site in the enzyme (4). Subsequent massive movement of t...