Template Nucleotide Moieties Required for De Novo Initiation of RNA Synthesis by a Recombinant Viral RNA-Dependent RNA Polymerase

Abstract

Virology 253:1-7, 1999). Using RNAs containing nucleotide analogs, we found that the N3 and C4-amino group at the initiation cytidine were required for RNA synthesis. However, the ribose C2-hydroxyl of the initiating cytidylate can accept several modifications and retain the ability to direct synthesis. The only unacceptable modification is a protonated C2-amino group. Quite strikingly, the recognition of the functional groups for the initiation cytidylate and other template nucleotides are different. For example, a C5-methyl group in cytidine can direct RNA synthesis at all template positions except at the initiation cytidylate and C2-amino modifications are tolerated better after the ؉11 position. When a 4-thiouracil (4sU) base analog that allows only imperfect base pairing with the nascent RNA is placed at different positions in the template, the efficiency of synthesis is correlated with the calculated stability of the template-nascent RNA duplex adjacent to the position of the 4sU. These results define the requirements for the specific interactions required for the initiation of RNA synthesis and will be compared to the mechanisms of initiation by other RNA-dependent and DNA-dependent RNA polymerases.De novo (primer-independent) initiation is an important mechanism for viral RNA replication that requires the specific and appropriate interactions of at least four components: (i) an RNA template with a virus-specific initiation nucleotide at or near the 3Ј end; (ii) an initiation nucleoside triphosphate (NTP); (iii) a second NTP; and (iv) an RNA-dependent RNA polymerase (RdRp). Improper recognition may reduce or inhibit efficient RNA synthesis. Little is known about specific recognition between RdRp and RNA template moieties for de novo initiation of viral RNA synthesis. Furthermore, the interactions may change as the polymerase undergoes conformational changes during synthesis (11,32).Bovine viral diarrhea virus (BVDV) is a member of the genus Pestivirus in the Flaviviridae family, which includes human and animal pathogens, such as Hepatitis C virus (HCV), Dengue virus, and Yellow fever virus (10,34,55). BVDV is an enveloped virus containing a single-stranded positive-sense RNA genome (55). In addition to being an important pathogen of livestock (51), BVDV is a model system for HCV infection and for building chimeric viruses with HCV (14,31,61,68).The BVDV RNA genome is translated as a polyprotein through a cap-independent mechanism after infection. The polyprotein is further processed into individual structural and nonstructural proteins by a combination of cellular and viral proteases (10). The structural proteins are located in the Nterminal portion of the polyprotein, while those associated with replication are present in the C-terminal portion (68). Among the nonstructural proteins, NS5B is an RdRp that can synthesize RNA (3, 69). Double-stranded, RNase-resistant replicative-form RNA and partially double-stranded replication intermediates have been observed early in infection in cells infected by BVDV. ...

Factors regulating template switch in vitro by viral RNA-dependent RNA polymerases: Implications for RNA–RNA recombination

“…Preliminary evidence suggests that the BVDV RdRp transits to processive synthesis between the synthesis of 10-to 12-phosphodiester bonds (ref. 23 and M.-J.K., unpublished results), a value similar to that reported for the BMV replicase (33,34). The internal template switch observed with RecO may be caused by the TC not being committed to elongation by position ϩ10.…”

“…1A). Both templates lack a 5Ј phosphate but can direct de novo initiation of RNA synthesis by the BVDV RdRp (22,23). We found that (-)21-3Јdd and d(-)21 produced RNAs that are multiples of 21 nucleotides in length whose abundance increased with template concentration (Fig.…”

Similar Structural Basis for Membrane Localization and Protein Priming by an RNA-dependent RNA Polymerase

“…4). For mammalian positive-strand RNA viruses such as poliovirus and hepatitis C, two mechanisms are suspected: de novo initiation (5)(6)(7)(8) and protein priming (9) from the genomic ends.…”

Análisis Funcional De Proteínas Codificadas Por El Virus De La Rotura Del Color De La Flor Del Pelargonium

“…A los elementos cis que intervienen en la replicación se les atribuyen diversas funciones, entre ellas: (1) participar en la formación de los complejos replicativos Wu et al, 2009], (2) actuar como promotores de la replicación promoviendo la síntesis de novo de la cadena complementaria [Kim et al, 2000;Kao et al, 2001], (3) regular la síntesis asimétrica de las cadenas de polaridad positiva y negativa [Ball, 2007], (4) intervenir en la transcripción de los distintos sgRNAs, si procede [Kim & Hemenway, 1999;Grdzelishvili et al, 2005] y, (5) funcionar como chaperonas de RNA [McCormack & Simon, 2004]. como mRNA permitiendo la traducción de las replicasas virales.…”

“…Del mismo modo, se han intentado identificar factores del huésped potencialmente implicados en replicación utilizando técnicas de entrecruzamiento con luz UV, empleando la 5´ o 3´ UTR virales y extractos celulares. En algún caso esta estrategia ha permitido identificar proteínas con capacidad de unirse a estas secuencias reguladoras [Sriskanda et al, 1996;Kim et al, 2000], pero su efectividad ha sido, en general, limitada.…”