Complementación trans- genética establece nuevo modelo para la transcripción y replicación del ARN del virus de la Influenza

Resumen
El genoma de los virus de la influenza A comprende 8 segmentos de cadenas simples de ARN de polaridad negativa. Cada una está incluida en una partícula de ribonucleoproteína (vRNP) que contiene al complejo de la polimerasa y un número de monómeros de nucleoproteínas (NP). La replicación del ARN viral se lleva a cabo por la formación de una RNP complementaria de polaridad positiva (cRNP) que sirve de intermediario para generar muchas progenies de vRNPs. El inicio de la transcripción se lleva a cabo por un mecanismo de secuestro de cubierta por el cual la polimerasa roba un oligonucleótido con cubierta celular y lo utiliza como base para copiar la plantilla de la vRNP. El final de la transcripción ocurre prematuramente con la señal de poliadenilación la que es copiada repetidamente para generar un poliA 3´.
http://www.plospathogens.org/article/info%3Adoi%2F10.1371%2Fjournal.ppat.1000462

Here we studied the mechanisms of the viral RNA replication and transcription. We used efficient systems for recombinant RNP transcription/replication in vivo and well-defined polymerase mutants deficient in either RNA replication or transcription to address the roles of the polymerase complex present in the template RNP and newly synthesised polymerase complexes during replication and transcription. The results of trans-complementation experiments showed that soluble polymerase complexes can synthesise progeny RNA in trans and become incorporated into progeny vRNPs, but only transcription in cis could be detected. These results are compatible with a new model for virus RNA replication, whereby a template RNP would be replicated in trans by a soluble polymerase complex and a polymerase complex distinct from the replicative enzyme would direct the encapsidation of progeny vRNA. In contrast, transcription of the vRNP would occur in cis and the resident polymerase complex would be responsible for mRNA synthesis and polyadenylation.
Author Summary
The influenza A viruses produce annual epidemics and occasional pandemics of respiratory disease. There is great concern about a potential new pandemic being caused by presently circulating avian influenza viruses, and hence increasing interest in understanding how the virus replicates its genome. This comprises eight molecules of RNA, each one bound to a polymerase complex and encapsidated by multiple copies of the nucleoprotein, in the form of ribonucleoprotein complexes (RNPs). These structures are responsible for virus RNA replication and transcription but the detailed mechanisms of these processes are not fully understood. We report here the results of genetic complementation experiments using proficient in vitro and in vivo recombinant systems for transcription and replication, and polymerase point mutants that are either transcription-defective or replication-defective. These results are compatible with a new model for virus replication whereby a polymerase distinct from that present in the parental RNP is responsible for RNA replication in trans and the progeny RNP is associated to a polymerase distinct from that performing replication. In contrast, transcription is carried out in cis by the polymerase resident in the RNP.
Citation: Jorba N, Coloma R, Ortín J (2009) Genetic trans-Complementation Establishes a New Model for Influenza Virus RNA Transcription and Replication. PLoS Pathog 5(5): e1000462. doi:10.1371/journal.ppat.1000462
Editor: Sean P. J. Whelan, Harvard Medical School, United States of America
Received: January 9, 2009; Accepted: April 30, 2009; Published: May 29, 2009