Genetic trans-complementation establishes a new model for influenza virus RNA transcription and replication.
The influenza A viruses genome comprises eight single-stranded RNA segments of negative polarity. Each one is included in a ribonucleoprotein particle (vRNP) containing the polymerase complex and a number of nucleoprotein (NP) monomers. Viral RNA replication proceeds by formation of a complementary RNP of positive polarity (cRNP) that serves as intermediate to generate many progeny vRNPs. Transcription initiation takes place by a cap-snatching mechanism whereby the polymerase steals a cellular capped oligonucleotide and uses it as primer to copy the vRNP template. Transcription termination occurs prematurely at the polyadenylation signal, which the polymerase copies repeatedly to generate a 3'-terminal polyA. 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.
Jorba
Núria
N
Centro Nacional de Biotecnología (CSIC) and CIBER de Enfermedades Respiratorias, Campus de Cantoblanco, Madrid, Spain.
Coloma
Rocío
R
Ortín
Juan
J
D012321:DNA-Directed RNA Polymerases / Q000235:genetics; D005816:Genetic Complementation Test; D009980:Influenza A virus / Q000235:genetics* / Q000502:physiology; D012367:RNA, Viral / Q000235:genetics*; D012261:Ribonucleoproteins / Q000235:genetics; D014158:Transcription, Genetic / Q000235:genetics*; D014779:Virus Replication / Q000235:genetics*
D012367:RNA, Viral; D012261:Ribonucleoproteins; D012321:DNA-Directed RNA Polymerases