Complexes of Sendai virus NP-P and P-L proteins are required for defective interfering particle genome replication in vitro.

We present evidence that the formation of NP-P and P-L protein complexes is essential for replication of the genome of Sendai defective interfering (DI-H) virus in vitro, using extracts of cells expressing these viral proteins from plasmids. Optimal replication of DI-H nucleocapsid RNA required extracts of cells transfected with critical amounts and ratios of each of the plasmids and was three- to fivefold better than replication with a control extract prepared from a natural virus infection. Extracts in which NP and P proteins were coexpressed supported replication of the genome of purified DI-H virus which contained endogenous polymerase proteins, but extracts in which NP and P were expressed separately and then mixed were inactive. Similarly, the P and L proteins must be coexpressed for biological activity. The replication data thus suggest that two protein complexes, NP-P and P-L, are required for nucleocapsid RNA replication and that these complexes must form during or soon after synthesis of the proteins. Biochemical evidence in support of the formation of each complex includes coimmunoprecipitation of both proteins of each complex with an antibody specific for one component and cosedimentation of the subunits of each complex. We propose that the P-L complex serves as the RNA polymerase and NP-P is required for encapsidation of newly synthesized RNA.     

N protein alone satisfies the requirement for protein synthesis during RNA replication of vesicular stomatitis virus.

Genomic replication of the negative-strand RNA viruses is dependent upon protein synthesis. To examine the requirement for protein synthesis in replication, we developed an in vitro system that supports the genome replication of defective interfering particles of the negative-strand rhabdovirus vesicular stomatitis virus (VSV), as a function of protein synthesis (Wertz, J. Virol. 46:513-522, 1983). The system consists of defective interfering nucleocapsid templates and an mRNA-dependent reticulocyte lysate to support protein synthesis. We report here an analysis of the requirement for individual viral proteins in VSV replication. Viral mRNAs purified by hybridization to cDNA clones were used to direct the synthesis of individual proteins in the in vitro system. By this method, it was demonstrated that the synthesis of the VSV nucleocapsid protein, N, alone, resulted in the replication of genome-length RNA by both defective interfering intracellular nucleocapsids and virion-derived nucleocapsids. Neither the viral phosphoprotein, NS, nor the matrix protein, M, supported RNA replication. The amount of RNA replication for a given amount of N protein was the same in reactions in which either all of the VSV proteins or only N protein were synthesized. In addition, RNA replication products synthesized in reactions containing only newly made N protein assembled with the N protein to form nucleocapsids. These results demonstrate that the major nucleocapsid protein (N) can by itself fulfill the requirement for protein synthesis in RNA replication and allow complete replication, i.e., initiation and elongation, as well as encapsidation of genome-length progeny RNA.     

Small Interfering RNA Targeting M2 Gene Induces Effective and Long Term Inhibition of Influenza A Virus Replication

RNA interference (RNAi) provides a powerful new means to inhibit viral infection specifically. However, the selection of siRNA-resistant viruses is a major concern in the use of RNAi as antiviral therapeutics. In this study, we conducted a lentiviral vector with a H1-short hairpin RNA (shRNA) expression cassette to deliver small interfering RNAs (siRNAs) into mammalian cells. Using this vector that also expresses enhanced green fluorescence protein (EGFP) as surrogate marker, stable shRNA-expressing cell lines were successfully established and the inhibition efficiencies of rationally designed siRNAs targeting to conserved regions of influenza A virus genome were assessed. The results showed that a siRNA targeting influenza M2 gene (siM2) potently inhibited viral replication. The siM2 was not only effective for H1N1 virus but also for highly pathogenic avian influenza virus H5N1. In addition to its M2 inhibition, the siM2 also inhibited NP mRNA accumulation and protein expression. A long term inhibition effect of the siM2 was demonstrated and the emergence of siRNA-resistant mutants in influenza quasispecies was not observed. Taken together, our study suggested that M2 gene might be an optimal RNAi target for antiviral therapy. These findings provide useful information for the development of RNAi-based prophylaxis and therapy for human influenza virus infection.