Promoter analysis of influenza virus RNA polymerase

Abstract

Influenza virus polymerase, which was prepared depleted of viral RNA, was used to copy small RNA templates prepared from plasmid-encoded sequences. Template constructions containing only the 3' end of genomic RNA were shown to be efficiently copied, indicating that the promoter lay solely within the 15-nucleotide 3' terminus. Sequences not specific for the influenza virus termini were not copied, and, surprisingly, RNAs containing termini identical to those from plus-sense cRNA were copied at low levels. The specificity for recognition of the virus sense promoter was further defined by site-specific mutagenesis. It was also found that increased levels of viral protein were required in order to catalyze both the cap endonucleaseprimed and primer-free RNA synthesis from these model templates, as well as from genomic-length RNAs. This finding indicates that the reconstituted system has catalytic properties very similar to those of native viral ribonucleoprotein complexes.

The influenza virus hemagglutinin cytoplasmic tail is not essential for virus assembly or infectivity.

“…Generation of transfectant virus containing an HA that lacks a cytoplasmic tail To generate a rescued (transfectant) influenza virus that contains an HA with an altered cytoplasmic tail, two plasmids were constructed: pT7HA and pT7HAtail-. In using the reverse genetics system, the general principles established by Parvin et al (1989), Luytjes et al (1989 and Enami and Palese (1991) were adopted. pT7HA contained the cDNA for the influenza virus A/Udom/72 HA.…”

Molecular anatomy of viral RNA-directed RNA polymerases

“…The dissociation of P proteins can be achieved by centrifugation in CsTFA. The solubilized RNA polymerase is composed of each one of the three P proteins [33] and exhibits RNA synthesis activity dependent on exogenously added templates [62]. Clear evidence for the requirement of all three P proteins for enzymatic activity was given by enzyme reconstitution experiments using individual P proteins, which were isolated from virions by SDS-gel electrophoresis followed by thioredoxin renaturation [81] or purified from insect cells expressing cDNA for each P protein carried on baculovirus vectors [43].…”

Many ways to make an influenza virus - review of influenza virus reverse genetics methods

“…The purification of the viral proteins was technically demanding and not highly reproducible. 13,14 A variation of this method used recombinant NP, expressed in insect cells using a baculovirus vector encoding the viral NP, instead of purified viral proteins from virus preparations. 15 Nucleic acid-based RNP reconstitution in cells Ribonucleoprotein complex can also be reconstituted within cells, by transfecting cells with a plasmid expressing a viral-like RNA.…”