Structural and Functional Characterization of an Influenza Virus RNA Polymerase-Genomic RNA Complex

Abstract

The replication and transcription of influenza A virus are carried out by ribonucleoproteins (RNPs) containing each genomic RNA segment associated with nucleoprotein monomers and the heterotrimeric polymerase complex. These RNPs are responsible for virus transcription and replication in the infected cell nucleus. Here we have expressed, purified, and analyzed, structurally and functionally, for the first time, polymerase-RNA template complexes obtained after replication in vivo. These complexes were generated by the cotransfection of plasmids expressing the polymerase subunits and a genomic plasmid expressing a minimal template of positive or negative polarity. Their generation in vivo was strictly dependent on the polymerase activity; they contained mainly negativepolarity viral RNA (vRNA) and could transcribe and replicate in vitro. The three-dimensional structure of the monomeric polymerase-vRNA complexes was similar to that of the RNP-associated polymerase and distinct from that of the polymerase devoid of template. These results suggest that the interaction with the template is sufficient to induce a significant conformation switch in the polymerase complex.The influenza A viruses are members of the family Orthomyxoviridae whose genome comprises eight single-stranded RNA segments of negative polarity. The extreme genetic and antigenic diversity and the segmented nature of their genome are the basis of the yearly epidemics and occasional pandemics of influenza respiratory disease. The influenza A viruses are at evolutionary equilibrium in their natural reservoir, which includes several wild avian species, and from this reservoir, viruses or viral genes can occasionally be transferred to new influenza virus strains and result in the infection of mammals, including humans (22, 68). Although fears have been generated regarding the generation of a new pandemic by highly pathogenic H5N1 viruses, which have caused sporadic cases in humans (50), a new H1N1 virus of swine origin produced worldwide outbreaks (41) and led the WHO to declare a pandemic situation (http://www.who.int/csr/disease/swineflu/4th _meeting_ihr/en/index.html).The replication and transcription of the influenza A virus genome take place in the nucleus of infected cells and are carried out by macromolecular complexes called ribonucleoproteins (RNPs) that include each one of the virus RNA segments associated with monomers of the nucleoprotein (NP) and with the RNA-dependent RNA polymerase (reviewed in reference 12). This virus enzyme is formed by three subunits (PB1, PB2, and PA) and is responsible for both transcription and replication, as mutations in any of its subunits can lead to alterations in either process (3,13,14,16,17,33,51). For transcription, the virus polymerase recognizes cellular cap-containing newly synthesized cellular RNA polymerase II (Pol II) transcripts in a template-dependent manner (6, 31) and generates capped oligonucleotides that serve as primers to copy the RNP template (52, 54). The virus mRNAs are polyadenylated by the ...

The influenza virus RNA synthesis machine

“…3). 34,[60][61][62] These studies documented that the polymerase has a quite compact structure and is probably a flexible complex. Thus, comparing the isolated heterotrimeric complex with the RNP-bound or template-bound complexes indicates that the polymerase shows considerable conformational changes upon interaction with other viral (and presumably also cellular) partners.…”

“…Although early in vitro studies indicated that the polymerase complex dissociates from the cap-structure soon after cap-snatching, 81 no in vivo studies have 62 the polymerase complex present in the recombinant RNP (middle, yellow), 34 and a polymerase-vRNA complex (bottom, blue). 61 …”

“…The large differences in cRNA and 115,116 and in vivo, 88,102 although replication of short RNA templates can proceed in the absence of NP. 61,117 The incorporation of NP into new vRNPs is facilitated by the activity of cellular factors UAP56 and Tat-SF1, that may act as chaperones. 118,119 Viral polymerase should also be required stoichiometrically, as the end product is not vRNA but a vRNP.…”

Influenza A Virus Multiplication and the Cellular SUMOylation System

“…Several different 3D conformations have been observed using cryo-and negative staining electron microscopy for the purified recombinant influenza viral polymerases in the presence or absence of vRNA, NP, and both [64][65][66][67]. Those studies demonstrated that the polymerase has a globular hollow conformation that becomes compacted upon association with vRNA alone or vRNA in the presence of NP oligomers [64][65][66][67].…”

“…Those studies demonstrated that the polymerase has a globular hollow conformation that becomes compacted upon association with vRNA alone or vRNA in the presence of NP oligomers [64][65][66][67]. Additionally, PB1, PB2, and NP have been identified as the main viral proteins mediating the RNA-dependent interaction of the viral polymerase and the RNA-protein complex formed between NP and vRNA [65].…”