Defective Assembly of Influenza A Virus due to a Mutation in the Polymerase Subunit PA

Abstract

The RNA-dependent RNA polymerase of influenza A virus is composed of three subunits that together synthesize all viral mRNAs and also replicate the viral genomic RNA segments (vRNAs) through intermediates known as cRNAs. Here we describe functional characterization of 16 site-directed mutants of one polymerase subunit, termed PA. In accord with earlier studies, these mutants exhibited diverse, mainly quantitative impairments in expressing one or more classes of viral RNA, with associated infectivity defects of varying severity. One PA mutant, however, targeting residues 507 and 508, caused only modest perturbations of RNA expression yet completely eliminated the formation of plaque-forming virus. Polymerases incorporating this mutant, designated J10, proved capable of synthesizing translationally active mRNAs and of replicating diverse cRNA or vRNA templates at levels compatible with viral infectivity. Both the mutant protein and its RNA products were appropriately localized in the cytoplasm, where influenza virus assembly occurs. Nevertheless, J10 failed to generate infectious particles from cells in a plasmid-based influenza virus assembly assay, and hemagglutinating material from the supernatants of such cells contained little or no nuclease-resistant genomic RNA. These findings suggest that PA has a previously unrecognized role in assembly or release of influenza virus virions, perhaps influencing core structure or the packaging of vRNAs or other essential components into nascent influenza virus particles.The influenza A virus encodes a heterotrimeric RNA-dependent RNA polymerase whose subunits are designated PB1, PB2, and PA. Localized in the nuclei of infected cells, this trimeric complex is essential for transcribing all virally encoded mRNAs and for replicating the eight negative-sense singlestranded RNA segments (vRNAs) that make up the viral genome. To carry out transcription, the polymerase endonucleolytically cleaves short, 5Ј-capped oligonucleotides from cellular mRNAs to be used as primers, which are then elongated using all but a few nucleotides from any given vRNA as a template. Replication, by contrast, is achieved through a primer-independent, two-step process in which the polymerase first copies a vRNA template to produce a full-length cRNA and then uses this cRNA as the template for new vRNA synthesis.Although all three polymerase subunits are required for viral infectivity, each of the known substrate-binding or catalytic activities of the complex appears to reside primarily in either the PB1 or PB2 subunit. The precise functions of PA are less certain. PA is 716 amino acids long and differs from the other two subunits in having an overall negative charge. Mutational studies from several laboratories have begun to delineate the functional architecture of PA and its roles in the influenza virus life cycle. Sequences near its N terminus (within residues 124 to 246) have been found to target PA into nuclei (21), whereas C-terminal residues 668 to 692 mediate its incorporation into the polymeras...

Reassortment and Mutation of the Avian Influenza Virus Polymerase PA Subunit Overcome Species Barriers

“…Polymerase activity assays were performed with human 293T cells expressing a luciferase-based viral reporter construct, the viral nucleoprotein (NP), and the polymerase subunits PB1, PB2, and PA (28,36). Reassortants were created by the replacement of one subunit of the polymerase or NP with that from a human isolate.…”

“…Vectors expressing proteins from A/WSN/1933 were a kind gift of E. Fodor. The vRNA-luciferase reporter plasmid pHH21-vNA-luc contains the luciferase-encoding sequence in the antisense orientation in the context of the vNA gene segment from the A/WSN/33 isolate (36). PA proteins were cloned into the bidirectional pBD vector for virus rescue.…”

“…PA proteins were cloned into the bidirectional pBD vector for virus rescue. Antibodies recognizing PB1, PB2, and PA were created in rabbits (28,36). 293T, MDCK, A549, and DF1 cells were maintained in Dulbecco's modified Eagle's medium (DMEM) plus 10% fetal bovine serum (FBS).…”

The PA Protein Directly Contributes to the Virulence of H5N1 Avian Influenza Viruses in Domestic Ducks

“…The PA protein is a component of the RNA-dependent polymerase complex and affects both transcription and replication (7,29,31). We investigated whether the two influenza viruses have different polymerase activities by using a minigenome assay in DEFs.…”

Oligomerization of the influenza virus polymerase complex in vivo

“…However, an additional possibility could be considered for the requirement of polymerasepolymerase interactions in vivo. Thus, the process of encapsidation of the segmented genome into virus particles might require multiple contacts between the polymerase complexes present in the virion RNPs and, indeed, a mutation in the PA subunit has been described that is defective in particle formation (Regan et al, 2006). Influenza virus polymerase oligomerization…”