Biochemical characterization of avian influenza viral polymerase containing PA or PB2 subunit from human influenza A virus

Adaptive mutations in viral polymerase, which is composed of PB1, PB2, and PA, of avian influenza viruses are major genetic determinants of the host range. In this study, to elucidate the molecular mechanism of mammalian adaptation of avian viral polymerase, we performed cell-based vRNP reconstitution assays and biochemical analyses using purified recombinant viral polymerase complexes. We found that avian viral polymerase from A/duck/Pennsylvania/10,218/84 (DkPen) enhances the viral polymerase activity in mammalian cells by replacing the PA or PB2 gene with that from human influenza virus A/WSN/33 (WSN). Chimeric constructs between DkPen PA and WSN PA showed that the N-terminal endonuclease domain of WSN PA was essential for the mammalian adaptation of DkPen viral polymerase. We also found that the cap-snatching activity of purified DkPen viral polymerase was more than 5 times weaker than that of WSN in vitro in a PB2 Glu627-dependent manner. However, the cap-snatching activity of DkPen viral polymerase was hardly increased by replacing DkPen PA to WSN PA. These results suggest that the activity of viral genome replication may be enhanced in the DkPen reassortant containing WSN PA.     

Involvement of the N-terminal portion of influenza virus RNA polymerase subunit PB1 in nucleotide recognition

The influenza virus PB1 protein functions as a catalytic subunit of the viral RNA-dependent RNA polymerase and contains the highly conserved motifs of RNA-dependent RNA polymerases together with putative nucleotide-binding sites. PB1 also binds to viral genomic RNAs and its replicative intermediates through the promoter regions. The detail function and interplay between functional domains are not clarified although a part of structures and functions of PB1 have been clarified. In this study, we analyzed the function of PB1 subunit in the sense of nucleotide recognition using ribavirin, which is a nucleoside analog and inhibits viral RNA synthesis of many RNA viruses including influenza virus. We screened ribavirin-resistant PB1 mutants from randomly mutated PB1 cDNA library using a mini-replicon assay, and we identified a single mutation at the amino acid position 27 of PB1 as an important residue for the nucleotide recognition.     

Structural insight into the essential PB1–PB2 subunit contact of the influenza virus RNA polymerase

Influenza virus RNA‐dependent RNA polymerase is a multi‐functional heterotrimer, which uses a ‘cap‐snatching’ mechanism to produce viral mRNA. Host cell mRNA is cleaved to yield a cap‐bearing oligonucleotide, which can be extended using viral genomic RNA as a template. The cap‐binding and endonuclease activities are only activated once viral genomic RNA is bound. This requires signalling from the RNA‐binding PB1 subunit to the cap‐binding PB2 subunit, and the interface between these two subunits is essential for the polymerase activity. We have defined this interaction surface by protein crystallography and tested the effects of mutating contact residues on the function of the holo‐enzyme. This novel interface is surprisingly small, yet, it has a crucial function in regulating the 250 kDa polymerase complex and is completely conserved among avian and human influenza viruses.     

Artificial hybrids of influenza A virus RNA polymerase reveal PA subunit modulates its thermal sensitivity

Background

Influenza A virus can infect a variety of different hosts and therefore has to adapt to different host temperatures for its efficient viral replication. Influenza virus codes for an RNA polymerase of 3 subunits: PB1, PB2 and PA. It is well known that the PB2 subunit is involved in temperature sensitivity, such as cold adaptation. On the other hand the role of the PA subunit in thermal sensitivity is still poorly understood.

Methodology/Principal Findings

To test which polymerase subunit(s) were involved in thermal stress we reconstituted artificial hybrids of influenza RNA polymerase in ribonucleoprotein (RNP) complexes and measured steady-state levels of mRNA, cRNA and vRNA at different temperatures. The PA subunit was involved in modulating RNP activity under thermal stress. Residue 114 of the PA subunit was an important determinant of this activity.

Conclusions/Significance

These findings suggested that influenza A virus may acquire an RNA polymerase adapted to different body temperatures of the host by reassortment of the RNA polymerase genes.     

Mutational analyses of the influenza A virus polymerase subunit PA reveal distinct functions related and unrelated to RNA polymerase activity

Influenza A viral polymerase is a heterotrimeric complex that consists of PA, PB1, and PB2 subunits. We previously reported that a di-codon substitution mutation (G507A-R508A), denoted J10, in the C-terminal half of PA had no apparent effect on viral RNA synthesis but prevented infectious virus production, indicating that PA may have a novel role independent of its polymerase activity. To further examine the roles of PA in the viral life cycle, we have now generated and characterized additional mutations in regions flanking the J10 site from residues 497 to 518. All tested di-codon mutations completely abolished or significantly reduced viral infectivity, but they did so through disparate mechanisms. Several showed effects resembling those of J10, in that the mutant polymerase supported normal levels of viral RNA synthesis but nonetheless failed to generate infectious viral particles. Others eliminated polymerase activity, in most cases by perturbing the normal nuclear localization of PA protein in cells. We also engineered single-codon mutations that were predicted to pack near the J10 site in the crystal structure of PA, and found that altering residues K378 or D478 each produced a J10-like phenotype. In further studies of J10 itself, we found that this mutation does not affect the formation and release of virion-like particles per se, but instead impairs the ability of those particles to incorporate each of the eight essential RNA segments (vRNAs) that make up the viral genome. Taken together, our analysis identifies mutations in the C-terminal region of PA that differentially affect at least three distinct activities: protein nuclear localization, viral RNA synthesis, and a trans-acting function that is required for efficient packaging of all eight vRNAs.     

cis-Acting packaging signals in the influenza virus PB1, PB2, and PA genomic RNA segments

The influenza A virus genome consists of eight negative-sense RNA segments. The cis-acting signals that allow these viral RNA segments (vRNAs) to be packaged into influenza virus particles have not been fully elucidated, although the 5' and 3' untranslated regions (UTRs) of each vRNA are known to be required. Efficient packaging of the NA, HA, and NS segments also requires coding sequences immediately adjacent to the UTRs, but it is not yet known whether the same is true of other vRNAs. By assaying packaging of genetically tagged vRNA reporters during plasmid-directed influenza virus assembly in cells, we have now mapped cis-acting sequences that are sufficient for packaging of the PA, PB1, and PB2 segments. We find that each involves portions of the distal coding regions. Efficient packaging of the PA or PB1 vRNAs requires at least 40 bases of 5' and 66 bases of 3' coding sequences, whereas packaging of the PB2 segment requires at least 80 bases of 5' coding region but is independent of coding sequences at the 3' end. Interestingly, artificial reporter vRNAs carrying mismatched ends (i.e., whose 5' and 3' ends are derived from different vRNA segments) were poorly packaged, implying that the two ends of any given vRNA may collaborate in forming specific structures to be recognized by the viral packaging machinery.     

Inhibition of the protease activity of influenza virus RNA polymerase PA subunit by viral matrix protein

Influenza virus PA is a subunit of RNA-dependent RNA polymerase. We demonstrated that PA has a unique chymotrypsin-like serine protease activity with Ser624 as an active site. To obtain further insight into the role of the protease activity of PA in viral proliferation, we examined the interaction between PA and matrix protein (M1). Both M1 purified from virion and hexa-histidine-tagged M1 expressed in Escherichia coli bound to PA. Hexa-histidine-tagged M1 pulled down PA. The interaction of PA with M1 was sensitive to ionic strength, suggesting that the interaction is formed by electrostatic force. Using Suc-Leu-Leu-Val-Tyr-MCA, a specific substrate for PA protease, M1 was demonstrated to inhibit the amidolytic activity of PA, whereas M1 did not inhibit that of chymotrypsin or trypsin at all. These results suggest that M1 binds to and inhibits the amidolytic activity of PA.     

Sulfatide is required for efficient replication of influenza A virus

Sulfatide is abundantly expressed in various mammalian organs, including the intestines and trachea, in which influenza A viruses (IAVs) replicate. However, the function of sulfatide in IAV infection remains unknown. Sulfatide is synthesized by two transferases, ceramide galactosyltransferase (CGT) and cerebroside sulfotransferase (CST), and is degraded by arylsulfatase A (ASA). In this study, we demonstrated that sulfatide enhanced IAV replication through efficient translocation of the newly synthesized IAV nucleoprotein (NP) from the nucleus to the cytoplasm, by using genetically produced cells in which sulfatide expression was down-regulated by RNA interference against CST mRNA or overexpression of the ASA gene and in which sulfatide expression was up-regulated by overexpression of both the CST and CGT genes. Treatment of IAV-infected cells with an antisulfatide monoclonal antibody (MAb) or an anti-hemagglutinin (HA) MAb, which blocks the binding of IAV and sulfatide, resulted in a significant reduction in IAV replication and accumulation of the viral NP in the nucleus. Furthermore, antisulfatide MAb protected mice against lethal challenge with pathogenic influenza A/WSN/33 (H1N1) virus. These results indicate that association of sulfatide with HA delivered to the cell surface induces translocation of the newly synthesized IAV ribonucleoprotein complexes from the nucleus to the cytoplasm. Our findings provide new insights into IAV replication and suggest new therapeutic strategies.     

Mutational analysis identifies functional domains in the influenza A virus PB2 polymerase subunit.

A collection of influenza virus PB2 mutant genes was prepared, including N-terminal deletions, C-terminal deletions, and single-amino-acid insertions. These mutant genes, driven by a T7 promoter, were expressed by transfection into COS-1 cells infected with a vaccinia virus encoding T7 RNA polymerase. Mutant proteins accumulated to levels similar to that of wild-type PB2. Immunofluorescence analyses showed that the C-terminal region of the protein is essential for nuclear transport and that internal sequences affect nuclear localization, confirming previous results (J. Mukaijawa and D. P. Nayak, J. Virol. 65:245-253, 1991). The biological activity of these mutants was tested by determining their capacity to (i) reconstitute RNA polymerase activity in vivo by cotransfection with proteins NP, PB1, and PA and a virion-like RNA encoding the cat gene into vaccinia virus T7-infected COS-1 cells and (ii) complete with the wild-type PB2 activity. In addition, when tested at different temperatures in vivo, two mutant PB2 proteins showed a temperature-sensitive phenotype. The lack of interference shown by some N-terminal deletion mutants and the complete interference obtained with a C-terminal deletion mutant encoding only 124 amino acids indicated that this protein domain is responsible for interaction with another component of the polymerase, probably PB1. To further characterize the mutants, their ability to induce in vitro synthesis of viral cRNA or mRNA was tested by using ApG or beta-globin mRNA as a primer. One of the mutants, 1299, containing an isoleucine insertion at position 299, was able to induce cRNA and mRNA synthesis in ApG-primed reactions but required a higher beta-globin mRNA concentration than wild-type PB2 for detection of in vitro synthesis. This result suggested that mutant I299 has diminished cap-binding activity.     

Distinct regions of influenza virus PB1 polymerase subunit recognize vRNA and cRNA templates.

The influenza virus RNA polymerase is a heterotrimer comprising the PB1, PB2 and PA subunits. PB1 is the core of the complex and accounts for the polymerase activity. We have studied the interaction of PB1 with model cRNA template by in vitro binding and Northwestern analyses. The binding to model cRNA was specific and showed an apparent Kd of approximately 7x10(-8) M. In contrast to the interaction with vRNA, PB1 was able to bind equally the 5' and 3' arm of the cRNA panhandle. The N-terminal 139 amino acids of PB1 and sequences between positions 267 and 493 proved positive for binding to cRNA, whereas the interaction with vRNA template previously was mapped to the N- and C-terminal regions. Competition experiments using the 5' and 3' arms of either the vRNA or cRNA panhandle indicated that the N-terminal binding site is shared by both templates. The data indicate that the PB1 RNA-binding sites are constituted by: (i) residues located at the N-terminus (probably common for vRNA and cRNA binding) and, either (ii) residues from the central part of PB1 (for cRNA) or (iii) residues from the C-terminal region of PB1 (for vRNA), and suggest that PB1 undergoes a conformational change upon binding to cRNA versus vRNA templates.