Association of the Influenza Virus RNA Polymerase Subunit PB2 with the Host Chaperonin CCT

Abstract

The RNA polymerase of influenza A virus is a host range determinant and virulence factor. In particular, the PB2 subunit of the RNA polymerase has been implicated as a crucial factor that affects cell tropism as well as virulence in animal models. These findings suggest that host factors associating with the PB2 protein may play an important role during viral replication. In order to identify host factors that associate with the PB2 protein, we purified recombinant PB2 from transiently transfected mammalian cells and identified copurifying host proteins by mass spectrometry. We found that the PB2 protein associates with the cytosolic chaperonin containing TCP-1 (CCT), stress-induced phosphoprotein 1 (STIP1), FK506 binding protein 5 (FKBP5), ␣-and ␤-tubulin, Hsp60, and mitochondrial protein p32. Some of these binding partners associate with each other, suggesting that PB2 might interact with these proteins in multimeric complexes. More detailed analysis of the interaction of the PB2 protein with CCT revealed that PB2 associates with CCT as a monomer and that the CCT binding site is located in a central region of the PB2 protein. PB2 proteins from various influenza virus subtypes and origins can associate with CCT. Silencing of CCT resulted in reduced viral replication and reduced PB2 protein and viral RNA accumulation in a ribonucleoprotein reconstitution assay, suggesting an important function for CCT during the influenza virus life cycle. We propose that CCT might be acting as a chaperone for PB2 to aid its folding and possibly its incorporation into the trimeric RNA polymerase complex.Influenza A viruses, members of the family of Orthomyxoviridae, contain a segmented RNA genome of negative polarity. The genomic RNA segments together with the three subunits of the viral RNA-dependent RNA polymerase (PB1, PB2, and PA protein) and the nucleoprotein (NP) form viral ribonucleoprotein complexes (vRNPs). The PB1 subunit is the polymerase itself, while the PB2 and PA subunits are involved in the generation of 5Ј capped RNA primers through binding to and endonucleolytic cleavage of host pre-mRNAs (8,10,11,41,61). After the virus enters the cell via endocytosis, vRNPs are released into the cytoplasm and transported into the nucleus. In the nucleus, vRNPs catalyze the synthesis of viral mRNAs and complementary RNAs (cRNA) which, in turn, are used as templates for the synthesis of vRNAs. The newly formed vRNPs in association with other viral proteins (M1 and nonstructural protein 2/nuclear export factor [NS2/NEP]) are transported into the cytoplasm and subsequently to the cell membrane, where the assembly process takes place, followed by the release of progeny virions by budding (44).The PB1, PB2, and PA proteins are synthesized in the cytoplasm whereupon PB1 and PA form a dimeric complex that is transported into the nucleus. In the nucleus the dimer assembles with the PB2 subunit, which is transported separately (7,14). RanBP5 was identified as a factor that is involved in the import of the PB1-PA dimer into the nucleus ...

Interactome Analysis of the Influenza A Virus Transcription/Replication Machinery Identifies Protein Phosphatase 6 as a Cellular Factor Required for Efficient Virus Replication

“…Hsp90 has been shown to interact with PB1 and PB2 to stimulate viral RNA synthesis in vitro (87) and to facilitate nuclear import (88). Members of the CCT chaperonin have been proposed to aid in the folding of PB2 for its incorporation into a trimeric RdRP (20). The high abundance of molecular chaperones in the interactome reflects the importance of protein folding, proteome integrity, and proteostasis for the biological function of viral proteins.…”

“…Furthermore, these methods do not distinguish between those factors that are required for virus replication due to direct interaction with a viral component and those factors that can contribute to virus replication indirectly. Additional techniques have been employed with the aim of identifying host factors that interact with the viral transcription/replication machinery, including yeast two-hybrid screens (15,16), complementation assays (17), and AP-MS (18)(19)(20)(21). However, these studies have been limited either by the number of cellular factors that can be cloned and screened or because the identifications were not carried out in the context of the infected cell.…”

Comprehensive Proteomic Analysis of Influenza Virus Polymerase Complex Reveals a Novel Association with Mitochondrial Proteins and RNA Polymerase Accessory Factors

“…In addition to published factors (10,14,17,31,33,35,43,45,48,52,59,61,66), such as chaperonins and members of the Ran signaling pathway, we found a novel association of PA with mitochondrial proteins. Particularly, we found a strong association of PA with the apoptosis-inducing factor (AIFM1), suggesting a potential role of PA during apoptosis.…”

“…Other major virulence factors of the influenza A viruses (IAVs) are the well-studied PB1-F2 protein and the PB2 protein. Both have been described to have roles in the induction of apoptosis during influenza virus infection (9,14,21,46,69). PB2 has also been found to interact with IPS-1 at the mitochondria, where it may be involved in controlling virus-induced apoptosis as well as regulating innate antiviral immune responses (21,29).…”

“…Watanabe et al (67) compiled a list of 128 proteins that affected influenza virus replication in at least two out of the six screens. These included proteins involved in the nuclear import of viral ribonucleoproteins (vRNPs), such as importin family members and nuclear pore complex proteins.Other studies have attempted to directly identify proteins that interact with either the influenza virus polymerase or the viral ribonucleoprotein complex, using a yeast two-hybrid system or proteomics approaches such as fractionation by SDS-PAGE followed by mass spectrometry (MS) (10,12,14,20,25,27,33,41,43,45,46,49,61,65,66). However, these approaches have been performed only at relatively small scale, and few interacting proteins have been identified to date.…”