The human H5N1 influenza A virus polymerase complex is active in vitro over a broad range of temperatures, in contrast to the WSN complex, and this property can be attributed to the PB2 subunit

Self Cite

The 2009 pandemic influenza virus (pH1N1) is a swine-origin reassortant containing human, avian, and swine influenza genes. We have previously shown that the polymerase complex of the pH1N1 strain A/California/04/2009 (Cal) is highly active in mammalian 293T cells, despite the avian origin of both its PA and PB2. In this study, we analyzed the polymerase residues that are responsible for high pH1N1 polymerase activity in the mammalian host. Characterization of polymerase complexes containing various combinations of Cal and avian influenza virus A/chicken/Nanchang/3-120/01 (H3N2) (Nan) by reporter gene assay indicates that Cal PA, but not PB2, is a major contributing factor to high Cal polymerase activity in 293T cells. In particular, Cal PA significantly activates the otherwise inactive Nan polymerase at 37 and 39°C but not at the lower temperature of 34°C. Further analysis using site-directed mutagenesis showed that the Cal PA residues 85I, 186S, and 336M contribute to enhanced activity of the Cal polymerase. Recombinant A/WSN/33 (H1N1) (WSN) viruses containing Nan NP and polymerase (PA, PB1, PB2) genes with individual mutations in PA at residues 85, 186, and 336 produced higher levels of viral protein than the virus containing wild-type (WT) Nan PA. Interestingly, compared to the WT, the virus containing the 85I mutation grew faster in human A549 cells and the 336M mutation most significantly enhanced pathogenicity in a mouse model, among the three PA mutations tested. Our results suggest that multiple mutations in PA, which were rarely present in previous influenza isolates, are involved in mammalian adaptation and pathogenicity of the 2009 pH1N1.

Section: Resultssupporting

confidence: 73%

PA Residues in the 2009 H1N1 Pandemic Influenza Virus Enhance Avian Influenza Virus Polymerase Activity in Mammalian Cells

Bussey

Desmet

Mattiacio

et al. 2011

Self Cite

“…BradelTretheway et al showed that the avian VN04 polymerase complex is transcriptionally active up to 42°C, whereas the human WSN polymerase complex lost activity at 39°C or higher. Through reassortant RNP molecules, they discovered that this activity is attributable to the PB2 gene (17). Kashiwagi et al also used VN04 and WSN reassortant RNP complexes, but they showed that thermal sensitivity is dependent on the species of origin of the PA gene (18).…”

Section: Discussionmentioning

confidence: 99%

The Temperature-Sensitive and Attenuation Phenotypes Conferred by Mutations in the Influenza Virus PB2, PB1, and NP Genes Are Influenced by the Species of Origin of the PB2 Gene in Reassortant Viruses Derived from Influenza A/California/07/2009 and A/WSN/33 Viruses

Broadbent

Santos

Godbout

et al. 2014

Live attenuated influenza vaccines in the United States are derived from a human virus that is temperature sensitive (ts), characterized by restricted (>100-fold) replication at 39°C. The ts genetic signature (ts sig) has been mapped to 5 loci in 3 genes: PB1 (391E, 581G, and 661T), PB2 (265S), and NP (34G). However, when transferred into avian and swine influenza viruses, only partial ts and attenuation phenotypes occur. ts sig was restricted >100-fold compared to that at 33°C. Reassortant CA07 ts sig viruses were generated with individual polymerase gene segments from WSN, and vice versa. Only ts sig viruses with a PB2 gene segment derived from WSN were restricted in replication >100-fold at 39°C. In ferrets, the CA07 ts sig virus replicated in the upper and lower respiratory tract, but the replication of a reassortant CA07 ts sig virus with a WSN PB2 gene was severely restricted in the lungs. Taken together, these data suggest that the origin of the PB2 gene segment influences the ts phenotype in vitro and attenuation in vivo. This could have implications for the design of novel live vaccines against animal origin influenza viruses. IMPORTANCELive attenuated influenza vaccines (LAIVs) on temperature-sensitive (ts) backbones derived from animal origin influenza viruses are being sought for use in the poultry and swine industries and to protect people against animal origin influenza. However, inserting the ts genetic signature from a licensed LAIV backbone fails to fully attenuate these viruses. Our data indicate this is associated with the presence of a PB2 gene segment derived from an avian influenza virus. We show that a reassortant 2009 pandemic H1N1 virus with the ts signature from a licensed LAIV donor virus is ts in vitro and attenuated in vivo when the PB2 gene is derived from a human origin virus but not from an avian virus. Our study provides information that could benefit the rational design of alternative LAIV backbones against animal origin influenza viruses.

“…In this approach, we purified the H5N1 (Vietnam/1203/04 [VN/1203]) polymerase (3P) complex and its subunit components (PA and PA-PB1) from human lung epithelial cells (A549 cell line) infected with adenovirus (Ad) vectors encoding these proteins. This novel expression system allows for the expression and purification of high levels of polymerase proteins (2). We analyzed proteins in the purified fractions by mass spectrometry, either before or after nuclease treatment to remove copurified RNA (and to dissociate protein complexes held together through an RNA intermediate).…”

mentioning

confidence: 99%

“…The polymerase genes from the H1N1 virus of a recent pandemic (CA/04/09) were human codon optimized and assembled from synthetic oligonucleotides by EpochBiolabs (Missouri City, TX); protein identifiers (IDs) for polymerase genes of CA/04 were ACP44156.1, ACP41103.1, and ACP41102.1. The adenoviral vectors were constructed as described previously (2). Briefly, PA from each viral strain was tagged at the C terminus with a tandem affinity purification (TAP) tag, and the viral polymerase genes were cloned into adenoviral expression vectors as described for A/Vietnam/1203/04.…”

mentioning

confidence: 99%

“…The polymerase genes of the H1N1 (A/WSN/ 33) and H5N1 (A/Vietnam/1203/04) viruses were assembled as described previously (2). The polymerase genes from the H1N1 virus of a recent pandemic (CA/04/09) were human codon optimized and assembled from synthetic oligonucleotides by EpochBiolabs (Missouri City, TX); protein identifiers (IDs) for polymerase genes of CA/04 were ACP44156.1, ACP41103.1, and ACP41102.1.…”

mentioning

confidence: 99%

See 1 more Smart Citation

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

Bradel-Tretheway

Mattiacio

Krasnoselsky

et al. 2011

Self Cite

The trimeric RNA polymerase complex (3P, for PA-PB1-PB2) of influenza A virus (IAV) is an important viral determinant of pathogenicity and host range restriction. Specific interactions of the polymerase complex with host proteins may be determining factors in both of these characteristics and play important roles in the viral life cycle. To investigate this question, we performed a comprehensive proteomic analysis of human host proteins associated with the polymerase of the well-characterized H5N1 Vietnam/1203/04 isolate. We identified over 400 proteins by liquid chromatography-tandem mass spectrometry (LC-MS/MS), of which over 300 were found to bind to the PA subunit alone. The most intriguing and novel finding was the large number of mitochondrial proteins (ϳ20%) that associated with the PA subunit. These proteins mediate molecular transport across the mitochondrial membrane or regulate membrane potential and may in concert with the identified mitochondrion-associated apoptosis inducing factor (AIFM1) have roles in the induction of apoptosis upon association with PA. Additionally, we identified host factors that associated with the PA-PB1 (68 proteins) and/or the 3P complex (34 proteins) including proteins that have roles in innate antiviral signaling (e.g., ZAPS or HaxI) or are cellular RNA polymerase accessory factors (e.g., polymerase I transcript release factor [PTRF] or Supt5H). IAV strain-specific host factor binding to the polymerase was not observed in our analysis. Overall, this study has shed light into the complex contributions of the IAV polymerase to host cell pathogenicity and allows for direct investigations into the biological significance of these newly described interactions.The trimeric RNA (3P) influenza virus polymerase complex (PA-PB1-PB2) assembles in the nucleus, where influenza virus replication occurs. Several events inside the host cell must take place in order to support viral replication, such as the regulation of early antiviral host responses, host protein shutoff, and nuclear transport of viral RNA and proteins (21,29,63). These events require the viral RNA polymerase to interact with cellular factors, some of which may also be important for host range specificity but have yet to be identified (17,44). A range of RNA interference (RNAi)-based, genome-wide screens have been published recently that identified host factors involved in influenza virus replication (4,22,31,35,52,56). Although the degree of overlap between these studies was minimal, similar biological pathways and functional classifications were identified, suggesting their importance in the influenza virus infectious cycle. 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 po...