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Conformational polymorphism of m7GTP in crystal structure of the PB2 middle domain from human influenza A virus.

ABSTRACT: Influenza pandemics with human-to-human transmission of the virus are of great public concern. It is now recognized that a number of factors are necessary for human transmission and virulence, including several key mutations within the PB2 subunit of RNA-dependent RNA polymerase. The structure of the middle domain in PB2 has been revealed with or without m(7)GTP, thus the middle domain is considered to be novel target for structure-based drug design. Here we report the crystal structure of the middle domain of H1N1 PB2 with or without m(7)GTP at 1.9 Å and 2.0 Å resolution, respectively, which has two mutations (P453H, I471T) to increase electrostatic potential and solubility. Here we report the m(7)GTP has unique conformation differ from the reported structure. 7-methyl-guanine is fixed in the pocket, but particularly significant change is seen in ribose and triphosphate region: the buried 7-methyl-guanine indeed binds in the pocket forming by H357, F404, E361 and K376 but the triphosphate continues directly to the outer domain. The presented conformation of m(7)GTP may be a clue for the anti-influenza drug-design.

SUBMITTER: Tsurumura T

PROVIDER: S-EPMC3843726 | biostudies-literature | 2013

REPOSITORIES: biostudies-literature

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Conformational polymorphism of m7GTP in crystal structure of the PB2 middle domain from human influenza A virus.

Tsurumura Toshiharu T Qiu Hao H Yoshida Toru T Tsumori Yayoi Y Hatakeyama Dai D Kuzuhara Takashi T Tsuge Hideaki H

PloS one 20131129 11

Influenza pandemics with human-to-human transmission of the virus are of great public concern. It is now recognized that a number of factors are necessary for human transmission and virulence, including several key mutations within the PB2 subunit of RNA-dependent RNA polymerase. The structure of the middle domain in PB2 has been revealed with or without m(7)GTP, thus the middle domain is considered to be novel target for structure-based drug design. Here we report the crystal structure of the m ...[more]

PMID: 24312396

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Project description:The RNA genome of influenza A viruses is transcribed and replicated by the viral RNA-dependent RNA polymerase, composed of the subunits PA, PB1, and PB2. High-resolution structural data revealed that the polymerase assembles into a central polymerase core and several auxiliary highly flexible, protruding domains. The auxiliary PB2 cap-binding and the PA endonuclease domains are both involved in cap snatching, but the role of the auxiliary PB2 627 domain, implicated in host range restriction of influenza A viruses, is still poorly understood. In this study, we used structure-guided truncations of the PB2 subunit to show that a PB2 subunit lacking the 627 domain accumulates in the cell nucleus and assembles into a heterotrimeric polymerase with PB1 and PA. Furthermore, we showed that a recombinant viral polymerase lacking the PB2 627 domain is able to carry out cap snatching, cap-dependent transcription initiation, and cap-independent ApG dinucleotide extension in vitro, indicating that the PB2 627 domain of the influenza virus RNA polymerase is not involved in core catalytic functions of the polymerase. However, in a cellular context, the 627 domain is essential for both transcription and replication. In particular, we showed that the PB2 627 domain is essential for the accumulation of the cRNA replicative intermediate in infected cells. Together, these results further our understanding of the role of the PB2 627 domain in transcription and replication of the influenza virus RNA genome.IMPORTANCE Influenza A viruses are a major global health threat, not only causing disease in both humans and birds but also placing significant strains on economies worldwide. Avian influenza A virus polymerases typically do not function efficiently in mammalian hosts and require adaptive mutations to restore polymerase activity. These adaptations include mutations in the 627 domain of the PB2 subunit of the viral polymerase, but it still remains to be established how these mutations enable host adaptation on a molecular level. In this report, we characterize the role of the 627 domain in polymerase function and offer insights into the replication mechanism of influenza A viruses.

Project description:UnlabelledSuccessful replication of influenza virus requires the coordinated expression of viral genes and replication of the genome by the viral polymerase, composed of the subunits PA, PB1, and PB2. Polymerase activity is regulated by both viral and host factors, yet the mechanisms of regulation and how they contribute to viral pathogenicity and tropism are poorly understood. To characterize these processes, we created a series of mutants in the 627 domain of the PB2 subunit. This domain contains a conserved "P[F/P]AAAPP" sequence motif and the well-described amino acid 627, whose identity regulates host range. A lysine present at position 627 in most mammalian viral isolates creates a basic face on the domain surface and confers high-level activity in humans compared to the glutamic acid found at this position in avian isolates. Mutation of the basic face or the P[F/P]AAAPP motif impaired polymerase activity, assembly of replication complexes, and viral replication. Most of these residues are required for general polymerase activity, whereas PB2 K586 and R589 were preferentially required for function in human versus avian cells. Thus, these data identify residues in the 627 domain and other viral proteins that regulate polymerase activity, highlighting the importance of the surface charge and structure of this domain for virus replication and host adaptation.ImportanceInfluenza virus faces barriers to transmission across species as it emerges from its natural reservoir in birds to infect mammals. The viral polymerase is an important regulator of this process and undergoes discrete changes to adapt to replication in mammals. Many of these changes occur in the polymerase subunit PB2. Here we describe the systematic analysis of a key region in PB2 that controls species-specific polymerase activity. We report the importance of conserved residues that contribute to the overall charge of the protein as well as those that likely affect protein structure. These findings provide further insight into the molecular events dictating species-specific polymerase function and viral replication.

Project description:Phylogenetic analysis of 20 influenza A virus PB2 genes showed that PB2 genes have evolved into the following four major lineages: (i) equine/Prague/56 (EQPR56); (ii and iii) two distinct avian PB2 lineages, one containing FPV/34 and H13 gull virus strains and the other containing North American avian and recent equine strains; and (iv) human virus strains joined with classic swine virus strains (i.e., H1N1 swine virus strains related to swine/Iowa/15/30). The human virus lineage showed the greatest divergence from its root relative to other lineages. The estimated nucleotide evolutionary rate for the human PB2 lineage was 1.82 x 10(-3) changes per nucleotide per year, which is within the range of published estimates for NP and NS genes of human influenza A viruses. At the amino acid level, PB2s of human viruses have accumulated 34 amino acid changes over the past 55 years. In contrast, the avian PB2 lineages showed much less evolution, e.g., recent avian PB2s showed as few as three amino acid changes relative to the avian root. The completion of evolutionary analyses of the PB1, PB2, PA and NP genes of the ribonucleoprotein (RNP) complex permits comparison of evolutionary pathways. Different patterns of evolution among the RNP genes indicate that the genes of the complex are not coevolving as a unit. Evolution of the PB1 and PB2 genes is less correlated with host-specific factors, and their proteins appear to be evolving more slowly than NP and PA. This suggests that protein functional constraints are limiting the evolutionary divergence of PB1 and PB2 genes. The parallel host-specific evolutionary pathways of the NP and PA genes suggest that these proteins are coevolving in response to host-specific factors. PB2s of human influenza A viruses share a common ancestor with classic swine virus PB2s, and the pattern of evolution suggests that the ancestor was an avian virus PB2. This same pattern of evolution appears in the other genes of the RNP complex. Antigenic studies of HA and NA proteins and sequence comparisons of NS and M genes also suggest a close ancestry for these genes in human and classic swine viruses. From our review of the evolutionary patterns of influenza A virus genes, we propose the following hypothesis: the common ancestor to current strains of human and classic swine influenza viruses predated the 1918 human pandemic virus and was recently derived from the avian host reservoir.

Dataset Information

Conformational polymorphism of m7GTP in crystal structure of the PB2 middle domain from human influenza A virus.

Publications

Conformational polymorphism of m7GTP in crystal structure of the PB2 middle domain from human influenza A virus.

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