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Influenza A viruses with different amino acid residues at PB2-627 display distinct replication properties in vitro and in vivo: revealing the sequence plasticity of PB2-627 position.

ABSTRACT: Sequence analyses of influenza PB2 sequences indicate that the 627 position almost exclusively contains either lysine (K) or glutamic acid (E), suggesting a high sequence constraint at this genetic marker. Here, we used a site-directed random mutagenesis method to demonstrate that PB2-627 position has a high sequence plasticity. Recombinant viruses carrying various amino acid residues at this position are viable in cell cultures. These PB2-627 mutants showed various polymerase activities and replication kinetics in mammalian and avian cells as well as pathogenicity in mice. Serially passaging these mutants in MDCK cells generated some compensatory PB2 mutations that can restore polymerase activities of the PB2-627 mutants. Of these, PB2-D309N was identified as a novel one. Besides showing that influenza virus can tolerate a wide range of amino acid residues at the PB2-627 position, this study also demonstrates a potential strategy to identify novel mutations that can enhance viral polymerase.

SUBMITTER: Chin AWH

PROVIDER: S-EPMC4252771 | biostudies-literature | 2014 Nov

REPOSITORIES: biostudies-literature

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Influenza A viruses with different amino acid residues at PB2-627 display distinct replication properties in vitro and in vivo: revealing the sequence plasticity of PB2-627 position.

Chin Alex W H AWH Li Olive T W OTW Mok Chris K P CKP Ng Miko K W MKW Peiris Malik M Poon Leo L M LLM

Virology 20140928

Sequence analyses of influenza PB2 sequences indicate that the 627 position almost exclusively contains either lysine (K) or glutamic acid (E), suggesting a high sequence constraint at this genetic marker. Here, we used a site-directed random mutagenesis method to demonstrate that PB2-627 position has a high sequence plasticity. Recombinant viruses carrying various amino acid residues at this position are viable in cell cultures. These PB2-627 mutants showed various polymerase activities and rep ...[more]

PMID: 25262472

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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:Poultry exposure is a major risk factor for human H7N9 zoonotic infections, for which the mode of transmission remains unclear. We studied the transmission of genetically related poultry and human H7N9 influenza viruses differing by four amino acids, including the host determinant PB2 residue 627. A/Silkie chicken/HK/1772/2014 (SCk1772) and A/HK/3263/14 (HK3263) replicated to comparable titers in chickens, with superior oropharyngeal over cloacal shedding; both viruses transmitted efficiently among chickens via direct contact but inefficiently via the airborne route. Interspecies transmission via the airborne route was observed for ferrets exposed to the SCk1772- or HK3263-infected chickens, while low numbers of copies of influenza viral genome were detected in the air, predominantly at particle sizes larger than 4 ?m. In ferrets, the human isolate HK3263 replicated to higher titers and transmitted more efficiently via direct contact than SCk1772. We monitored "intrahost" and "interhost" adaptive changes at PB2 residue 627 during infection and transmission of the Sck1772 that carried E627 and HK3263 that carried V/K/E polymorphism at 60%, 20%, and 20%, respectively. For SCk1772, positive selection for K627 over E627 was observed in ferrets during the chicken-to-ferret or ferret-to-ferret transmission. For HK3263 that contained V/K/E polymorphism, mixed V627 and E627 genotypes were transmitted among chickens while either V627 or K627 was transmitted to ferrets with a narrow transmission bottleneck. Overall, our results suggest direct contact as the main mode for H7N9 transmission and identify the PB2-V627 genotype with uncompromised fitness and transmissibility in both avian and mammalian species.We studied the modes of H7N9 transmission, as this information is crucial for developing effective control measures for prevention. Using chicken (SCk1772) and human (HK3263) H7N9 isolates that differed by four amino acids, including the host determinant PB2 residue 627, we observed that both viruses transmitted efficiently among chickens via direct contact but inefficiently via the airborne route. Chicken-to-ferret transmission via the airborne route was observed, along with the detection of viral genome in the air at low copy numbers. In ferrets, HK3263 transmitted more efficiently than SCk1772 via direct contact. During the transmission of SCk1772 that contained E and HK3263 that contained V/K/E polymorphism at PB2 residue 627, positive selections of E627 and K627 were observed in chickens and ferrets, respectively. In addition, PB2-V627 was transmitted and stably maintained in both avian and mammalian species. Our results support applying intervention strategies that minimize direct and indirect contact at the poultry markets during epidemics.

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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.

Dataset Information

Influenza A viruses with different amino acid residues at PB2-627 display distinct replication properties in vitro and in vivo: revealing the sequence plasticity of PB2-627 position.

Publications

Influenza A viruses with different amino acid residues at PB2-627 display distinct replication properties in vitro and in vivo: revealing the sequence plasticity of PB2-627 position.

Similar Datasets

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