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Variations outside the conserved motifs of PB1 catalytic active site may affect replication efficiency of the RNP complex of influenza A virus.

ABSTRACT: PB1 functions as the catalytic subunit of influenza virus RNA polymerase complex and plays an essential role in viral RNA transcription and replication. To determine plasticity in the PB1 enzymatic site and map catalytically important residues, 658 mutants were constructed, each with one to seven mutations in the enzymatic site of PB1. The polymerase activities of these mutants were quantified using a minigenome assay, and polymerase activity-associated residues were identified using sparse learning. Results showed that polymerase activities are affected by the residues not only within the conserved motifs, but also across the inter-motif regions of PB1, and the latter are primarily located at the base of the palm domain, a region that is conserved in avian PB1 but with high sequence diversity in swine PB1. Our results suggest that mutations outside the PB1 conserved motifs may affect RNA replication and could be associated with influenza virus host adaptation.

SUBMITTER: Waters K

PROVIDER: S-EPMC8579824 | biostudies-literature | 2021 Jul

REPOSITORIES: biostudies-literature

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Variations outside the conserved motifs of PB1 catalytic active site may affect replication efficiency of the RNP complex of influenza A virus.

Waters Kaitlyn K Wan Hamilton J HJ Han Lei L Xue Jianli J Ykema Matthew M Tao Yizhi J YJ Wan Xiu-Feng XF

Virology 20210409

PB1 functions as the catalytic subunit of influenza virus RNA polymerase complex and plays an essential role in viral RNA transcription and replication. To determine plasticity in the PB1 enzymatic site and map catalytically important residues, 658 mutants were constructed, each with one to seven mutations in the enzymatic site of PB1. The polymerase activities of these mutants were quantified using a minigenome assay, and polymerase activity-associated residues were identified using sparse lear ...[more]

PMID: 33887645

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Project description:BackgroundMutations in the PB1 subunit of RNA-dependent RNA polymerase (RdRp) of influenza A virus can affect replication fidelity. Before the influenza A/H1N1 pandemic in 2009, most human influenza A/H1N1 viruses contained the avian-associated residue, serine, at position 216 in PB1. However, near the onset of the 2009 pandemic, human viruses began to acquire the mammalian-associated residue, glycine, at PB1-216, and PB1-216G became predominant in human viruses thereafter.MethodsUsing entropy-based analysis algorithm, we have previously identified several host-specific amino-acid signatures that separated avian and swine viruses from human influenza viruses. The presence of these host-specific signatures in human influenza A/H1N1 viruses suggested that these mutations were the result of adaptive genetic evolution that enabled these influenza viruses to circumvent host barriers, which resulted in cross-species transmission. We investigated the biological impact of this natural avian-to-mammalian signature substitution at PB1-216 in human influenza A/H1N1 viruses.ResultsWe found that PB1-216G viruses had greater mutation potential, and were more sensitive to ribavirin than PB1-216S viruses. In oseltamivir-treated HEK293 cells, PB1-216G viruses generated mutations in viral neuraminidase at a higher rate than PB1-216S viruses. By contrast, PB1-216S viruses were more virulent in mice than PB1-216G viruses. These results suggest that the PB1-S216G substitution enhances viral epidemiological fitness by increasing the frequency of adaptive mutations in human influenza A/H1N1 viruses.ConclusionsOur results thus suggest that the increased adaptability and epidemiological fitness of naturally arising human PB1-216G viruses, which have a canonical low-fidelity replicase, were the biological mechanisms underlying the replacement of PB1-216S viruses with a high-fidelity replicase following the emergence of pdmH1N1. We think that continued surveillance of such naturally occurring PB1-216 variants among others is warranted to assess the potential impact of changes in RdRp fidelity on the adaptability and epidemiological fitness of human A/H1N1 influenza viruses.

Project description:BackgroundPlant HD-Zip transcription factors are modular proteins in which a homeodomain is associated to a leucine zipper. Of the four subfamilies in which they are divided, the tested members from subfamily I bind in vitro the same pseudopalindromic sequence CAAT(A/T)ATTG and among them, several exhibit similar expression patterns. However, most experiments in which HD-Zip I proteins were over or ectopically expressed under the control of the constitutive promoter 35S CaMV resulted in transgenic plants with clearly different phenotypes. Aiming to elucidate the structural mechanisms underlying such observation and taking advantage of the increasing information in databases of sequences from diverse plant species, an in silico analysis was performed. In addition, some of the results were also experimentally supported.ResultsA phylogenetic tree of 178 HD-Zip I proteins together with the sequence conservation presented outside the HD-Zip domains allowed the distinction of six groups of proteins. A motif-discovery approach enabled the recognition of an activation domain in the carboxy-terminal regions (CTRs) and some putative regulatory mechanisms acting in the amino-terminal regions (NTRs) and CTRs involving sumoylation and phosphorylation. A yeast one-hybrid experiment demonstrated that the activation activity of ATHB1, a member of one of the groups, is located in its CTR. Chimerical constructs were performed combining the HD-Zip domain of one member with the CTR of another and transgenic plants were obtained with these constructs. The phenotype of the chimerical transgenic plants was similar to the observed in transgenic plants bearing the CTR of the donor protein, revealing the importance of this module inside the whole protein.ConclusionsThe bioinformatical results and the experiments conducted in yeast and transgenic plants strongly suggest that the previously poorly analyzed NTRs and CTRs of HD-Zip I proteins play an important role in their function, hence potentially constituting a major source of functional diversity among members of this subfamily.

Project description:UnlabelledThe replication of plus-strand RNA virus genomes is mediated by virally encoded RNA-dependent RNA polymerases (RdRps). We have investigated the role of the C-proximal region in the RdRp of tomato bushy stunt virus (TBSV) in mediating viral RNA synthesis. TBSV is the prototype species in the genus Tombusvirus, family Tombusviridae, and its RdRp is responsible for replicating the viral genome, transcribing two subgenomic mRNAs, and supporting replication of defective interfering RNAs. Comparative sequence analysis of the RdRps of tombusvirids identified three highly conserved motifs in their C-proximal regions, and these sequences were subsequently targeted for mutational analysis in TBSV. The results revealed that these motifs are important for (i) synthesizing viral genomic RNA and subgenomic mRNAs, (ii) facilitating plus- and/or minus-strand synthesis, and (iii) modulating trans-replication of a defective interfering RNA. These motifs were also found to be conserved in other plant viruses as well as in a fungal and insect virus. The collective findings are discussed in relation to viral RNA synthesis and taxonomy.ImportanceLittle is currently known about the structure and function of the viral polymerases that replicate the genomes of RNA plant viruses. Tombusviruses, the prototype of the tombusvirids, have been used as model plus-strand RNA plant viruses for understanding many of the steps in the infectious process; however, their polymerases remain poorly characterized. To help address this issue, the function of the C-terminal region of the polymerase of a tombusvirus was investigated. Three conserved motifs were identified and targeted for mutational analysis. The results revealed that these polymerase motifs are important for determining what type of viral RNA is produced, facilitating different steps in viral RNA production, and amplifying subgenomic RNA replicons. Accordingly, the C-terminal region of the tombusvirus polymerase is needed for a variety of fundamental activities. Furthermore, as these motifs are also present in distantly related viruses, the significance of these results extends beyond tombusvirids.

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Variations outside the conserved motifs of PB1 catalytic active site may affect replication efficiency of the RNP complex of influenza A virus.

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Variations outside the conserved motifs of PB1 catalytic active site may affect replication efficiency of the RNP complex of influenza A virus.

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