Functional Genomics Reveals Linkers Critical for Influenza Virus Polymerase

Abstract

Influenza virus mRNA synthesis by the RNA-dependent RNA polymerase involves binding and cleavage of capped cellular mRNA by the PB2 and PA subunits, respectively, and extension of viral mRNA by PB1. However, the mechanism for such a dynamic process is unclear. Using high-throughput mutagenesis and sequencing analysis, we have not only generated a comprehensive functional map for the microdomains of individual subunits but also have revealed the PA linker to be critical for polymerase activity. This PA linker binds to PB1 and also forms ionic interactions with the PA C-terminal channel. Nearly all mutants with five-amino-acid insertions in the linker were nonviable. Our model further suggests that the PA linker plays an important role in the conformational changes that occur between stages that favor capped mRNA binding and cleavage and those associated with viral mRNA synthesis. IMPORTANCEThe RNA-dependent RNA polymerase of influenza virus consists of the PB1, PB2, and PA subunits. By combining genome-wide mutagenesis analysis with the recently discovered crystal structure of the influenza polymerase heterotrimer, we generated a comprehensive functional map of the entire influenza polymerase complex. We identified the microdomains of individual subunits, including the catalytic domains, the interaction interfaces between subunits, and nine linkers interconnecting different domains. Interestingly, we found that mutants with five-amino-acid insertions in individual linkers were nonviable, suggesting the critical roles these linkers play in coordinating spatial relationships between the subunits. We further identified an extended PA linker that binds to PB1 and also forms ionic interactions with the PA C-terminal channel.

DNA-Binding Properties of African Swine Fever Virus pA104R, a Histone-Like Protein Involved in Viral Replication and Transcription

“…(C) The specific recognition of the serum used was tested against whole infected-cell extracts by Western blotting. pA104R-DNA interactions were not detected under high-ionic-strength conditions (Ն2 M NaCl), indicating that pA104R-binding activity involves ion pair formation, as reported for other viral DNA-binding proteins (26)(27)(28).…”

Human CD8+ T cell cross-reactivity across influenza A, B and C viruses

“…Genome-wide mutational analysis, has shown that IAV cannot tolerate substitutions in these motifs 46 . More interesting, however, is the IBV-wide cross-reactivity conferred by the BHA 543 peptide.…”

“…the universal A2/PB1 413 and two IBV-specific (A2/BHA 543-551 and A2/NS1 266-274 hereafter A2/BHA 543 and A2/BNS1 266 ) epitopes. To further understand the role of the universal A2/PB1 413 + CD8 + T cells in the immunodominance hierarchy following either IAV or IBV infection, we established IAV-or IBV-specific CD8 + T cell lines in vitro from PBMC of healthy adults (n=11) and assessed tetramer-specific CD8 + T cell responses against IAV epitopes (A2/M1 58-66 (A2/M1 58 ) , A2/PA [46][47][48][49][50][51][52][53][54] (A2/PA 64 ) , A2/PB1 413 ) and IBV epitopes (A2/BHA 543 , A2/BNS1 266 , A2/PB1 413 ). Consistent with our IFNγ staining ( Fig.…”

Comparative pathogenicity of H5N6 subtype highly pathogenic avian influenza viruses in chicken, Pekin duck and Muscovy duck

“…Regarding the highly pathogenic nature of HE144 in Pekin duck, several amino acid differences in PB2, PB1, PA and HA were observed when we compared the genome of HE144 with the consensus sequences of the other viruses we studied. One of these differences was at the PA‐arch domain of PA that binds to vRNA (Wang et al., ). A H5N1 HPAIV possessing D383 in the PA‐arch domain of PA killed ducks and D383 in PA affected polymerase activity and decreased the accumulation of PA in the nucleus (Song et al., ).…”

“…Regarding the highly pathogenic nature of HE144 in Pekin duck, several amino acid differences in PB2, PB1, PA and HA were observed when we compared the genome of HE144 with the consensus sequences of the other viruses we studied. One of these differences was at the PA-arch domain of PA that binds to vRNA (Wang et al, 2015).…”