Identification of Amino Acid Residues Required for Inhibition of Host Gene Expression by Influenza Virus A/Viet Nam/1203/2004 H5N1 PA-X

Abstract

PA-X is a non-structural protein of influenza A virus (IAV), which is encoded by the polymerase acidic (PA) N-terminal region that contains a C-terminal +1 frameshifted sequence. IAV PA-X protein modulates virus-induced host innate immune responses and viral pathogenicity via suppression of host gene expression or cellular shutoff, through cellular mRNA cleavage. Highly pathogenic avian influenza viruses (HPAIV) of the H5N1 subtype naturally infect different avian species, they have an enormous economic impact in the poultry farming, and they also have zoonotic and pandemic potential, representing a risk to human public health. In the present study, we describe a novel bacteria-based approach to identify amino acid residues in the PA-X protein of the HPAIV A/Viet Nam/1203/2004 H5N1 that are important for its ability to inhibit host protein expression or cellular shutoff activity. Identified PA-X mutants displayed a reduced shutoff activity as compared to that of the wild-type (WT) A/Viet Nam/1203/2004 H5N1 PA-X protein. Notably, this new bacteria-based screening allowed us to identify amino acid residues widely distributed over the entire N-terminal region of PA-X. Furthermore, we found that some of the residues affecting A/Viet Nam/1203/2004 H5N1 PA-X host shutoff activity also affect PA polymerase activity in a minigenome assay. This information could be used for the rational design of new and more effective compounds with antiviral activity against IAV. Moreover, our results demonstrate the feasibility of using this bacteria-based approach to identify amino acid residues important for the activity of viral proteins to inhibit host gene expression. IMPORTANCE Highly pathogenic avian influenza viruses (HPAIV) continue to pose a huge threat to global animal and human health. Despite of the limited genome size of Influenza A virus (IAV), the virus encodes eight main viral structural proteins and multiple accessory non-structural proteins, depending on the IAV type, subtype or strain. One of the IAV accessory proteins, PA-X, is encoded by the polymerase acidic (PA) protein and is involved in pathogenicity through the modulation of IAV-induced host inflammatory and innate immune responses. However, the molecular mechanism(s) of IAV PA-X regulation of the host immune response is not well understood. In this work, we used, for the first time, a bacteria-based approach for the identification of amino acids important for the ability of IAV PA-X to induce host shutoff activity and describe novel residues relevant for its ability to inhibit host gene expression, and their contribution in PA polymerase activity.

Amino acid residues involved in inhibition of host gene expression by influenza A/Brevig Mission/1/1918 PA-X

“…Our results indicate that all the amino acid changes contributed to decrease 1918 PA-X's ability to induce cellular shutoff (Figures 5 and 6). Similarly, in a previous work using an H5N1 IAV strain, we also found that amino acid mutations H146Y, and L187P, present in the double mutants, as well as F76L, found in a single mutant, decrease the ability of H5N1 PA-X to inhibit host gene expression [36].…”

“…Empty and 1918 WT PA-X-expressing pCAGGS plasmids were included as controls. Unsurprisingly, 1918 WT PA-X was not detected in transfected cells by immunofluorescence because its ability to inhibit host gene expression, including its own expression ( Figure 4 ) [12,25,27,32–36]. However, 1918 PA-X containing F76L, L109R, T123A, R168G, T98I/P103S and H146Y/L187P mutations were detectable under immunofluorescence because these 1918 PA-X mutants were affected in its shutoff capabilities ( Figure 3 ).…”

Amino Acid Residues Involved in Inhibition of Host Gene Expression by Influenza A/Brevig Mission/1/1918 PA-X

“…Our results indicate that all the amino acid changes contributed to a decrease in 1918 PA-X's ability to induce cellular shutoff (Figures 5 and 6). Similarly, in a previous work using an H5N1 IAV strain, we also found that amino acid mutations, H146Y and L187P, present in the double mutants, as well as F76L, found in a single mutant, decrease the ability of H5N1 PA-X to inhibit host gene expression [40].…”

Mutation L319Q in the PB1 Polymerase Subunit Improves Attenuation of a Candidate Live-Attenuated Influenza A Virus Vaccine

“…To evaluate the effect of WT and mutant PB2 and PB1 proteins on viral polymerase activity, a MG assay was performed as previously described ( 42 , 53 ). Briefly, human 293T cells (5 × 10 5 cells/well, 12-well plate format, triplicates) were transiently co-transfected in suspension, using Lipofectamine 2000 (Invitrogen), with 125 ng of each ambisense pDZ plasmids (pDZ-PB2 or PB2 LAIV , -PB1 or PB1 LAIV or -PB1 L319Q or pDZ-PB1 LAIV+L319Q , -PA, - and NP), together with 250 ng of two reporter viral (v)RNA-like expression pPOL-I plasmids encoding GFP or Gluc driven by a human RNA polymerase I promoter ( 53 , 71 ). A Cluc-encoding plasmid under the simian virus 40 promoter (SV40-Cluc, 50 ng) was included to normalize transfection efficiencies ( 72 , 73 ).…”