Biochemical characterization of avian influenza viral polymerase containing PA or PB2 subunit from human influenza A virus

Pham, Phu Tran Vinh; Turan, Kadir; Nagata, Kyosuke; Kawaguchi, Atsushı

doi:10.1016/j.micinf.2018.04.003

Cited by 8 publications

(15 citation statements)

References 30 publications

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“…The results show that the gene transcription profiles in the cells that synthesize three polymerase subunits (PB2, PB1, and PA) as a complex were closer to the transcript profiles of the virus-infected cells. In our previous study (Pham et al, 2018), the PA component of the viral RdRP composed of different combinations of WSN and/or DkPen polymerase subunits was found the most determinative subunit for enzyme activity in mammalian HEK293 cells. Therefore, the effects of PA protein on host cell transcript level in HEK293 cells were also evaluated.…”

Section: Discussionmentioning

confidence: 83%

“…This result supports the hypothesis that DkPen PA has higher endonuclease activity than that of WSN PA in the cells. Although the cap-snatching activity assay performed under in vitro conditions shows that WSN virus PA protein has higher activity (Pham et al, 2018), it is thought that this result may be different in natural conditions within the cells.…”

Section: Discussionmentioning

confidence: 99%

“…The expression plasmids constructed in previous studies (Turan et al, 2004;Pham et al, 2018) were used for the transient synthesis of influenza A virus RdRP subunits (PB2 + PB1 + PA) or only PA protein in transfected HEK293 cells. To determine the effects of the influenza A virus RNA polymerase subunit PA protein in transcription and/or translation in HEK293 cells, the pSEAP (Clontech) reporter plasmid and the pCHA-ACTB plasmid (Gelmez, 2019) encoding HA-tagged human beta-actin protein were used.…”

Section: Plasmid Vectorsmentioning

confidence: 99%

“…To determine the effects of the influenza A virus RNA polymerase subunit PA protein in transcription and/or translation in HEK293 cells, the pSEAP (Clontech) reporter plasmid and the pCHA-ACTB plasmid (Gelmez, 2019) encoding HA-tagged human beta-actin protein were used. The plasmid vectors coding chimeric PA proteins (consisting of amino-terminal moiety of WSN PA and carboxyl-terminal moiety of DkPen PA, or vice versa) were constructed with subcloning using pCAGGS-PA(W) and pCAGGS-PA(D) plasmids (Pham et al, 2018). For this purpose, plasmid DNA was first digested with BsmI restriction enzyme (New England Biolabs Ltd., Hitchin, UK) via a cut site in the vector and then DraIII (New England Biolabs Ltd.) enzyme was used.…”

Section: Plasmid Vectorsmentioning

confidence: 99%

“…K577E mutation in PB1 protein, another subunit of the viral RdRP, is also known to provide adaptation of the H9N2 virus to mammalian cells (Kamiki et al, 2018). In a study carried out with influenza A virus minireplicons, it was revealed that the RdRP of avian type influenza A/duck/ Pennsylvania/10218/84 (DkPen) has very low activity compared to enzymes of the human type A/WSN/33 (WSN), and the N-terminal endonuclease domain of the PA subunit of the RdRP is essential for mammalian adaptation (Pham et al, 2018). Another viral protein, the nonstructural NS1, has also been shown to be important for the adaptation of avian influenza A H9N2 subtype (A/ Ck/Korea/163/04H9N2) to mammalian cells (Park et al, 2015) The virus-host specificity and pathogenicity do not depend only on viral factors, as the host's intracellular defense mechanisms also play an important role.…”

Section: Introductionmentioning

confidence: 99%

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Expression profiles of interferon-related genes in cells infected with influenza A viruses or transiently transfected with plasmids encoding viral RNA polymerase

Çağlayan¹,

Turan²

2021

Turk J Biol

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Influenza A viruses frequently change their genetic characteristics, which leads to the emergence of new viruses. Consequently, elucidation of the relationship between influenza A virus and host cells has a great importance to cope with viral infections. In this study, it was aimed to determine expression profiles of interferon response genes in human embryonic kidney 293 (HEK293) cells infected with human (A/WSN-H1N1) and avian influenza A viruses (duck/Pennsylvania/10218/84/H5N2) or transfected with plasmids encoding viral RdRP subunits and, to obtain clues about the genes that may be important for the viral pathogenesis. The HEK293 cells cultured in a 12-well plate were infected with influenza A viruses or transfected with plasmids encoding viral polymerase. Total RNA extraction and cDNA preparation were carried out with commercial kits. Qiagen 96-well-RT 2 Profiler PCR Array plates designated for interferons response genes were used for quantitation of the transcripts. The relative quantities of transcripts were normalized with STAT3 gen, and the results were evaluated. Quantitative RT-PCR results showed that there are substantial differences of the interferon response gene transcription in cells infected with viruses or transfected with plasmids. A higher number of interferon-related genes were found to be downregulated in the cells infected with DkPen compared to WSN. On the other hand, significant differences in the expression profiles of interferon response genes were observed in the cells expressing viral PA protein. In particular, avian influenza PA protein was found to cause more aggressive changes on the transcript levels. Human and avian influenza A viruses cause a substantial change in interferon response gene expression in HEK293 cells. However, a higher number of genes were downregulated in the cells infected with avian influenza DkPen compared to WSN. It has been also concluded that the viral PA protein is one of the important viral factors affecting the transcript level of host genes.

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Section: Discussionmentioning

confidence: 83%

Section: Discussionmentioning

confidence: 99%

Section: Plasmid Vectorsmentioning

confidence: 99%

Section: Plasmid Vectorsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 3 more Smart Citations

Expression profiles of interferon-related genes in cells infected with influenza A viruses or transiently transfected with plasmids encoding viral RNA polymerase

Çağlayan¹,

Turan²

2021

Turk J Biol

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An in silico prediction of interaction models of influenza A virus PA and human C14orf166 protein from yeast‐two‐hybrid screening data

Çağlayan

Turan

2023

Proteins

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The human C14orf166 protein, also known as RNA transcription, translation, and transport factor, shows positive modulatory activity on the cellular RNA polymerase II enzyme. This protein is a component of the tRNA‐splicing ligase complex and is involved in RNA metabolism. It also functions in the nucleo‐cytoplasmic transport of RNA molecules. The C14orf166 protein has been reported to be associated with some types of cancer. It has been shown that the C14orf166 protein binds to the influenza A virus RNA polymerase PA subunit and has a stimulating effect on viral replication. In this study, candidate interactor proteins for influenza A virus PA protein were screened with a Y2H assay using HEK293 Matchmaker cDNA. The C14orf166 protein fragments in different sizes were found to interact with the PA. The three‐dimensional structures of the viral PA and C14orf166 proteins interacting with the PA were generated using the I‐TASSER algorithm. The interaction models between these proteins were predicted with the ClusPro protein docking algorithm and analyzed with PyMol software. The results revealed that the carboxy‐terminal end of the C14orf166 protein is involved in this interaction, and it is highly possible that it binds to the carboxy‐terminal of the PA protein. Although amino acid residues in the interaction area of the PA protein with the C14orf166 showed distribution from 450th to 700th position, the intense interaction region was revealed to be at amino acid positions 610–630.

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Human sorting nexin 2 protein interacts with Influenza A virus PA protein and has a negative regulatory effect on the virus replication

et al. 2021

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Biochemical characterization of avian influenza viral polymerase containing PA or PB2 subunit from human influenza A virus

Cited by 8 publications

References 30 publications

Expression profiles of interferon-related genes in cells infected with influenza A viruses or transiently transfected with plasmids encoding viral RNA polymerase

Expression profiles of interferon-related genes in cells infected with influenza A viruses or transiently transfected with plasmids encoding viral RNA polymerase

An in silico prediction of interaction models of influenza A virus PA and human C14orf166 protein from yeast‐two‐hybrid screening data

Human sorting nexin 2 protein interacts with Influenza A virus PA protein and has a negative regulatory effect on the virus replication

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