TY - JOUR
T1 - Nucleoprotein of influenza a virus negatively impacts antiapoptotic protein api5 to enhance e2f1-dependent apoptosis and virus replication
AU - Mayank, A. K.
AU - Sharma, S.
AU - Nailwal, H.
AU - Lal, S. K.
N1 - Funding Information:
Acknowledgements. We would like to thank Dr. Nicholas J Dyson (Harvard Medical School, USA) for providing us pLPC-Flag-API5 plasmid, Dinh Duy Khang (Institute of Biotechnology, Hanoi, Vietnam) for providing the influenza virus (A/Hatay isolate) cDNA, Dr Paul Digard (CDC, USA) for providing us with luciferase reporter assay plasmids, and Dr Vijaya Pandey for critical reviewing of the manuscript and for useful comments on the experimental design. We thank Ms Purnima Kumar for helping us in the confocal experiments and Mr Ravi Kumar for helping us in tissue culture experiments. This study was supported by internal funds from ICGEB, a research grant from the Department of Biotechnology, India. Adarsh Kumar Mayank and Shipra Sharma were supported by a fellowship from Indian Council for Medical Research, New Delhi, India. All virus infections were carried out in Biosafety Lab-2 facilities at ICGEB.
Funding Information:
We would like to thank Dr. Nicholas J Dyson (Harvard Medical School, USA) for providing us pLPC-Flag-API5 plasmid, Dinh Duy Khang (Institute of Biotechnology, Hanoi, Vietnam) for providing the influenza virus (A/Hatay isolate) cDNA, Dr Paul Digard (CDC, USA) for providing us with luciferase reporter assay plasmids, and Dr Vijaya Pandey for critical reviewing of the manuscript and for useful comments on the experimental design. We thank Ms Purnima Kumar for helping us in the confocal experiments and Mr Ravi Kumar for helping us in tissue culture experiments. This study was supported by internal funds from ICGEB, a research grant from the Department of Biotechnology, India. Adarsh Kumar Mayank and Shipra Sharma were supported by a fellowship from Indian Council for Medical Research, New Delhi, India. All virus infections were carried out in Biosafety Lab-2 facilities at ICGEB.
Publisher Copyright:
© 2015, Nature Publishing Group. All rights reserved.
Copyright:
Copyright 2019 Elsevier B.V., All rights reserved.
PY - 2015
Y1 - 2015
N2 - Apoptosis of host cells profoundly influences virus propagation and dissemination, events that are integral to influenza A virus (IAV) pathogenesis. The trigger for activation of apoptosis is regulated by an intricate interplay between cellular and viral proteins, with a strong bearing on IAV replication. Though the knowledge of viral proteins and mechanisms employed by IAV to induce apoptosis has advanced considerably of late, we know relatively little about the repertoire of host factors targeted by viral proteins. Thus, identification of cellular proteins that are hijacked by the virus will help us not only to understand the molecular underpinnings of IAV-induced apoptosis, but also to design future antiviral therapies. Here we show that the nucleoprotein (NP) of IAV directly interacts with and suppresses the expression of API5, a host antiapoptotic protein that antagonizes E2F1-dependent apoptosis. siRNA-mediated depletion of API5, in NP-overexpressed as well as IAV-infected cells, leads to upregulation of apoptotic protease activating factor 1 (APAF1), a downstream modulator of E2F1-mediated apoptosis, and cleavage of caspases 9 and 3, although a reciprocal pattern of these events was observed on ectopic overexpression of API5. In concordance with these observations, annexin V and 7AAD staining assays exhibit downregulation of early and late apoptosis in IAV-infected or NP-transfected cells on overexpression of API5. Most significantly, while overexpression of API5 decreases viral titers, cellular NP protein as well as mRNA levels in IAV-infected A549 cells, silencing of API5 expression causes a steep rise in the same parameters. From the data reported in this manuscript, we propose a proapoptotic role for NP in IAV pathogenesis, whereby it suppresses expression of antiapoptotic factor API5, thus potentiating the E2F1-dependent apoptotic pathway and ensuring viral replication.
AB - Apoptosis of host cells profoundly influences virus propagation and dissemination, events that are integral to influenza A virus (IAV) pathogenesis. The trigger for activation of apoptosis is regulated by an intricate interplay between cellular and viral proteins, with a strong bearing on IAV replication. Though the knowledge of viral proteins and mechanisms employed by IAV to induce apoptosis has advanced considerably of late, we know relatively little about the repertoire of host factors targeted by viral proteins. Thus, identification of cellular proteins that are hijacked by the virus will help us not only to understand the molecular underpinnings of IAV-induced apoptosis, but also to design future antiviral therapies. Here we show that the nucleoprotein (NP) of IAV directly interacts with and suppresses the expression of API5, a host antiapoptotic protein that antagonizes E2F1-dependent apoptosis. siRNA-mediated depletion of API5, in NP-overexpressed as well as IAV-infected cells, leads to upregulation of apoptotic protease activating factor 1 (APAF1), a downstream modulator of E2F1-mediated apoptosis, and cleavage of caspases 9 and 3, although a reciprocal pattern of these events was observed on ectopic overexpression of API5. In concordance with these observations, annexin V and 7AAD staining assays exhibit downregulation of early and late apoptosis in IAV-infected or NP-transfected cells on overexpression of API5. Most significantly, while overexpression of API5 decreases viral titers, cellular NP protein as well as mRNA levels in IAV-infected A549 cells, silencing of API5 expression causes a steep rise in the same parameters. From the data reported in this manuscript, we propose a proapoptotic role for NP in IAV pathogenesis, whereby it suppresses expression of antiapoptotic factor API5, thus potentiating the E2F1-dependent apoptotic pathway and ensuring viral replication.
UR - http://www.scopus.com/inward/record.url?scp=85006016518&partnerID=8YFLogxK
U2 - 10.1038/cddis.2015.360
DO - 10.1038/cddis.2015.360
M3 - Article
C2 - 26673663
AN - SCOPUS:85006016518
VL - 6
JO - Cell Death & Disease
JF - Cell Death & Disease
SN - 2041-4889
IS - 12
M1 - e2018
ER -
