Influenza A Virus Proteins NS1 and Hemagglutinin Along with M2 Are Involved in Stimulation of Autophagy in Infected Cells

Abstract

The NS1 protein of influenza A virus is known to downregulate apoptosis early in infection in order to support virus replication (O. P. Zhirnov, T. E. Konakova, T. Wolff, and H. D. Klenk, J. Virol. 76: [1617][1618][1619][1620][1621][1622][1623][1624][1625] 2002). In the present study, we analyzed the development of autophagy, another mechanism to protect cells from degradation that depends on NS1 expression. To this end, we compared autophagy in cells infected with wild-type (WT) influenza virus and virus lacking the NS1 gene (delNS1 virus). The results show that in WT-infected cells but not in delNS1 virus-infected cells, synthesis of the autophagy marker LC3-II, the lipidated form of microtubule light chain-associated protein LC3, is stimulated and that LC3-II accumulates in a perinuclear zone enriched with double-layered membrane vesicles characteristic of autophagosomes. Transfection experiments revealed that NS1 expressed alone was unable to upregulate autophagy, whereas hemagglutinin (HA) and M2 were. Proteolytic cleavage of HA increased autophagy. Taken together, these observations indicate that NS1 stimulates autophagy indirectly by upregulating the synthesis of HA and M2. Thus, it appears that NS1, besides downregulating apoptosis, is involved in upregulation of autophagy and that it supports the survival of infected cells by both mechanisms. Autophagy is a mechanism that allows cell survival under stress conditions, such as amino acid starvation, energy deprivation, and virus infection (1). It is a constitutive process that involves recycling of cytosolic proteins and organelles by lysosomal degradation and an immune response by virus protein degradation and extracellular presentation of degraded molecules to immune recognition (2, 3). Autophagy thereby provides nutrients for maintenance and repair of vital functions of the host, including immune defense. Autophagy is mediated by autophagosomes, specialized double-membrane vesicles originating from the Golgi apparatus, endoplasmic reticulum, and mitochondria, and it is regulated by the interaction of more than 30 autophagy-related cellular genes (Atgs) (4). Two stages can be discriminated in the autophagy process: an initial stage of autophagosome formation and a terminal effector stage (maturation) involving fusion of autophagosomes with lysosomes and degradation of the intra-autophagosomal content. Phosphatidylinositol-3-kinase class III (PI3K-III), beclin (Atg6), and LC3 (Atg8) are important factors involved in autophagy (5). Viruses have developed strategies either to counteract autophagy or to employ the autophagic pathway for their own benefit (1, 3).Influenza A virus modulates autophagy (6-8) as well as apoptosis, another pathway regulating survival of infected cells (8). To allow host cell survival and to promote viral replication, apoptosis is downregulated early after infection by the interaction of the PI3K (class I)-AKT complex with the virus protein NS1 (9-12). As a consequence, rapid apoptosis develops in cells infected with influenza vir...

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