Autophagy Promotes Replication of Influenza A Virus In Vitro

Abstract

Influenza A virus (IAV) infection could induce autophagosome accumulation. However, the impact of the autophagy machinery on IAV infection remains controversial. Here, we showed that induction of cellular autophagy by starvation or rapamycin treatment increases progeny virus production, while disruption of autophagy using a small interfering RNA (siRNA) and pharmacological inhibitor reduces progeny virus production. Further studies revealed that alteration of autophagy significantly affects the early stages of the virus life cycle or viral RNA synthesis. Importantly, we demonstrated that overexpression of both the IAV M2 and NP proteins alone leads to the lipidation of LC3 to LC3-II and a redistribution of LC3 from the cytosol to punctate vesicles indicative of authentic autophagosomes. Intriguingly, both M2 and NP colocalize and interact with LC3 puncta during M2 or NP transfection alone and IAV infection, leading to an increase in viral ribonucleoprotein (vRNP) export and infectious viral particle formation, which indicates that the IAV-host autophagy interaction plays a critical role in regulating IAV replication. We showed that NP and M2 induce the AKT-mTOR-dependent autophagy pathway and an increase in HSP90AA1 expression. Finally, our studies provided evidence that IAV replication needs an autophagy pathway to enhance viral RNA synthesis via the interaction of PB2 and HSP90AA1 by modulating HSP90AA1 expression and the AKT-mTOR signaling pathway in host cells. Collectively, our studies uncover a new mechanism that NP-and M2-mediated autophagy functions in different stages of virus replication in the pathogenicity of influenza A virus. IMPORTANCE Autophagy impacts the replication cycle of many viruses. However, the role of the autophagy machinery in IAV replication remains unclear. Therefore, we explored the detailed mechanisms utilized by IAV to promote its replication. We demonstrated that IAV NP-and M2-mediated autophagy promotes IAV replication by regulating the AKT-mTOR signaling pathway and HSP90AA1 expression. The interaction of PB2 and HSP90AA1 results in the increase of viral RNA synthesis first; subsequently the binding of NP to LC3 favors vRNP export, and later the interaction of M2 and LC3 leads to an increase in the production of infectious viral particles, thus accelerating viral progeny production. These findings improve our understanding of IAV pathogenicity in host cells.

Activation of the PI3K-AKT Pathway by Old World Alphaviruses

“…Some viruses, such as ZIKV and rotavirus require the process early in infection [43][44][45][46], whereas this is detrimental for others. Conversely, some viruses, such as influenza A virus induce autophagy late after infection to increase replication [47]. Finally, there are viruses in which autophagy does not appear to influence replication.…”

Escaping the Lion’s Den: redirecting autophagy for unconventional release and spread of viruses

“…Although LC3 is not integrated into the budding IAV virions, it appears that the presence of LC3 at the plasma membrane is able to modulate viral envelope composition by remobilizing appropriate resources to the cell surface during budding, to assist in virion stability via unknown mechanisms. Apart from these mechanisms, Wang et al [47] suggested another possible molecular mechanism for M2 to assist IAV in evading the autophagy machinery, by involving the host factor heat shock protein 90a (HSP90AA1). HSP90AA1 is an essential chaperone that maintains PI3K/AKT activity by binding to AKT and interacting with PB2 to facilitate viral RNA synthesis, viral ribonucleoprotein (vRNP) nuclear import, and viral assembly [48][49][50].…”

“…HSP90AA1 is an essential chaperone that maintains PI3K/AKT activity by binding to AKT and interacting with PB2 to facilitate viral RNA synthesis, viral ribonucleoprotein (vRNP) nuclear import, and viral assembly [48][49][50]. The authors found that in addition to colocalizing and interacting with LC3 in the perinuclear region, which results in enhanced vRNP export and infectious virion formation, M2 and NP also increased HSP90AA1 expression and regulated the AKT-mTOR pathway to mediate autophagosome accumulation [47]. Therefore, at early stages of infection, NP and M2 induced autophagy to facilitate viral RNA synthesis via increased levels of HSP90AA1 that bind to PB2 and by enhancing vRNP export through binding of NP to LC3.…”

High Expression of IL-36γ in Influenza Patients Regulates Interferon Signaling Pathway and Causes Programmed Cell Death During Influenza Virus Infection

“…only presents in late stages (Supplemental Figure 2C). Wang et al reported that influenza virus can induce autophagosome accumulation and autophagy promotes replication of Influenza virus in turn (22). Pharmacological inhibition of autophagy could decrease influenza viral yields (23).…”