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Acid phosphatase 2 (ACP2) is required for membrane fusion during influenza virus entry.

ABSTRACT: Influenza viruses exploit host factors to successfully replicate in infected cells. Using small interfering RNA (siRNA) technology, we identified six human genes required for influenza A virus (IAV) replication. Here we focused on the role of acid phosphatase 2 (ACP2), as its knockdown showed the greatest inhibition of IAV replication. In IAV-infected cells, depletion of ACP2 resulted in a significant reduction in the expression of viral proteins and mRNA, and led to the attenuation of virus multi-cycle growth. ACP2 knockdown also decreased replication of seasonal influenza A and B viruses and avian IAVs of the H7 subtype. Interestingly, ACP2 depletion had no effect on the replication of Ebola or hepatitis C virus. Because ACP2 is known to be a lysosomal acid phosphatase, we assessed the role of ACP2 in influenza virus entry. While neither binding of the viral particle to the cell surface nor endosomal acidification was affected in ACP2-depleted cells, fusion of the endosomal and viral membranes was impaired. As a result, downstream steps in viral entry were blocked, including nucleocapsid uncoating and nuclear import of viral ribonucleoproteins. Our results established ACP2 as a necessary host factor for regulating the fusion step of influenza virus entry.

SUBMITTER: Lee J

PROVIDER: S-EPMC5341025 | biostudies-literature | 2017 Mar

REPOSITORIES: biostudies-literature

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Acid phosphatase 2 (ACP2) is required for membrane fusion during influenza virus entry.

Lee Jihye J Kim Jinhee J Son Kidong K d'Alexandry d'Orengiani Anne-Laure Pham Humg AP Min Ji-Young JY

Scientific reports 20170308

Influenza viruses exploit host factors to successfully replicate in infected cells. Using small interfering RNA (siRNA) technology, we identified six human genes required for influenza A virus (IAV) replication. Here we focused on the role of acid phosphatase 2 (ACP2), as its knockdown showed the greatest inhibition of IAV replication. In IAV-infected cells, depletion of ACP2 resulted in a significant reduction in the expression of viral proteins and mRNA, and led to the attenuation of virus mul ...[more]

PMID: 28272419

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Project description:Influenza A virus (IAV) entry is a multistep process that requires the interaction of the virus with numerous host factors. In this study, we demonstrate that prolidase (PEPD) is a cellular factor required by IAV for successful entry into target cells. PEPD was selected as a candidate during an entry screen performed on nonvalidated primary hits from previously published genome-wide small interfering RNA (siRNA) screens. siRNA-mediated depletion of PEPD resulted in the decreased growth of IAV during mono- and multicycle growth. This growth defect was independent of cell type or virus strain. Furthermore, IAV restriction was apparent as early as 3 h postinfection, and experiments in the absence of protein biosynthesis revealed that the nuclear import of viral ribonucleoprotein complexes (vRNPs) was already blocked in the absence of PEPD. These results led us to investigate which step during entry was affected. Receptor expression, IAV attachment, or IAV internalization was not dependent on the presence of PEPD. However, when looking at the distribution of incoming IAV particles in PEPD-knockdown cells, we found a localization pattern that differed from that in control cells: IAV mostly localized to the cell periphery, and consequently, viral particles displayed reduced colocalization with early and late endosome markers and fusion between viral and endosomal membranes was strongly reduced. Finally, experiments using a competitive inhibitor of PEPD catalytic activity suggested that the enzymatic function of the dipeptidase is required for its proviral effect on IAV entry. In sum, this study establishes PEPD as a novel entry factor required for early endosomal trafficking of IAV.Influenza A virus (IAV) continues to be a constant threat to public health. As IAV relies on its host cell for replication, the identification of host factors required by the virus is of importance. First, such studies often reveal novel functions of cellular factors and can extend our knowledge of cellular processes. Second, we can further our understanding of processes that are required for the entry of IAV into target cells. Third, the identification of host factors that contribute to IAV entry will increase the number of potential targets for the development of novel antiviral drugs that are of urgent need. Our study identifies prolidase (PEPD) to be a novel entry factor required by IAV for correct routing within the endosomal compartment following virus internalization. Thereby, we link PEPD, which has been shown to play a role during collagen recycling and growth factor signaling, to early events of viral infection.

Project description:Herpesviruses-ubiquitous pathogens that cause persistent infections-have some of the most complex cell entry mechanisms. Entry of the prototypical herpes simplex virus 1 (HSV-1) requires coordinated efforts of 4 glycoproteins, gB, gD, gH, and gL. The current model posits that the glycoproteins do not interact before receptor engagement and that binding of gD to its receptor causes a "cascade" of sequential pairwise interactions, first activating the gH/gL complex and subsequently activating gB, the viral fusogen. But how these glycoproteins interact remains unresolved. Here, using a quantitative split-luciferase approach, we show that pairwise HSV-1 glycoprotein complexes form before fusion, interact at a steady level throughout fusion, and do not depend on the presence of the cellular receptor. Based on our findings, we propose a revised "conformational cascade" model of HSV-1 entry. We hypothesize that all 4 glycoproteins assemble into a complex before fusion, with gH/gL positioned between gD and gB. Once gD binds to a cognate receptor, the proximity of the glycoproteins within this complex allows for efficient transmission of the activating signal from the receptor-activated gD to gH/gL to gB through sequential conformational changes, ultimately triggering the fusogenic refolding of gB. Our results also highlight previously unappreciated contributions of the transmembrane and cytoplasmic domains to glycoprotein interactions and fusion. Similar principles could be at play in other multicomponent viral entry systems, and the split-luciferase approach used here is a powerful tool for investigating protein-protein interactions in these and a variety of other systems. IMPORTANCE Herpes simplex virus 1 (HSV-1) infects the majority of humans for life and can cause diseases ranging from painful sores to deadly brain inflammation. No vaccines or curative treatments currently exist. HSV-1 infection of target cells requires coordinated efforts of four viral glycoproteins. But how these glycoproteins interact remains unclear. Using a quantitative protein interaction assay, we found that HSV-1 glycoproteins form receptor-independent complexes and interact at a steady level. We propose that the 4 proteins form a complex, which could facilitate transmission of the entry-triggering signal from the receptor-binding component to the membrane fusogen component through sequential conformational changes. Similar principles could be applicable across other multicomponent protein systems. A revised model of HSV-1 entry could facilitate the development of therapeutics targeting this process.

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Acid phosphatase 2 (ACP2) is required for membrane fusion during influenza virus entry.

Publications

Acid phosphatase 2 (ACP2) is required for membrane fusion during influenza virus entry.

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