Prolidase Is Required for Early Trafficking Events during Influenza A Virus Entry

Abstract

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. IMPORTANCEInfluenza 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.

Flavivirus Antagonism of Type I Interferon Signaling Reveals Prolidase as a Regulator of IFNAR1 Surface Expression

“…Further, the observed basal expression of antiviral ISGs may confer some resistance to viral infection, while simultaneously increasing susceptibility to bacterial infections, as recently suggested for two ISGs, oxysterol 25-hydroxycholesterol (Reboldi et al, 2014) and the methyltransferase Setdb2 (Schliehe et al, 2014). PEPD is also required for early trafficking events of influenza virus in endosomes (Pohl et al, 2014) which may explain the lack of severe influenza in PD patients, although PEPD was clearly antiviral in the context of IFN-I-dependent inhibition of TBEV replication. Therefore, the roles of PEPD in innate immunity and virus infection are multi-faceted.…”

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“…During endosome maturation the incoming viruses can gain exclusive exposure to cues that are not available on the plasma membrane or in the cytosol . This is supported by the notion that defects in endosome or macropinosome maturation on the pathway from the plasma membrane to lysosomes inhibits the productive entry of many viruses . Along the same lines, virus entry can be inhibited by perturbations of microtubule‐mediated vesicular traffic, conversion of Rab5 early to Rab7 late endosomes, or the formation of intralumenal vesicles .…”