A Capsid-Encoded PPxY-Motif Facilitates Adenovirus Entry

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Abstract
Viruses use cellular machinery to enter and infect cells. In this study we address the cell entry mechanisms of nonenveloped adenoviruses (Ads). We show that protein VI, an internal capsid protein, is rapidly exposed after cell surface attachment and internalization and remains partially associated with the capsid during intracellular transport. We found that a PPxY motif within protein VI recruits Nedd4 E3 ubiquitin ligases to bind and ubiquitylate protein VI. We further show that this PPxY motif is involved in rapid, microtubule-dependent intracellular movement of protein VI. Ads with a mutated PPxY motif can efficiently escape endosomes but are defective in microtubule-dependent trafficking toward the nucleus. Likewise, depletion of Nedd4 ligases attenuates nuclear accumulation of incoming Ad particles and infection. Our data provide the first evidence that virus-encoded PPxY motifs are required during virus entry, which may be of significance for several other pathogens. Viruses exploit cellular functions during entry and exit of cells. To redirect cellular functions for their own purpose, viruses encode high-affinity binding sites for key-cellular factors. One such domain is the PPxY motif, which is present in structural proteins of several, mainly enveloped viruses. This motif binds to ubiquitin ligases of the Nedd4 family and recruits their function to sites of virus budding from cells. Here we show that adenoviruses also encode a PPxY motif in the internal structural protein VI and that the PPxY motif has an unprecedented function in virus entry. Adenoviruses with mutations in the protein VI PPxY motif undergo normal endosomal uptake and membrane penetration but have reduced infectivity, altered intracellular targeting and lack efficient gene-delivery. We also find that protein VI is ubiquitylated by Nedd4 ligases in a PPxY dependent manner following partial capsid disassembly and displays rapid intracellular movement. Depletion of Nedd4 ligases also alters virus movement within cells during entry and reduces viral infectivity. Given that PPxY motifs are important for virus exit our findings might have uncovered an additional function for PPxY motifs in virus entry, potentially expanding the significance of PPxY motifs and functionally related domains for viral replication.