A Capsid-Encoded PPxY-Motif Facilitates Adenovirus Entry

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.

Endosomophagy clears disrupted early endosomes but not virus particles during virus entry into cells     

“…These results were in agreement with earlier notions that HAdV escapes quickly from endosomes and is transported by dynein/dynactin mediated processes toward the nucleus [50] [51] [52]. Protein VI on the other hand likely remains membrane associated, and may be sorted separately for degradation [26] [53]. Remarkably, visual inspection of the images suggested that the number of HAdV-C5 particles, or protein VI puncta, exceeded the number of mCherry-Gal3 foci (Fig.…”

The Amphipathic Helix of Adenovirus Capsid Protein VI Contributes to Penton Release and Postentry Sorting

“…Next, the membrane-lytic capacity upon entry of the three viruses was determined. Cells were incubated with 150, 1,500, or 15,000 pp/c in the presence of the non-cell-permeable translation inhibitor ␣-sarcin (18,26). The release of ␣-sarcin into the cytosol following endocytic uptake is entirely dependent on the membrane-lytic activity of the virus.…”

“…Bacterial artificial chromosome (BAC) mutagenesis was used to generate recombinant adenoviruses following a protocol described previously (26,39), with the following modifications: bp 1 to 3513 of the HAdV-C5 genome, including the left inverted terminal repeat (ITR) and the whole E1 region, was replaced by a flippase recognition target (FRT) site and maintained as a BAC to perform lambda red recombination essentially as described in reference 39. Briefly, BAC mutagenesis is achieved by first replacing the target sequence in the viral genome with a selectable marker (GalK/Amp) through homologous recombination.…”

“…Upon entry, HAdV-C5-PVI-L40Q virions were found to associate with the endosomal compartment for prolonged times compared to wild-type (wt) virions (25). It was shown that in intact virions, protein VI remains inaccessible to protein VI-specific antibodies (26). Following uptake into cells, protein VI is liberated and thus becomes accessible to antibody staining, providing direct evidence for entry-dependent disassembly and protein VI release (26).…”

“…It was shown that in intact virions, protein VI remains inaccessible to protein VI-specific antibodies (26). Following uptake into cells, protein VI is liberated and thus becomes accessible to antibody staining, providing direct evidence for entry-dependent disassembly and protein VI release (26). A recent study using cellular markers of membrane damage provided the first experimental system to show in vivo visualization of the membrane lysis step of AdV entry, demonstrating that membrane damage coincided with protein VI deployment from the AdV capsid (16,22,23).…”