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.

No abstract

The protease of the porcine endogenous retrovirus (PERV) subtypes A/B and C was recombinantly expressed in Escherichia coli as proteolytically active enzyme and characterized. The PERV Gag precursor was also recombinantly produced and used as the substrate in an in vitro enzyme assay in parallel with synthetic nonapeptide substrates designed according to cleavage site sequences identified in the PERV Gag precursor. The proteases of all PERV subtypes consist of 127 amino acid residues with an M r of 14,000 as revealed by determining the protease N and C termini. The PERV proteases have a high specificity for PERV substrates and do not cleave human immunodeficiency virus (HIV)-specific substrates, nor are they inhibited by specific HIV protease inhibitors. Among the known retroviral proteases, the PERV proteases resemble most closely the protease of the murine leukemia retrovirus.Organ xenotransplantation from pigs may be one possibility to solve the shortage of human organ donors. An unanswered question is, however, the risk of viral infection by porcine endogenous retroviruses (PERVs) upon xenografting (3,5,7,10,11,16). One way to minimize this problem might be the option of designing an effective antiviral chemotherapy against PERV, comparable to that presently employed against human immunodeficiency virus (HIV) infection. Here we have characterized the PERV protease as a target for protease inhibitors (PIs) that might be employed as antiviral agents for chemotherapy.Three main subtypes of PERV, A, B, and C, are known to date (14). All are classical C-type retroviruses (e.g., 1, 2, 15) and are genetically similar but not identical. We have revealed the coding sequences for the proteases of all three PERV subtypes, produced active proteases by recombinant methods in Escherichia coli, and established an in vitro assay system for PERV protease activity.Coding sequence of the PERV protease. For recombinant expression of the PERV protease, the boundaries of its coding sequence had to be revealed. Possible N and C termini of the functional PERV protease coding sequence were predicted by multiple alignment of the translated sequences of gag and pol regions of PERV subtypes A/B (DuxDL3791 [J. Blusch et al., unpublished data]) and C (PERV-MSL [1]) with the protease sequence of Moloney murine leukemia virus (MMLV [8,20]), the phylogenetically closest C-type retrovirus with a known protease protein sequence (Fig. 1).The alignment indicated that the C terminus of the PERV protease seemed to be highly similar to that of the murine leukemia virus protease; the N terminus of PERV protease, however, showed significant differences (Fig. 1). The N terminus of MMLV protease is known to overlap into the C-terminal Gag open reading frame (ORF) by 4 amino acid residues (T L D D) (21). Thus, the gag stop (amber) codon TAG (X in Fig. 1) is part of the protease coding sequences and can be read through by a Gln suppressor tRNA. An alignment of the protease N terminus emerged only in the way shown in Fig. 1; the L*A was provided as the r...

No abstract

No abstract