1.1.1. Adenovirus

Adenoviruses (Ad) are among the best-studied non-enveloped viruses for membrane penetration. Their ~90 nm capsids enter target cells following receptor binding through clathrin-mediated endocytosis. Most Ads rapidly escape the endosomal compartment to avoid lysosomal sorting and use cytosolic motors to reach the nucleus for replication. Adenovirus endosome penetration was shown through co-uptake of non-membrane permeable substrates [10,11]. Observations made with the thermosensitive mutant virus Ad2/5 *ts1* showed that membrane penetration is an essential step in the infection process [12–14]. This *ts1* mutant fails to package the adenoviral protease into the capsid. This failure prevents capsid maturation by cleavage of precursor proteins, resulting in hyperstable and non-infectious particles [15–17]. Adenovirus capsid maturation is required for the release of the internal membrane lytic capsid protein VI upon virus entry. What triggers protein VI release from the capsid is not known, but it may involve disassembly cues during initial cell binding [14,18–21]. Protein VI encodes an amphipathic helix with membrane binding and lytic activity [13,22] and mutations in the amphipathic helix strongly reduced both its membrane lytic activity and viral infectivity [23]. Adenovirus inflicted membrane damage creates openings large enough for cytosolic delivery of 70 kDa dextrans or 25 nm parvovirus particles [10,11]. Electron microscopy images of Ad particles in partially disrupted endosomes show large physical openings in the endosome plugged by the virus [3]. High-resolution fluorescence microscopy images sugges<sup>t</sup> localized protein VI release from the capsid at the membrane damage site [24]. Ceramides have been proposed to increase protein VI membrane affinity, showing the importance of the local lipid composition at the membrane penetration site [19]. Protein VI release also plays a role in virus escape from endosomes by counteracting cellular autophagy, which is normally mounted in response to membrane damage as detailed in Section 2 [3].

**Figure 1.** Virus-inflicted membrane damage. After binding to cell-surface receptors, viruses are internalized through endocytosis. Once in the endosome, adenovirus capsid undergoes partial disassembly and releases protein VI. The increase of ceramide concentration enhances the binding of protein VI to the endosomal membrane and its subsequent rupture. Polyomavirus-containing endosomes are targeted to the endoplasmic reticulum (ER) where the virus undergoes conformational changes to penetrate the ER-membrane and escape to the cytosol. Parvovirus and reovirus require a pH drop and the action of endosomal cathepsins to induce conformational rearrangements, disrupt the endosome, and reach the cytosol. After endocytosis and conformational changes, picornaviruses rely on a cellular lipid-modifying enzyme (PLA2G16) to facilitate the translocation of its genome via selective pores across the endosomal membrane. See Section 1 for further details. Abbreviations: ER, endoplasmic reticulum; Hsc70, Heat shock cognate 71 kDa protein; Hsp105, Heat shock protein 105 kDa; PLA2G16, phospholipase A2 group XVI.
