How non-enveloped viruses overcome host cell membranes is poorly understood. Here, we show that after endocytosis and transport to the endoplasmic reticulum (ER), but before crossing the ER membrane to the cytosol, incoming simian virus 40 particles are structurally remodelled leading to exposure of the amino-terminal sequence of the minor viral protein VP2. These hydrophobic sequences anchor the virus to membranes. A negatively charged residue, Glu 17, in the α-helical, membrane-embedded peptide is essential for infection, most likely by introducing an 'irregularity' recognized by the ER-associated degradation (ERAD) system for membrane proteins. Using a siRNA-mediated screen, the lumenal chaperone BiP and the ER-membrane protein BAP31 (both involved in ERAD) were identified as being essential for infection. They co-localized with the virus in discrete foci and promoted its ER-to-cytosol dislocation. Virus-like particles devoid of VP2 failed to cross the membrane. The results demonstrated that ERAD-factors assist virus transport across the ER membrane.

During cell entry, non-enveloped viruses undergo partial uncoating to expose membrane lytic proteins for gaining access to the cytoplasm. We report that adenovirus uses membrane piercing to induce and hijack cellular wound removal processes that facilitate further membrane disruption and infection. Incoming adenovirus stimulates calcium influx and lysosomal exocytosis, a membrane repair mechanism resulting in release of acid sphingomyelinase (ASMase) and degradation of sphingomyelin to ceramide lipids in the plasma membrane. Lysosomal exocytosis is triggered by small plasma membrane lesions induced by the viral membrane lytic protein-VI, which is exposed upon mechanical cues from virus receptors, followed by virus endocytosis into leaky endosomes. Chemical inhibition or RNA interference of ASMase slows virus endocytosis, inhibits virus escape to the cytosol, and reduces infection. Ceramide enhances binding of protein-VI to lipid membranes and protein-VI-induced membrane rupture. Thus, adenovirus uses a positive feedback loop between virus uncoating and lipid signaling for efficient membrane penetration.

Endocytosis is the most prevalent entry port for viruses into cells, but viruses must escape from the lumen of endosomes to ensure that viral genomes reach a site for replication and progeny formation. Endosomal escape also helps viruses bypass endolysosomal degradation and presentation to certain Toll-like intrinsic immunity receptors. The mechanisms for cytosolic delivery of nonenveloped viruses or nucleocapsids from enveloped viruses are poorly understood, in part because no quantitative assays are readily available which directly measure the penetration of viruses into the cytosol. Following uptake by clathrin-mediated endocytosis or macropinocytosis, the nonenveloped adenoviruses penetrate from endosomes to the cytosol, and they traffic with cellular motors on microtubules to the nucleus for replication. In this report, we present a novel single-cell imaging assay which quantitatively measures individual cytosolic viruses and distinguishes them from endosomal viruses or viruses at the plasma membrane. Using this assay, we showed that the penetration of human adenoviruses of the species C and B occurs rapidly after virus uptake. Efficient penetration does not require acidic pH in endosomes. This assay is versatile and can be adapted to other adenoviruses and members of other nonenveloped and enveloped virus families.