After binding to its cell surface receptor ganglioside GM1, simian virus 40 (SV40) is endocytosed by lipid raft-mediated endocytosis and slowly transported to the endoplasmic reticulum, where partial uncoating occurs. We analyzed the intracellular pathway taken by the virus in HeLa and CV-1 cells by using a targeted small interfering RNA (siRNA) silencing screen, electron microscopy, and live-cell imaging as well as by testing a variety of cellular inhibitors and other perturbants. We found that the virus entered early endosomes, late endosomes, and probably endolysosomes before reaching the endoplasmic reticulum and that this pathway was part of the infectious route. The virus was especially sensitive to a variety of perturbations that inhibited endosome acidification and maturation. Contrary to our previous models, which postulated the passage of the virus through caveolin-rich organelles that we called caveosomes, we conclude that SV40 depends on the classical endocytic pathway for infectious entry.To enter their host cells, the majority of animal viruses take advantage of endocytic mechanisms offered by the cell (23, 39). Penetration into the cytosol usually occurs from endosomes, often triggered by the low lumenal pH. However, there are viruses that deviate from this standard itinerary. These viruses include members of the polyomavirus family, such as mouse polyomavirus (mPy) and simian virus 40 (SV40). These viruses are nonenveloped DNA viruses that replicate in the nucleus. The interest and importance of this virus family are rapidly growing with the increasing number of human polyomaviruses identified. The most recently discovered human pathogens include KI polyomavirus (KIPyV), WU polyomavirus (WUPyV), and Merkel cell polyomavirus (MCPyV) (1,17,20). MCPyV is associated with the aggressive neuroendocrine skin cancer Merkel cell carcinoma.Most polyomaviruses bind to gangliosides on the cell surface and are internalized into small tight-fitting vesicles devoid of a clathrin coat (26,28,30,34,55,59). Instead of using endosomes for penetration, they travel to the lumen of the endoplasmic reticulum (ER), in which they are activated by lumenal thiol oxidoreductases and chaperones before penetrating into the cytosol or possibly directly into the nucleoplasm (28,37,46,53).In this study, we focus on SV40, a virus that binds to GM1 and is internalized via caveola/lipid raft-dependent endocytic mechanisms (2,11,45,57,59). Some virus particles are endocytosed via caveolae, and others enter through a parallel clathrin-and caveolin-independent mechanism. Uptake is slow and nonsynchronous, with transfer into the ER and penetration through the ER membrane occurring several hours after the initial endocytosis (53).Exactly where SV40 spends the intervening hours is not clear. Unlike ER-targeted bacterial toxins such as cholera toxin and Shiga toxin, the virus particles are not observed in the trans-Golgi network or the cisternae of the Golgi complex. Some of them are seen in early endosomes (EEs) by confocal and electron ...
