While clathrin-mediated infection by many enveloped viruses has been well-characterized, the infectious entry of the non-enveloped viruses has received less attention. Simian Virus 40 (SV40), is a non-enveloped oncogenic virus which must pass from the plasma membrane to the nucleus for productive infection. Early electron microscopic studies revealed uptake of SV40 within close-fitting uncoated membranes later shown to be caveolin-rich caveolae. SV40 virions were also shown to accumulate in cisternae of the endoplasmic reticulum (ER) but no other morphological intermediates of infection were identified. After arrival at the ER, viral translocation into the cytoplasm and nuclear entry via nuclear pore complexes is required for productive infection. Endocytosis of many bacterial toxins also results in their delivery to the ER followed by escape into the cytoplasm, the site of toxin action. One of these, cholera toxin, has been studied extensively and is found to follow an endocytic route involving transit through endosomes and the Golgi compartment. In this case, retrograde transport of the toxin from Golgi compartment to ER occurs by COPI-dependent vesicle traffic. The similarities in intracellular targeting of the toxin and SV40 are suggestive of a common route of endocytic uptake to the ER and subsequent delivery to the cytoplasm. This thesis presents a comparative study of the two pathways, characterizing the effects of previously identified inhibitors of cholera toxin uptake and toxicity on SV40 infection.
Chapter 3 investigates the initial entry of both cholera toxin and SV40, examining the dependence of both events on the GTPase, dynamin, and addressing the question of SV40 delivery to early endosomes subsequent to virus internalization. Both toxin internalization to early endosomes and SV40 infection were found to be inhibited by microinjection of antibodies to dynamin. However, treatments such as exposure to the drug, Brefeldin A, and incubation at 20°C, that accumulate cholera toxin in EEAl- positive endosomes by inhibiting traffic out of this compartment, appeared to elicit a potent inhibition of initial endocytosis of the virus. This was indicative of distinct mechanisms of entry of SV40 and cholera toxin, despite the dependence of both pathways on dynamin function.
Chapter 4 addresses the question of virus transit through the Golgi compartment en route to the ER. A number of inhibitors of COPI-dependent retrograde transport (Brefeldin A, a GTP-restricted Arfl mutant and microinjected antibodies to ßC0P) were found to greatly abrogate SV40 infection as well as inhibiting Golgi-to-ER transport of cholera toxin. While these findings could be suggestive of Arfl/COPI-dependent retrograde trafficking steps in SV40 infectious entry, they may also indicate a dependence of SV40 infection on a functional secretory pathway since ER-to-Golgi transport was also indirectly inhibited by these agents. Expression of the GTP-restricted form of Sari, the GTPase responsible for COPII-dependent anterograde transport, also inhibited both retrograde transport of cholera toxin and SV40 infection, confirming the interdependence of retrograde and anterograde transport between the Golgi and ER. In addition to this phenomenon, expression of the Arf1 mutant also selectively affected several endocytic pathways which were not similarly affected by the Sari mutant. Although Arf1 is not known to function in endocytosis at the plasma membrane, indirect inhibition of the function of other plasma membrane Arf proteins by GTP-restricted Arf1 remains an intriguing possibility.
In Chapter 5 of this thesis, a novel inhibitor of cholera toxin previously shown to inhibit late steps in the toxic pathway is similarly shown to inhibit late steps in SV40 infection. Although actions of the benzyloxycarbonyl dipeptide, Cbz-gly-phe-NH2, described in previous studies of the drug include both inhibition of metalloendoproteases and independently, depletion of intracellular Ca2+stores, no evidence for the latter was observed in our studies. Rather, inhibition of metalloendoproteases appeared to be the mechanism of action since an unrelated metalloendoprotease inhibitor, Cbz-L-Phe, having none of the Ca2+-disrupting effects of Cbz-gly-phe-NH2, similarly inhibited Golgi-to-ER delivery of cholera toxin and late steps in SV40 infection. These findings suggest an important role for metalloendoproteases in latter steps of SV40 infectious entry and identify Cbz-gly-phe- NH2 as a promising tool for dissecting the molecular mechanisms involved in this process.
In summary, the work presented in this thesis identifies novel inhibitors of the SV40 infectious pathway which also interfere with toxic internalization of cholera toxin, and demonstrates either similar or differential mechanisms of inhibition of the two pathways by each inhibitor. Taken together, these studies challenge pre-existing dogma by suggesting distinct mechanisms of initial entry for cholera toxin and SV40 but indicate similar processes in the latter steps of both pathways mediating delivery to, and exit from, the ER.