EFFECT OF BAFILOMYCIN A1 AND NOCODAZOLE ON ENDOCYTIC TRANSPORT IN HELA-CELLS - IMPLICATIONS FOR VIRAL UNCOATING AND INFECTION

Bafilomycin Al (baf), a specific inhibitor of vacuolar proton ATPases, is commonly employed to demonstrate the requirement of low endosomal pH for viral uncoating. However, in certain cell types baf also affect s the transport of endocytosed material from early to late endocytic c ompartments. To characterize the endocytic route in HeLa cells that ar e frequently used to study early events in viral infection, we used S- 35-labeled human rhinovirus serotype 2 (HRV2) together with various fl uid-phase markers. These virions are taken up via receptor-mediated en docytosis and undergo a conformational change to C-antigenic particles at a pH of <5.6, resulting in release of the genomic RNA and ultimate ly in infection (E. Prchla, E. Kuechler, D. Blaas, and R. Fuchs, J. Vi rol. 68:3713-3723, 1994). As revealed by fluorescence microscopy and s ubcellular fractionation of microsomes by free-flow electrophoresis (F FE), baf arrests the transport of all markers in early endosomes. In c ontrast, the microtubule-disrupting agent nocodazole was found to inhi bit transport by accumulating marker in endosomal carrier vesicles (EC V), a compartment intermediate between early and late endosomes. Accor dingly, lysosomal degradation of HRV2 was suppressed, whereas its conf ormational change and infectivity remained unaffected by this drug. An alysis of the subcellular distribution of HRV2 and fluid-phase markers in the presence of nocodazole by FFE revealed no difference from the control incubation in the absence of nocodazole. ECV and late endosome s thus have identical electrophoretic mobilities, and intraluminal pHs of <5.6 and allow uncoating of HRV2. As bafilomycin not only dissipat es the low endosomal pH but also blocks transport from early to late e ndosomes in HeLa cells, its inhibitory effect on viral infection could in part also be attributed to trapping of virus in early endosomes wh ich might lack components essential for uncoating. Consequently, inhib ition of viral uncoating by bafilomycin cannot be taken to indicate a low pH requirement only.