Markers for trans-Golgi membranes and the intermediate compartment localize to induced membranes with distinct replication functions in flavivirus-infected cells

Replication of the flavivirus Kunjin virus is associated with virus-induced membrane structures within the cytoplasm of infected cells; these membrane s appear as packets of vesicles associated with the sites of viral RNA synt hesis and as convoluted membranes (CM) and paracrystalline arrays (PC) cont aining the components of the virus-specified protease (E. G. Westaway, J. M . Mackenzie, M. T. Kenney, M. K. Jones, and A. A. Khromykh, J. Virol. 71:66 50-6661, 1997). To determine the cellular origins of these membrane structu res, we compared the immunolabelling patterns of several cell markers in re lation to these sites by immunofluorescence and immunoelectron microscopy. A marker for the trans-Golgi membranes and the trans-Golgi network, 1,4-gal actosyltransferase (GalT), was redistributed to large foci in the cytoplasm of Kunjin virus-infected cells, partially coincident with immunofluorescen t foci associated with the putative sites of viral RNA synthesis. As determ ined by immunoelectron microscopy, the induced vesicle packets contained Ga lT, whereas the CM and PC contained a specific protein marker for the inter mediate compartment (ERGIC53). A further indicator of the role of cellular organelles in their biogenesis was the observation that the Golgi apparatus -disrupting agent brefeldin A prevented further development of immunofluore scent foci of induced membranes if added before the end of the latent perio d but that once formed, these membrane foci were resistant to brefeldin A d ispersion. Reticulum membranes emanating from the induced CM and PC were al so labelled with the rough endoplasmic reticulum marker anti-protein disulf ide isomerase and were obviously redistributed during infection. This is th e first report identifying trans-Golgi membranes and the intermediate compa rtment as the apparent sources of the flavivirus-induced membranes involved in events of replication.