The entry mechanisms of viruses into cells are critical for infection and subsequent pathogenesis. For -influenza virus, many of the aspects of the entry pathway under viral control are well understood, but how the virus interacts with the cellular trafficking machinery for internalization and endocytosis remains unclear. The virus is known to bind to a sialic acid-containing receptor and to fuse out of a low-pH intracellular compartment, but how the virus traverses the cell between these two events, and which cellular control mechanisms regulate these events are unknown. Transit through the cellular endocytic pathway is regulated by GTPases such as dynamin and specific Rab proteins. The effects on virus entry of specific inhibition of these molecules will be studied in this proposal. Experiments will be performed both by light microscopy and at the ultra-structural level by electron microscopy. GTPase function will be inhibited by use of dominant-negative mutants and by microinjection of inhibitory antibodies. We have shown that influenza virus entry into cells is inhibited by a specific inhibitor of cellular protein kinase C (PKC). This may represent a key control point for the virus in its route of entry into a host cell. The mechansim of action of the inhibitor and the role of PKC in virus endocytosis will be examined, in relation to the various endocytic pathways of the cell. Influenza virus is known to occur in nature as both a large filamentous form (100 nm by up to 2pm) and a small spherical form (100 nrn diameter). It is the filamentous forms that are thought to be important for infection in the human lung, but a study of the entry mechanism for this morphological form of the virus has never been carried out. Whereas entry of tissue culture-adapted spherical virus occurs via clathrin-coated pits, this would appear to be an inefficient route of entry for large filamentous viruses. Both filamentous and spherical viruses will be isolated and compared for their route of entry, to determine the entry mechanism for the clinically-important filamentous viruses. Influenza remains a major threat to human health. These studies will lead to a more complete understanding of the entry mechanism of the virus and may lead to the development of more effective anti-viral drugs.
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