| Despite the fact that Ebola virus encodes only seven structural proteins, the virus replicates rapidly, infects nearly all tissues, and induces systemic decimation of immune system function. To begin to more fully understand how a limited set of proteins can cause such complex disease processes, research was undertaken to determine the functions(s) of these polypeptides. Two phases of the viral life cycle, replication and particle formation, were chosen for analysis.; The process of virus budding was examined in detail. It was discovered that VP40 is capable of mediating its own release from mammalian cells in virus-like particle. A small, conserved PPXY motif at the protein's N-terminus was shown to be important in the budding process. In addition, VP40 which lacks this motif, and VP40 containing C-terminal truncations of up to 150 amino acids, were shown to inhibit VP40-induced particle formation in a dominant-negative manner when coexpressed with wild-type VP40. Flotation gradient experiments revealed that VP40 binds tightly to cellular membranes, Triton X-114 phase-partitioning analysis indicated that VP40 is a peripheral membrane protein.; There are significant differences in RNA synthesis between Ebola virus and a closely related family member, Marburg virus. Rapid screening for small molecules that affect nucleocapsid activity may prove useful in characterizing this complex and, in addition, could isolate novel drug candidates with inhibitory effects on Ebola virus replication. To make screening for anti-Zaire Ebola virus compounds practical in biosafety level 2 conditions, a virus-like minigenome system expressing firefly luciferase was optimized for screening. A Z' factor of 0.64 was obtained with a 96-well format and ∼1000 compounds were surveyed for their ability to inhibit minigenome-based reporter gene expression, seven were found that reduced luminescence by >75% while showing little or no cytotoxic effects.; Nucleocapsid complex formation and activity was also examined directly. The N-terminus 500 of L was shown to interact with VP35 as well as with itself. Deletion analysis within L (2-500) revealed two sites, residues 2-125 and residues 351-375, important for minigenome transcription. |
