The molecular interaction between ML336 and the VEEV non-structural proteins

Venezuelan Equine Encephalitis Virus (VEEV) is a positive sense RNA virus in the family Togaviridae. VEEV circulates in the Americas, causing occasional large scale epidemics. Our group has previously discovered and described the anti-VEEV compound ML336. We found that ML336 inhibits viral RNA synthesis during infection. This RNA synthesis inhibition is highly specific for VEEV, and ML336 has no effect on the closely related chikungunya virus, or on cellular RNA synthesis. We also found that this activity was maintained in a cell-free viral RNA synthesis system, supporting our hypothesis that ML336 is a direct acting antiviral compound. We recently discovered that treatment with ML336 reduces the amount of double stranded RNA present in infected cells. This reduction supports that ML336 is interfering with the synthesis of viral RNA. This was measured qualitatively with microscopy, and quantitatively with flow cytometry. We have also reported that resistant viral mutants emerge when grown in the presence of inhibitory compounds and these mutations mapped to the N terminal domains of both nsP2 and nsP4. This region of nsP2 has recently been shown to be a helical region which serves as an accessory domain to the viral helicase. However, the region of nsP4 in question currently lacks known function. Based on this genetic data we hypothesized that ML336 and related compounds interact with these two domains to interfere with the activity of the replicase complex. We are currently examining this interaction using ectopically expressed protein.