ROLE OF VP40 IN EBOLA ASSEMBLY

Abstract

The VP40 protein of Ebola virus (EBOV) is thought to be a key component in the assembly of infectious virions. If so, one might predict that when expressed alone, it would form virus-like particles. To test this possibility, we created a plasmid that expressed VP40 under the control of a CMV promoter. Transfection of mammalian cells with this construct showed that VP40 was synthesized as a single protein species that was eventually released into the culture medium. Pulse-chase experiments demonstrated that VP40 protein expressed alone had a rate of release that was comparable to infectious EBOV. Further analysis of the culture medium by density gradient centrifugation revealed that VP40 was released as two distinct forms: one with a density similar to that of authentic virions (1.15 g/cm³) and one with a density consistent with lipid membranes (1.05 g/cm³). Deletion mutants of VP40 identified areas within the protein that are necessary for the protein to mediate its release from cells. EM and immuno-EM studies of VP40-transfected cells showed that VP40 localized at or near the plasma membrane where it induced proliferation, or "ruffling", and formation of empty liposomes. These data suggest that VP40 has the capacity to associate with the host-cell plasma membrane and cause evaginations that are released from the cell as liposomes in the absence of the other viral components. However, with further investigation, we now know that this is not entirely true. VP40 does bind to the surface of the cell and induces proliferation, but the liposomes and "free" protein released from the cells are merely artifact. Electron microscopy (EM) of both isopycnic gradient peaks reveals fib -particles in the light peak and seemingly no particles in the heavy peak. However, additional isopycnic gradients in which the culture media was pre-treated with trypsin eliminated the light peak, and showed the small amount of VP40 protein in the heavy peak was resistant. This suggests that the particles seen in the light peak are artifact of EM or the sucrose gradient. In fact additional experiments show that VP40 is extremely inefficient in budding; rather plays a role in ruffling for viral pre-assembly.