Investigation of Ebola VP40 Assembly and Oligomerization in Live Cells Using Number and Brightness Analysis

Abstract

Ebola virus assembles and buds from the inner leaflet of the plasma membrane of mammalian cells, which is primarily attributed to its major matrix protein VP40. Oligomerization of VP40 has been shown to be essential to the life cycle of the virus including formation of virions from infected cells. To date, VP40 oligomerization has mainly been assessed by chemical cross-linking following cell fractionation studies with VP40 transfected cells. This has made it difficult to discern the spatial and temporal dynamics of VP40 oligomerization. To gain a better understanding of the VP40 assembly and oligomerization process in live cells, we have employed real-time imaging of enhanced green fluorescent protein tagged VP40. Here, we use both confocal and total internal reflection microscopy coupled with number and brightness analysis to show that VP40 oligomers are localized on the plasma membrane and are highly enriched at sites of membrane protrusion, consistent with sites of viral budding. These filamentous plasma membrane protrusion sites harbor VP40 hexamers, octamers, and higher order oligomers. Consistent with previous reports, abrogation of VP40 oligomerization through mutagenesis greatly diminished VP40 egress and also abolished membrane protrusion sites enriched with VP40. In sum, real-time single-molecule imaging of fluorescently labeled Ebola VP40 is able to resolve the spatial and temporal dynamics of VP40 oligomerization.

A cationic, C-terminal patch and structural rearrangements in Ebola virus matrix VP40 protein control its interactions with phosphatidylserine

“…In order to assess the localization of the VP40 monomer and octamer in live cells, mutants L117R and I307R were made in a previously utilized EGFP-VP40 construct (12,27). Constructs were transfected into HEK293 cells and imaged using confocal microscopy (See Figure 6).…”

“…VP40 is a peripheral membrane protein that regulates budding as well as virus structure and stability (6-8). VP40 is the most abundant protein contained in mature virions (9), and can associate with (10) and oligomerize on (8,(11)(12)(13)(14) the plasma membrane (PM) inner leaflet, where it acquires the viral lipid envelope.…”

“…Moreover, VP40 oligomers have been detected in VLPs and UVinactivated virions (7,8) and reside predominantly in filamentous structures emanating from the PM (12,13,28,29). Larger oligomeric structures of VP40 may also play a role in viral assembly and egress (1,12,14).…”

Inhibition of Ebola Virus by Anti-Ebola miRNAs in silico

“…Besides membrane association, VP40 also associates or otherwise interacts with host cell factors, including the endosomal sorting complex that supports transport (ESCRT) machinery [39,40], the vesicle coat II proteins (COPII) [41], as well as the protein actin [42,43]; these host cell factors, respectively, have been shown to enable VP40 budding, transport, and movement. Moreover, host cell protein kinases could contribute to Ebola infectivity since cAbl1 can phosphorylate Tyr 13 in VP40 [42].…”

“…Still we have inadequate understanding of how VP40 actually assembles on the plasma membrane before virion release occurs. Localization of VP40 in the plasma membrane is believed to be important since studies give evidence that hydrophobic residues located within the C-terminal domain, including Leu, 213 are essential in the localization and budding processes [43]. Detection of VP40 oligomers in VLPs and UV-inactivated virions has occurred [44][45][46]; they have been detected mainly in filamentous structures stemming from the plasma membrane [42].…”