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Packaging of actin into Ebola virus VLPs

DOI: 10.1186/1743-422x-2-92

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Abstract:

Ebola virus VP40 is known to bud from cells as a virus-like particle (VLP) independent of additional virus proteins [1-4]. The most efficient release of VP40 VLPs requires both host proteins (e.g. tsg101 and vps4), as well as additional virus proteins (e.g. glycoprotein [GP] and nucleoprotein [NP]) [5-7]. Cytoskeletal proteins have also been implicated in assembly and budding of various RNA-containing viruses [8-22]. Thus, we sought to determine whether cellular actin may be important for Ebola virus VP40 VLP budding.First, we sought to detect actin in budding VP40 VLPs. Human 293T cells were mock-transfected, or transfected with VP40 alone, VP40 + GP, VP40 + a mucin domain deletion mutant (GPΔM), or VP40 + secreted GP (sGP) (Fig. 1A). VP40 synthesis in all cell extracts is shown as an expression control (Fig. 1A, cells). As expected, VP40 alone was readily detected in budding VLPs; however, actin was weakly detectable in VLPs containing VP40 alone (Fig. 1A, VLPs, lane 2). Co-expression of either full-length wild type GP (lane 3), or GPΔM (lane 4) resulted in enhanced release of VP40. Similarly, release of cellular actin was also enhanced in VP40 VLPs containing full-length GP (lane 3), or GPΔM (lanes 4). In contrast, co-expression of sGP (lane 5) did not enhance release of either VP40 or actin (compare lanes 2 and 5). Both VP40 and actin were enhanced 5–6 fold (determined by phosphoimager analysis) in VLPs when GP or GPΔM were co-expressed along with VP40 compared to that when VP40 was expressed alone (data not shown). These results suggest that actin can be packaged in budding VP40 VLPs, and that co-expression of a membrane-anchored form of GP equally enhances release of both VP40 and actin. In addition, GP-mediated enhancement of VP40 VLP budding and actin packaging into VLPs is independent of the mucin-like domain of GP.To confirm that actin was indeed incorporated into VP40/GP VLPs and does not represent a cellular contaminant, protease protection (Fig. 1B) and

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