Packaging of actin into Ebola virus VLPs

Abstract

The actin cytoskeleton has been implicated in playing an important role assembly and budding of several RNA virus families including retroviruses and paramyxoviruses. In this report, we sought to determine whether actin is incorporated into Ebola VLPs, and thus may play a role in assembly and/or budding of Ebola virus. Our results indicated that actin and Ebola virus VP40 strongly co-localized in transfected cells as determined by confocal microscopy. In addition, actin was packaged into budding VP40 VLPs as determined by a functional budding assay and protease protection assay. Co-expression of a membrane-anchored form of Ebola virus GP enhanced the release of both VP40 and actin in VLPs. Lastly, disruption of the actin cytoskeleton with latrunculin-A suggests that actin may play a functional role in budding of VP40/GP VLPs. These data suggest that VP40 may interact with cellular actin, and that actin may play a role in assembly and/or budding of Ebola VLPs.

Figure 1

Packaging of actin into VLPs. A) Human 293T cells were mock-transfected (lane 1), or transfected with VP40 alone (lane 2), VP40 + GP (lane 3), VP40 + GPΔM (lane 4), or VP40 + sGP (lane 5). Radiolabeled VP40 was detected in cell extracts (cells) and in VLPs. Actin was detected in VLPs by immunoprecipitation using an anti-actin polyclonal Ab. B) VP40 VLP samples were untreated (lane 1), treated with trypsin alone (lane 2), or treated with trypsin + TX-100 (lane 3). VP40 and actin were detected by immunoprecipitation. C) Indirect immunofluorescence of VP40 (green) and actin (red) with the merged image shown in yellow.

Figure 2

Affect of Latrunculin-A on VLP budding. VLPs were isolated from mock-transfected cells or cells transfected with VP40 alone or VP40 + GP in theabsence, or presence of the indicated concentration of lat-A. VP40 (panel A) or actin (panel B) was detected by immunoprecipitation and quantitated by phosphoimager analysis of at least two independent experiments.