- Short report
- Open Access
Packaging of actin into Ebola virus VLPs
© Han and Harty; licensee BioMed Central Ltd. 2005
- Received: 05 August 2005
- Accepted: 20 December 2005
- Published: 20 December 2005
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.
- West Nile Virus
- Newcastle Disease Virus
- Equine Infectious Anemia Virus
- Cellular Actin
- Murine Mammary Tumor Virus
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.
To confirm that actin was indeed incorporated into VP40/GP VLPs and does not represent a cellular contaminant, protease protection (Fig. 1B) and flotation gradient analyses (data not shown) were performed. Radiolabeled VP40 VLPs were divided into equal aliquots and treated as indicated in Fig. 1B. Following treatment, β-actin and VP40 were detected by immunoprecipitation and analyzed by SDS-PAGE (Fig. 1B). As reported previously [2, 3, 6], VP40 was only degraded completely by trypsin in the presence of TX-100 (Fig. 1B lane 3). Similarly, actin was also only degraded completely by trypsin in the presence of TX-100 (lane 3). Treatment with trypsin alone was not sufficient to degrade either VP40 or actin (lane 2). These findings indicate that cellular actin is indeed packaged within Ebola virus VLPs. It should be noted that flotation gradients of purified VLPs were also utilized to demonstrate that actin, VP40, and GP co-purified together in the upper fractions (fractions 2 and 3) of the VLP gradient (data not shown). These findings are consistent with those presented above that actin is incorporated into budding VLPs.
We next sought to use immunofluorescence and confocal microscopy to determine whether VP40 colocalized with cellular actin in COS-1 cells (Fig. 1C). VP40 (green) is known to localize to the cell periphery and can be visualized in membrane fragments or blebs (VLPs) being released from the cell (Fig. 1C). Cellular actin (red) was detected by the use of a polyclonal anti-actin antibody (Santa Cruz Biotechnology, Inc.). Upon merging of the two images, VP40 and actin were found to colocalize (yellow) in many of the membrane fragments that likely represent the formation of VLPs (Fig. 1C). These results correlate with those described above to suggest that VP40 may interact with actin, and that actin may be incorporated into budding VLPs in a specific manner.
The mechanism by which GP enhances budding of VP40 VLPs remains unclear . Preliminary data from our lab suggests that GP does not enhance budding of VP40 via a direct protein-protein interaction (data not shown). An alternative possibility is that GP modifies the cell in a global manner that positively influences VP40 release. Indeed, GP is known to be cytotoxic and induces cell rounding and detachment [25–27]. Thus, GP expression likely induces significant changes to the cellular cytoskeleton during infection. Lat-A may be inhibiting the mechanism by which GP enhances budding of VP40 (Fig. 2). It remains to be determined whether actin directly interacts with VP40, or whether actin may directly interact with GP.
The actin cytoskeleton has been implicated in assembly and budding of Newcastle disease virus, HIV-1, Black Creek Canal Virus, fowlpox virus, West Nile virus, equine infectious anemia virus, and respiratory syncytial virus RSV [9, 10, 14, 18, 20, 23, 24]. Cellular actin has been detected in virion or virus-like particles of murine mammary tumor virus (MuMTV), Moloney murine leukemia virus (MoMuLV), HIV-1, and Sendai virus [11, 13, 15, 16, 28]. Ebola virus VP40 has recently been shown to associate with microtubules and enhance tubulin polymerization . Yonezawa et al. found that agents that inhibited microfilaments also inhibited entry and fusion of Ebola virus GP pseudotypes . These authors suggest that microtubules and microfilaments may play a role in trafficking Ebola virions from the cell surface to acidified vesicles for fusion.
Our data indicate that actin is indeed packaged into Ebola virus VLPs. Co-expression of a membrane-anchored form of GP enhances release of actin and VP40 by equivalent levels in VLPs. The mucin-like domain of GP was not necessary for enhancement of VP40 or actin release in VLPs. VP40 was found to co-localize with actin suggesting that VP40 may interact with actin and perhaps may utilize the actin network for assembly and budding VLPs from the plasma-membrane. Lat-A treatment resulted in a slight increase in budding of VP40 VLPs; however, the same concentrations of lat-A resulted in a slight decrease in budding of VP40/GP VLPs. Experiments are now underway to understand further the mechanism of action of lat-A and other actin depolymerizing drugs on Ebola VLP budding. In addition, we will attempt to determine whether actin binding proteins may be involved in VLP budding. Lastly, experiments are underway to determine whether actin plays a role in assembly and budding of live Ebola virus.
The authors wish to acknowledge members of the Harty lab for fruitful discussions and Shiho Irie for excellent technical support. This work was supported by NIH grant AI46499 to RNH.
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