Date Published: October 18, 2011
Publisher: Hindawi Publishing Corporation
Author(s): Yuliang Liu, Michael S. Lee, Mark A. Olson, Ronald N. Harty.
Virus-host interactions play key roles in promoting efficient egress of many RNA viruses, including Ebola virus (EBOV or “e”) and Marburg virus (MARV or “m”). Late- (L-) domains conserved in viral matrix proteins recruit specific host proteins, such as Tsg101 and Nedd4, to facilitate the budding process. These interactions serve as attractive targets for the development of broad-spectrum budding inhibitors. A major gap still exists in our understanding of the mechanism of filovirus budding due to the difficulty in detecting virus-host complexes and mapping their trafficking patterns in the natural environment of the cell. To address this gap, we used a bimolecular complementation (BiMC) approach to detect, localize, and follow the trafficking patterns of eVP40-Tsg101 complexes in live mammalian cells. In addition, we used the BiMC approach along with a VLP budding assay to test small molecule inhibitors identified by in silico screening for their ability to block eVP40 PTAP-mediated interactions with Tsg101 and subsequent budding of eVP40 VLPs. We demonstrated the potential broad spectrum activity of a lead candidate inhibitor by demonstrating its ability to block PTAP-dependent binding of HIV-1 Gag to Tsg101 and subsequent egress of HIV-1 Gag VLPs.
Filoviruses are human pathogens that cause severe hemorrhagic disease and are potential agents of bioterrorism [1, 2]. EBOV and MARV are BSL-4 agents and NIAID Category A priority pathogens due to their association with high fatality rates and lack of approved vaccines or antivirals . Filoviruses are enveloped, nonsegmented, negative-strand RNA viruses with an approximately 19.0-kilobase genome encoding the nucleoprotein (NP), VP35, matrix protein (VP40), attachment glycoprotein (GP), VP30, VP24, and RNA polymerase protein (L) . VP40 is the major component of virions, and expression of VP40 alone in mammalian cells is sufficient to generate extracellular virus-like particles (VLPs), which resemble authentic virions in overall morphology [4–10]. Late- (L-) domain motifs conserved in the VP40 proteins are critical for efficient egress of VLPs and virions, as they function by hijacking specific host proteins involved in vacuolar protein sorting (vps) pathways to facilitate the final step of virus-cell separation [3, 6, 10–14]. EBOV VP40 (eVP40) possesses two L-domain motifs (PTAP and PPEY) at its N-terminus (7-PTAPPEY-13) [4, 6] whereas MARV VP40 (mVP40) and NP (mNP) contain single PPPY and PTAP L-domain motifs, respectively [12, 15]. Various approaches such as protein affinity chromatography, GST-pulldowns, and yeast two-hybrid screens have been used successfully to detect these functionally relevant L-domain mediated virus-host interactions in vitro [6, 12, 15]. For example, the PTAP L-domain of eVP40 recruits host Tsg101, a component of the cellular ESCRT (endosomal sorting complex required for transport) pathway involved in sorting monoubiquitinated proteins into multivesicular bodies (MVBs) [3, 6, 10, 12, 15–22] whereas the PPEY motif of eVP40 mediates an interaction with host Nedd4 ubiquitin ligase  leading to ubiquitination of eVP40 and enhanced VLP egress [4, 10, 19, 23, 24]. Despite these in vitro studies, detection and visualization of these virus-host complexes, as well as the intracellular trafficking patterns of these complexes in the natural environment of the host cell remain elusive.
Filovirus-host interactions are important for efficient egress of virus particles; however, mechanistic details of the formation, dynamics, and trafficking of these virus-host complexes in the natural environment of the host cell have been elusive. In this report, we used a BiMC approach to visualize eVP40-Tsg101 complexes as they formed in the cell. The specificity of this interaction was confirmed by using an L-domain deletion mutant of eVP40 and by using Tsg101 specific siRNAs. Importantly, the NYFP-Tsg101 fusion protein was stably expressed in mammalian cells, and CYFP-VP40 fusion proteins retained their ability to bud independently from cells as VLPs in an L-domain-dependent manner. The BiMC approach is ideal for detecting weak and/or transient protein-protein interactions in living cells [27, 28]. We demonstrated that eVP40-Tsg101 complexes formed between 3–4 hours after transfection and colocalized at early times (6 hrs. p.t.) with pericentrin-B, an MTOC marker. We postulate that the initial eVP40-Tsg101 complexes may then migrate from the MTOC to the site of budding at the plasma membrane by 12–24 hours p.t. This working model correlates with previous reports which suggested that filovirus VP40 proteins may interact with and utilize the host cytoskeletal network during assembly and egress [43, 44].