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The VP40 matrix protein of Ebola virus buds from cells in the form of virus-like particles (VLPs) and plays a central role in virus assembly and budding. In this study, we utilized a functional budding assay and cotransfection experiments to examine the contributions of the glycoprotein (GP), nucleoprotein (NP), and VP24 of Ebola virus in facilitating release of VP40 VLPs. We demonstrate that VP24 alone does not affect VP40 VLP release, whereas NP and GP enhance release of VP40 VLPs, individually and to a greater degree in concert. We demonstrate further the following: (i) VP40 L domains are not required for GP-mediated enhancement of budding; (ii) the membrane-bound form of GP is necessary for enhancement of VP40 VLP release; (iii) NP appears to physically interact with VP40 as judged by detection of NP in VP40-containing VLPs; and (iv) the C-terminal 50 amino acids of NP may be important for interacting with and enhancing release of VP40 VLPs. These findings provide a more complete understanding of the role of VP40 and additional Ebola virus proteins during budding.Ebola virus is a member of the Filoviridae family and is associated with recurrent outbreaks of deadly hemorrhagic fevers (8). Currently, there are no approved vaccines, nor are there antiviral therapeutics to prevent or treat individuals infected with Ebola virus (16). A better understanding of the molecular aspects of Ebola virus replication will be necessary for successful development of specific treatments for Ebola virus infection.The VP40 matrix protein of Ebola virus is the most abundant virion protein and plays a key role in virus assembly and budding (12,14,28). For example, VP40 can bud as a filamentous virus-like particle (VLP) from mammalian cells in the absence of any other viral protein (12,20). The ability of VP40 to bud as a VLP is due, in part, to the presence of L domains present at the N terminus of the protein (12,14,17). Viral L domains are thought to serve as docking sites for interactions with specific cellular proteins, and the resultant virus-host interactions are believed to facilitate efficient virus budding (for a review, see reference 9). Although VP40 and other viral matrix proteins (e.g., Gag and M proteins of retroviruses and rhabdoviruses, respectively) can bud independently from cells, additional viral proteins are undoubtedly important for the budding process. For example, the G protein of vesicular stomatitis virus (VSV) has been shown to be important for efficient budding. More specifically, the membrane-proximal stem, transmembrane domain, and cytoplasmic tail of VSV G appear to confer this efficiency (21, 25). In addition, alterations to the cytoplasmic tails of glycoproteins of other RNA viruses, such as influenza A, simian virus 5, and rabies virus, appear to result in poor budding despite an intact matrix protein (15,19,23).In addition to VP40, the surface glycoprotein (GP), the nucleoprotein (NP), and the minor matrix protein (VP24) of Ebola virus have been implicated in virus assembly and budding (1,11,12)...

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Budding of filoviruses, arenaviruses, and rhabdoviruses is facilitated by subversion of host proteins, such as Nedd4 E3 ubiquitin ligase, by viral PPxY late (L) budding domains expressed within the matrix proteins of these RNA viruses. As L domains are important for budding and are highly conserved in a wide array of RNA viruses, they represent potential broad-spectrum targets for the development of antiviral drugs. To identify potential competitive blockers, we used the known Nedd4 WW domain-PPxY interaction interface as the basis of an in silico screen. Using PPxY-dependent budding of Marburg (MARV) VP40 virus-like particles (VLPs) as our model system, we identified small-molecule hit 1 that inhibited Nedd4-PPxY interaction and PPxY-dependent budding. This lead candidate was subsequently improved with additional structure-activity relationship (SAR) analog testing which enhanced antibudding activity into the nanomolar range. Current lead compounds 4 and 5 exhibit on-target effects by specifically blocking the MARV VP40 PPxY-host Nedd4 interaction and subsequent PPxY-dependent egress of MARV VP40 VLPs. In addition, lead compounds 4 and 5 exhibited antibudding activity against Ebola and Lassa fever VLPs, as well as vesicular stomatitis and rabies viruses (VSV and RABV, respectively). These data provide target validation and suggest that inhibition of the PPxY-Nedd4 interaction can serve as the basis for the development of a novel class of broad-spectrum, host-oriented antivirals targeting viruses that depend on a functional PPxY L domain for efficient egress. IMPORTANCEThere is an urgent and unmet need for the development of safe and effective therapeutics against biodefense and high-priority pathogens, including filoviruses (Ebola and Marburg) and arenaviruses (e.g., Lassa and Junin) which cause severe hemorrhagic fever syndromes with high mortality rates. We along with others have established that efficient budding of filoviruses, arenaviruses, and other viruses is critically dependent on the subversion of host proteins. As disruption of virus budding would prevent virus dissemination, identification of small-molecule compounds that block these critical viral-host interactions should effectively block disease progression and transmission. Our findings provide validation for targeting these virus-host interactions as we have identified lead inhibitors with broad-spectrum antiviral activity. In addition, such inhibitors might prove useful for newly emerging RNA viruses for which no therapeutics would be available.

Previous studies have indicated that the replication of the RNA genome of hepatitis delta virus (HDV) involves redirection of RNA polymerase II (Pol II), a host enzyme that normally uses DNA as a template. However, there has been some controversy about whether in one part of this HDV RNA transcription, a polymerase other than Pol II is involved. The present study applied a recently described cell system (293-HDV) of tetracycline-inducible HDV RNA replication to provide new data regarding the involvement of host polymerases in HDV transcription. The data generated with a nuclear run-on assay demonstrated that synthesis not only of genomic RNA but also of its complement, the antigenome, could be inhibited by low concentrations of amanitin specific for Pol II transcription. Subsequent studies used immunoprecipitation and rate-zonal sedimentation of nuclear extracts together with double immunostaining of 293-HDV cells, in order to examine the associations between Pol II and HDV RNAs, as well as the small delta antigen, an HDV-encoded protein known to be essential for replication. Findings include evidence that HDV replication is somehow able to direct the available delta antigen to sites in the nucleoplasm, almost exclusively colocalized with Pol II in what others have described as transcription factories.During the replication of human hepatitis delta virus (HDV), three RNAs are generated by RNA-directed RNA transcription and posttranscriptional processing. The genome and its exact complement, the antigenome, are 1,679-nucleotide circular RNAs that fold into a rod-like structure with 74% of their nucleotides base paired (20,38). It is considered that these RNAs are derived from longer than unit length primary transcripts that are processed to unit length by the HDV ribozymes and then ligated into circles (35). The third RNA is of the same polarity as the antigenome but only about 800 nucleotides in length. It has a defined 5Ј end that is capped and a defined 3Ј end that is polyadenylated (14). This mRNA contains the open reading frame for the only protein of HDV, the small delta antigen (␦Ag). This protein is essential for HDV replication but at 195 amino acids in length is too small to have polymerase activity (7).Several lines of evidence implicate the host RNA polymerase II (Pol II) as being required for the transcription of HDV RNAs (reviewed in references 21, 35, and 36). However, data from Lai and coworkers has been interpreted as evidence that the synthesis of antigenomic RNA is resistant to high doses of amanitin and therefore may be more consistent with being directed by RNA Pol I, the enzyme involved in the transcription of rRNAs (23,25,27). In order to resolve this controversy and also to obtain more information about HDV RNA-directed transcription, we have made use of the following experimental system.As previously described, we first established 293-␦Ag cells, a line of 293 cells in which the essential ␦Ag is provided by an integrated cDNA, with expression inducible by tetracycline (TET). These cells w...