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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.

Antiviral compounds that increase the resistance of host tissues represent an attractive class of therapeutic. Here, we show that squalamine, a compound previously isolated from the tissues of the dogfish shark ( Squalus acanthias) and the sea lamprey ( Petromyzon marinus) , exhibits broad-spectrum antiviral activity against human pathogens, which were studied in vitro as well as in vivo. Both RNA- and DNA-enveloped viruses are shown to be susceptible. The proposed mechanism involves the capacity of squalamine, a cationic amphipathic sterol, to neutralize the negative electrostatic surface charge of intracellular membranes in a way that renders the cell less effective in supporting viral replication. Because squalamine can be readily synthesized and has a known safety profile in man, we believe its potential as a broad-spectrum human antiviral agent should be explored.

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...

Background: Nipah virus (NiV) is a recently emerged paramyxovirus capable of causing fatal disease in a broad range of mammalian hosts, including humans. Together with Hendra virus (HeV), they comprise the genus Henipavirus in the family Paramyxoviridae. Recombinant expression systems have played a crucial role in studying the cell biology of these Biosafety Level-4 restricted viruses. Henipavirus assembly and budding occurs at the plasma membrane, although the details of this process remain poorly understood. Multivesicular body (MVB) proteins have been found to play a role in the budding of several enveloped viruses, including some paramyxoviruses, and the recruitment of MVB proteins by viral proteins possessing late budding domains (Ldomains) has become an important concept in the viral budding process. Previously we developed a system for producing NiV virus-like particles (VLPs) and demonstrated that the matrix (M) protein possessed an intrinsic budding ability and played a major role in assembly. Here, we have used this system to further explore the budding process by analyzing elements within the M protein that are critical for particle release.

Bluetongue virus (BTV) 1 is a member of the Orbivirus genus within the Reoviridae family of segmented double-stranded RNA viruses. BTV remains an agriculturally important veterinary pathogen transmitted primarily to sheep by biting midges. BTV encodes seven structural (VP1-7) and four nonstructural (NS1, NS2, NS3, and NS3A) proteins. NS3 is a 229-amino acid protein encoded by the viral S10 RNA segment, and NS3A is a 216-amino acid protein in which synthesis is initiated from an in-frame downstream AUG codon within the Ser-10 RNA segment (1, 2). Both proteins have been shown to be N-linked glycosylated and associated with lipid membranes in infected cells (2-5). Hyatt et al. (3,6,7) were the first to provide evidence that NS3/NS3A may be involved in the late stages of BTV assembly and that NS3/NS3A were required for efficient release of virus-like particles from BTV-infected cells. For example, electron microscopic studies suggested that NS3 was associated with areas of plasma membrane perturbation (3). Similar findings have been reported for the NS3 protein encoded by the closely related African horse sickness virus (5, 8, 9). More recently, an elegant study by Beaton et al. (10) demonstrated that NS3 of BTV facilitates virus release by interacting with specific host-trafficking proteins at the plasma membrane. The authors suggest that NS3 functions as a bridge, linking progeny virions with cellular export machinery to promote virus release (10). Thus, NS3 appears to play a key role in virus assembly and release. We were initially intrigued by the presence of conserved PPXY and PSAP motifs within NS3 that are identical in sequence to late budding domains (L-domains) that we and others have identified in matrix proteins of RNA viruses (11). These L-domains have been shown to interact with specific host proteins, and the resultant virus-host interactions are thought to facilitate virus budding by an, as yet, undetermined mechanism. Results from our initial experiments indicated that the PPXY and PSAP motifs of NS3 did not appear to function as typical L-domains, in that these motifs did not promote the release of NS3-containing virus-like particles from mammalian cells. Interestingly, we did find that cells expressing NS3 were permeable to the translational inhibitor hygromycin B in a dose-dependent manner. This finding suggested that NS3 may be capable of permeabilizing or destabilizing lipid membranes; a property shared by a group of viral proteins termed viroporins (12). We went on to demonstrate that NS3 possesses additional characteristics commonly associated with viroporins. For example, NS3 was able to form homo-oligomers when expressed in mammalian cells. Furthermore, like other viroporins, NS3 is a transmembrane protein containing two transmembrane domains (TM1 and TM2). Mutations introduced into TM1 to disrupt the hydrophobic nature of this region abolished the ability of NS3 to permeabilize the plasma membrane. In contrast, the deletion of amino acids comprising TM2 did not result in disruption of NS3-i...