PMCC PMCC

Search tips
Search criteria

Advanced
Results 1-25 (35)
 

Clipboard (0)
None

Select a Filter Below

Journals
more »
Year of Publication
more »
1.  Small-Molecule Probes Targeting the Viral PPxY-Host Nedd4 Interface Block Egress of a Broad Range of RNA Viruses 
Journal of Virology  2014;88(13):7294-7306.
ABSTRACT
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.
IMPORTANCE There 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.
doi:10.1128/JVI.00591-14
PMCID: PMC4054416  PMID: 24741084
2.  A Host-Oriented Inhibitor of Junin Argentine Hemorrhagic Fever Virus Egress 
Journal of Virology  2014;88(9):4736-4743.
ABSTRACT
There are currently no U.S. Food and Drug Administration (FDA)-approved vaccines or therapeutics to prevent or treat Argentine hemorrhagic fever (AHF). The causative agent of AHF is Junin virus (JUNV); a New World arenavirus classified as a National Institute of Allergy and Infectious Disease/Centers for Disease Control and Prevention category A priority pathogen. The PTAP late (L) domain motif within JUNV Z protein facilitates virion egress and transmission by recruiting host Tsg101 and other ESCRT complex proteins to promote scission of the virus particle from the plasma membrane. Here, we describe a novel compound (compound 0013) that blocks the JUNV Z-Tsg101 interaction and inhibits budding of virus-like particles (VLPs) driven by ectopic expression of the Z protein and live-attenuated JUNV Candid-1 strain in cell culture. Since inhibition of the PTAP-Tsg101 interaction inhibits JUNV egress, compound 0013 serves as a prototype therapeutic that could reduce virus dissemination and disease progression in infected individuals. Moreover, since PTAP l-domain-mediated Tsg101 recruitment is utilized by other RNA virus pathogens (e.g., Ebola virus and HIV-1), PTAP inhibitors such as compound 0013 have the potential to function as potent broad-spectrum, host-oriented antiviral drugs.
IMPORTANCE There are currently no FDA-approved vaccines or therapeutics to prevent or treat Argentine hemorrhagic fever (AHF). The causative agent of AHF is Junin virus (JUNV); a New World arenavirus classified as an NIAID/CDC category A priority pathogen. Here, we describe a prototype therapeutic that blocks budding of JUNV and has the potential to function as a broad-spectrum antiviral drug.
doi:10.1128/JVI.03757-13
PMCID: PMC3993831  PMID: 24522922
3.  Virus Budding/Host Interactions 
Advances in Virology  2011;2011:963192.
doi:10.1155/2011/963192
PMCID: PMC3265320  PMID: 22312359
4.  Host IQGAP1 and Ebola Virus VP40 Interactions Facilitate Virus-Like Particle Egress 
Journal of Virology  2013;87(13):7777-7780.
We have identified host IQGAP1 as an interacting partner for Ebola virus (EBOV) VP40, and its expression is required for EBOV VP40 virus-like particle (VLP) budding. IQGAP1 is involved in actin cytoskeletal remodeling during cell migration and formation of filopodia. The physical interaction and the functional requirement for IQGAP1 in EBOV VP40 VLP egress link virus budding to the cytoskeletal remodeling machinery. Consequently, this interaction represents a novel target for development of therapeutics to block budding and transmission of filoviruses.
doi:10.1128/JVI.00470-13
PMCID: PMC3700276  PMID: 23637409
5.  Characterization of Filovirus Protein–Protein Interactions in Mammalian Cells Using Bimolecular Complementation 
The Journal of Infectious Diseases  2011;204(Suppl 3):S817-S824.
The virion protein 40 (VP40) and nucleoprotein (NP) of Ebola (EBOV) and Marburg viruses (MARV) play key roles during virion assembly and egress. The ability to detect interactions between VP40-VP40, VP40-NP, and NP-NP and follow these complexes as they traffic through mammalian cells would enhance our understanding of the molecular events leading to filovirus assembly and budding, and provide new insights into filovirus replication and pathogenesis. Here, we successfully employed a bimolecular complementation (BiMC) approach to visualize interactions between EBOV and MARV VP40-VP40, NP-NP, and VP40-NP proteins and localize these protein complexes in mammalian cells using confocal microscopy. We demonstrate that VP40-VP40 complexes localized predominantly at the plasma membrane, whereas VP40-NP and NP-NP complexes displayed a more dispersed pattern throughout the cytoplasm. As expected based on previous findings, efficient interactions between EBOV or MARV VP40-VP40 proteins were independent of L-domains PTAPPEY and PPPY, respectively. In contrast, the formation of EBOV or MARV VP40-VP40 complexes was dependent on the previously characterized LPLGVA and LPLGIM motifs of EBOV and MARV VP40 proteins, respectively, indicating that these motifs are important for VP40 oligomerization and subsequent budding. These results highlight the feasibility and usefulness of the BiMC approach as a strategy to further characterize both filovirus protein interactions as well as filovirus-host interactions in real time in the natural environment of the cell.
doi:10.1093/infdis/jir293
PMCID: PMC3189979  PMID: 21987757
6.  Bimolecular Complementation to Visualize Filovirus VP40-Host Complexes in Live Mammalian Cells: Toward the Identification of Budding Inhibitors 
Advances in virology  2011;2011:341816.
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.
doi:10.1155/2011/341816
PMCID: PMC3217271  PMID: 22102845
7.  Bimolecular Complementation to Visualize Filovirus VP40-Host Complexes in Live Mammalian Cells: Toward the Identification of Budding Inhibitors 
Advances in Virology  2011;2011:341816.
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.
doi:10.1155/2011/341816
PMCID: PMC3217271  PMID: 22102845
8.  Antiviral Activity of Innate Immune Protein ISG15 
Journal of Innate Immunity  2009;1(5):397-404.
The host innate immune response, including the production of type-I IFN, represents the primary line of defense against invading viral pathogens. Of the hundreds of IFN-stimulated genes (ISGs) discovered to date, ISG15 was one of the first identified and shown to encode a ubiquitin-like protein that functions, in part, as a modifier of protein function. Evidence implicating ISG15 as an innate immune protein with broad-spectrum antiviral activity continues to accumulate rapidly. This review will summarize recent findings on the innate antiviral activity of ISG15, with a focus on the interplay between ubiquitination and ISGylation pathways resulting in modulation of RNA virus assembly/budding. Indeed, ubiquitination is known to be proviral for some RNA viruses, whereas the parallel ISGylation pathway is known to be antiviral. A better understanding of the antiviral activities of ISG15 will enhance our fundamental knowledge of host innate responses to viral pathogens and may provide insight useful for the development of novel therapeutic approaches designed to enhance the immune response against such pathogens.
doi:10.1159/000226245
PMCID: PMC2725329  PMID: 19680460
Antiviral; Budding; Innate immunity; Interferon-stimulated gene 15; ISGylation; Ubiquitin; Ubiquitination
9.  Antiviral Activity of Innate Immune Protein ISG15 
Journal of innate immunity  2009;1(5):397-404.
The host innate immune response, including the production of type-I IFN, represents the primary line of defense against invading viral pathogens. Of the hundreds of IFN-stimulated genes (ISGs) discovered to date, ISG15 was one of the first identified and shown to encode a ubiquitin-like protein that functions, in part, as a modifier of protein function. Evidence implicating ISG15 as an innate immune protein with broad-spectrum antiviral activity continues to accumulate rapidly. This review will summarize recent findings on the innate antiviral activity of ISG15, with a focus on the interplay between ubiquitination and ISGylation pathways resulting in modulation of RNA virus assembly/budding. Indeed, ubiquitination is known to be proviral for some RNA viruses, whereas the parallel ISGylation pathway is known to be antiviral. A better understanding of the antiviral activities of ISG15 will enhance our fundamental knowledge of host innate responses to viral pathogens and may provide insight useful for the development of novel therapeutic approaches designed to enhance the immune response against such pathogens.
doi:10.1159/000226245
PMCID: PMC2725329  PMID: 19680460
Antiviral; Budding; Innate immunity; Interferon-stimulated gene 15; ISGylation; Ubiquitin; Ubiquitination
10.  In Vivo Replication and Pathogenesis of Vesicular Stomatitis Virus Recombinant M40 Containing Ebola Virus L-Domain Sequences 
Infectious diseases  2012;5:59-64.
The M40 VSV recombinant was engineered to contain overlapping PTAP and PPxY L-domain motifs and flanking residues from the VP40 protein of Ebola virus. Replication of M40 in cell culture is virtually indistinguishable from that of control viruses. However, the presence of the Ebola PTAP motif in the M40 recombinant enabled this virus to interact with and recruit host Tsg101, which was packaged into M40 virions. In this brief report, we compared replication and the pathogenic profiles of M40 and the parental virus M51R in mice to determine whether the presence of the Ebola L-domains and flanking residues altered in vivo characteristics of the virus. Overall, the in vivo characteristics of M40 were similar to those of the parental M51R virus, indicating that the Ebola sequences did not alter pathogenesis of VSV in this small animal model of infection.
doi:10.4137/IDRT.S10652
PMCID: PMC3686127  PMID: 23794798
VSV; Ebola; recombinant virus; budding; L-domain; pathogenesis; animal model
11.  Cytopathogenesis of Vesicular Stomatitis Virus Is Regulated by the PSAP Motif of M Protein in a Species-Dependent Manner 
Viruses  2012;4(9):1605-1618.
Vesicular stomatitis virus (VSV) is an important vector-borne pathogen of bovine and equine species, causing a reportable vesicular disease. The matrix (M) protein of VSV is multifunctional and plays a key role in cytopathogenesis, apoptosis, host protein shut-off, and virion assembly/budding. Our previous findings indicated that mutations of residues flanking the 37PSAP40 motif within the M protein resulted in VSV recombinants having attenuated phenotypes in mice. In this report, we characterize the phenotype of VSV recombinant PS > A4 (which harbors four alanines (AAAA) in place of the PSAP motif without disruption of flanking residues) in both mice, and in Aedes albopictus C6/36 mosquito and Culicoides sonorensis KC cell lines. The PS > A4 recombinant displayed an attenuated phenotype in infected mice as judged by weight loss, mortality, and viral titers measured from lung and brain samples of infected animals. However, unexpectedly, the PS > A4 recombinant displayed a robust cytopathic phenotype in insect C6/36 cells compared to that observed with control viruses. Notably, titers of recombinant PS > A4 were approximately 10-fold greater than those of control viruses in infected C6/36 cells and in KC cells from Culicoides sonorensis, a known VSV vector species. In addition, recombinant PS > A4 induced a 25-fold increase in the level of C3 caspase activity in infected C6/36 cells. These findings indicate that the PSAP motif plays a direct role in regulating cytopathogenicity in a species-dependent manner, and suggest that the intact PSAP motif may be important for maintaining persistence of VSV in an insect host.
doi:10.3390/v4091605
PMCID: PMC3499822  PMID: 23170175
vesicular stomatitis virus, VSV recombinant; M protein; apoptosis; cytopathic; CPE; insect; persistence; motif
12.  Viral and host proteins that modulate filovirus budding 
Future virology  2010;5(4):481-491.
The filoviruses, Ebola and Marburg, utilize a multifaceted mechanism for assembly and budding of infectious virions from mammalian cells. Growing evidence not only demonstrates the importance of multiple viral proteins for efficient assembly and budding, but also the exploitation of various host proteins/pathways by the virus during this late stage of filovirus replication, including endocytic compartments, vacuolar protein sorting pathways, ubiquitination machinery, lipid rafts and cytoskeletal components. Continued elucidation of these complex and orchestrated virus-host interactions will provide a fundamental understanding of the molecular mechanisms of filovirus assembly/budding and ultimately lead to the development of novel viral- and/or host-oriented therapeutics to inhibit filovirus egress and spread. This article will focus on the most recent studies on host interactions and modulation of filovirus budding and summarize the key findings from these investigations.
doi:10.2217/FVL.10.33
PMCID: PMC2922766  PMID: 20730024
budding; Ebola; filovirus; Marburg; VLP
13.  Evaluation of an Attenuated Vesicular Stomatitis Virus Vector Expressing Interferon-β for Use in Malignant Pleural Mesothelioma: Heterogeneity in Interferon Responsiveness Defines Potential Efficacy 
Human Gene Therapy  2009;21(1):51-64.
Abstract
Vesicular stomatitis virus (VSV) has shown promise as an oncolytic agent, although unmodified VSV can be neurotoxic. To avoid toxicity, a vector was created by introducing the interferon-β (IFN-β) gene (VSV.IFN-β). We conducted this study to determine the ability of VSV.IFN-β to lyse human cancer (mesothelioma) cells and to evaluate the potential of this recombinant virus for clinical translation. Four normal human mesothelial and 12 mesothelioma cell lines were tested for their susceptibility to VSV vectors in vitro. VSV.hIFN-β did not cause cytotoxicity in any normal lines. Only 4 of 12 lines were effectively lysed by VSV.hIFN-β. In the eight resistant lines, pretreatment with IFN-β prevented lysis of cells by VSV.GFP, and VSV infection or addition of IFN-β protein resulted in the upregulation of double-stranded RNA-dependent protein kinase (PKR), myxovirus resistance A (MxA), and 2′,5′-oligo-adenylate-synthetase (2′5′-OAS) mRNA. In the susceptible lines, there was no protection by pretreatment with IFN-β protein and no IFN- or VSV-induced changes in PKR, MxA, and 2′5′-OAS mRNA. This complete lack of IFN responsiveness could be explained by marked downregulation of interferon alpha receptors (IFNARs), p48, and PKR in both the mesothelioma cell lines and primary tumor biopsies screened. Presence of p48 in three tumor samples predicted responsiveness to IFN. Our data indicate that many mesothelioma tumors have partially intact IFN pathways that may affect the efficacy of oncolytic virotherapy. However, it may be feasible to prescreen individual susceptibility to VSV.IFN-β by immunostaining for the presence of p48 protein.
doi:10.1089/hum.2009.088
PMCID: PMC2829454  PMID: 19715403
14.  Conserved Motifs within Ebola and Marburg Virus VP40 Proteins Are Important for Stability, Localization, and Subsequent Budding of Virus-Like Particles ▿  
Journal of Virology  2009;84(5):2294-2303.
The filovirus VP40 protein is capable of budding from mammalian cells in the form of virus-like particles (VLPs) that are morphologically indistinguishable from infectious virions. Ebola virus VP40 (eVP40) contains well-characterized overlapping L domains, which play a key role in mediating efficient virus egress. L domains represent only one component required for efficient budding and, therefore, there is a need to identify and characterize additional domains important for VP40 function. We demonstrate here that the 96LPLGVA101 sequence of eVP40 and the corresponding 84LPLGIM89 sequence of Marburg virus VP40 (mVP40) are critical for efficient release of VP40 VLPs. Indeed, deletion of these motifs essentially abolished the ability of eVP40 and mVP40 to bud as VLPs. To address the mechanism by which the 96LPLGVA101 motif of eVP40 contributes to egress, a series of point mutations were introduced into this motif. These mutants were then compared to the eVP40 wild type in a VLP budding assay to assess budding competency. Confocal microscopy and gel filtration analyses were performed to assess their pattern of intracellular localization and ability to oligomerize, respectively. Our results show that mutations disrupting the 96LPLGVA101 motif resulted in both altered patterns of intracellular localization and self-assembly compared to wild-type controls. Interestingly, coexpression of either Ebola virus GP-WT or mVP40-WT with eVP40-ΔLPLGVA failed to rescue the budding defective eVP40-ΔLPLGVA mutant into VLPs; however, coexpression of eVP40-WT with mVP40-ΔLPLGIM successfully rescued budding of mVP40-ΔLPLGIM into VLPs at mVP40-WT levels. In sum, our findings implicate the LPLGVA and LPLGIM motifs of eVP40 and mVP40, respectively, as being important for VP40 structure/stability and budding.
doi:10.1128/JVI.02034-09
PMCID: PMC2820906  PMID: 20032189
15.  Functional characterization of Ebola virus L-domains using VSV recombinants 
Virology  2005;336(2):291-298.
VSV recombinants containing the overlapping L-domain sequences from Ebola virus VP40 (PTAPPEY) were recovered by reverse-genetics. Replication kinetics of M40-WT, M40-P24L, and M40-Y30A were indistinguishable from VSV-WT in BHK-21 cells, whereas the double mutant (M40-P2728A) was defective in budding. Insertion of the Ebola L-domain region into VSV M protein was sufficient to alter the dependence on host proteins for efficient budding. Indeed, M40 recombinants containing a functional PTAP motif specifically incorporated endogenous tsg101 into budding virions and were dependent on tsg101 expression for efficient budding. Thus, VSV represents an excellent negative-sense RNA virus model for elucidating the functional aspects and diverse host interactions associated with the L-domains of Ebola virus.
doi:10.1016/j.virol.2005.03.027
PMCID: PMC2929245  PMID: 15892969
Ebola virus; L-domains; VSV recombinants
16.  Interaction between Ebola Virus Glycoprotein and Host Toll-Like Receptor 4 Leads to Induction of Proinflammatory Cytokines and SOCS1 ▿ †  
Journal of Virology  2009;84(1):27-33.
Ebola virus initially targets monocytes and macrophages, which can lead to the release of proinflammatory cytokines and chemokines. These inflammatory cytokines are thought to contribute to the development of circulatory shock seen in fatal Ebola virus infections. Here we report that host Toll-like receptor 4 (TLR4) is a sensor for Ebola virus glycoprotein (GP) on virus-like particles (VLPs) and that resultant TLR4 signaling pathways lead to the production of proinflammatory cytokines and suppressor of cytokine signaling 1 (SOCS1) in a human monocytic cell line and in HEK293-TLR4/MD2 cells stably expressing the TLR4/MD2 complex. Ebola virus GP was found to interact with TLR4 by immunoprecipitation/Western blot analyses, and Ebola virus GP on VLPs was able to stimulate expression of NF-κB in a TLR4-dependent manner. Interestingly, we found that budding of Ebola virus VLPs was more pronounced in TLR4-stimulated cells than in unstimulated control cells. In sum, these findings identify the host innate immune protein TLR4 as a sensor for Ebola virus GP which may play an important role in the immunopathogenesis of Ebola virus infection.
doi:10.1128/JVI.01462-09
PMCID: PMC2798428  PMID: 19846529
17.  No exit: targeting the budding process to inhibit filovirus replication 
Antiviral research  2008;81(3):189-197.
The filoviruses, Ebola and Marburg, cause severe hemorrhagic fever in humans and nonhuman primates, with high mortality rates. Although the filovirus replication pathway is now understood in considerable detail, no antiviral drugs have yet been developed that directly inhibit steps in the replication cycle. One potential target is the filovirus VP40 matrix protein, the key viral protein that drives the budding process, in part by mediating specific virus-host interactions to facilitate the efficient release of virions from the infected cell. This review will summarize current knowledge of key structural and functional domains of VP40 believed to be necessary for efficient budding of virions and virus-like particles. A better understanding of the structure and function of these key regions of VP40 will be crucial, as they may represent novel and rational targets for inhibitors of filovirus egress.
doi:10.1016/j.antiviral.2008.12.003
PMCID: PMC2666966  PMID: 19114059
filovirus; Ebola virus; Marburg virus; viral budding; VP40 matrix protein; virus-like particles; antiviral therapy
18.  Cigarette smoking products suppress anti-viral effects of Type I interferon via phosphorylation-dependent downregulation of its receptor 
FEBS letters  2008;582(21-22):3206-3210.
While negative effect of smoking on the resistance to viral infections was known, the underlying mechanisms remained unclear. Here we report that products of cigarette smoking compromise the cellular anti-viral defenses by inhibiting the signaling induced by Type I interferon (IFN). Cigarette smoking condensate (but not pure nicotine) stimulated specific serine phosphorylation-dependent ubiquitination and degradation of the IFNAR1 subunit of the Type I IFN receptor leading to attenuation of IFN signaling and decreased resistance to viral infection. This resistance was restored in cells where phosphorylation-dependent degradation of IFNAR1 is abolished. We conclude that smoking compromises cellular anti-viral defenses via degradation of Type I IFN receptor and discuss the significance of this mechanism for efficacy of IFN-based therapies.
doi:10.1016/j.febslet.2008.08.013
PMCID: PMC2567056  PMID: 18722370
19.  PPEY Motif within the Rabies Virus (RV) Matrix Protein Is Essential for Efficient Virion Release and RV Pathogenicity ▿  
Journal of Virology  2008;82(19):9730-9738.
Late (L) domains containing the highly conserved sequence PPXY were first described for retroviruses, and later research confirmed their conservation and importance for efficient budding of several negative-stranded RNA viruses. Rabies virus (RV), a member of the Rhabdoviridae family, contains the sequence PPEY (amino acids 35 to 38) within the N terminus of the matrix (M) protein, but the functions of this potential L-domain in the viral life cycle, viral pathogenicity, and immunogenicity have not been established. Here we constructed a series of recombinant RVs containing mutations within the PPEY motif and analyzed their effects on viral replication and RV pathogenicity. Our results indicate that the first proline at position 35 is the most important for viral replication, whereas P36 and Y38 have a lesser but still noticeable impact. The reduction in viral replication was most likely due to inhibition of virion release, because initially no major impact on RV RNA synthesis was observed. In addition, results from electron microscopy demonstrated that the M4A mutant virus (PPEY→SAEA) displayed a more cell-associated phenotype than that of wild-type RV. Furthermore, all mutations within the PPEY motif resulted in reduced spread of the recombinant RVs as indicated by a reduction in focus size. Importantly, recombinant PPEY L-domain mutants were highly attenuated in mice yet still elicited potent antibody responses against RV G protein that were as high as those observed after infection with wild-type virus. Our data indicate that the RV PPEY motif has L-domain activity essential for efficient virus production and pathogenicity but is not essential for immunogenicity and thus can be targeted to increase the safety of rabies vaccine vectors.
doi:10.1128/JVI.00889-08
PMCID: PMC2546990  PMID: 18667490
20.  The YPLGVG sequence of the Nipah virus matrix protein is required for budding 
Virology Journal  2008;5:137.
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 (L-domains) 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.
Results
Using rationally targeted site-directed mutagenesis we show that a NiV M sequence YPLGVG is required for M budding and that mutation or deletion of the sequence abrogates budding ability. Replacement of the native and overlapping Ebola VP40 L-domains with the NiV sequence failed to rescue VP40 budding; however, it did induce the cellular morphology of extensive filamentous projection consistent with wild-type VP40-expressing cells. Cells expressing wild-type NiV M also displayed this morphology, which was dependent on the YPLGVG sequence, and deletion of the sequence also resulted in nuclear localization of M. Dominant-negative VPS4 proteins had no effect on NiV M budding, suggesting that unlike other viruses such as Ebola, NiV M accomplishes budding independent of MVB cellular proteins.
Conclusion
These data indicate that the YPLGVG motif within the NiV M protein plays an important role in M budding; however, involvement of any specific components of the cellular MVB sorting pathway in henipavirus budding remains to be demonstrated. Further investigation of henipavirus assembly and budding may yet reveal a novel mechanism(s) of viral assembly and release that could be applicable to other enveloped viruses or have therapeutic implications.
doi:10.1186/1743-422X-5-137
PMCID: PMC2625347  PMID: 19000317
21.  Role for Amino Acids 212KLR214 of Ebola Virus VP40 in Assembly and Budding▿  
Journal of Virology  2007;81(20):11452-11460.
Ebola virus VP40 is able to produce virus-like particles (VLPs) in the absence of other viral proteins. At least three domains within VP40 are thought to be required for efficient VLP release: the late domain (L-domain), membrane association domain (M-domain), and self-interaction domain (I-domain). While the L-domain of Ebola VP40 has been well characterized, the exact mechanism by which VP40 mediates budding through the M- and I-domains remains unclear. To identify additional domains important for VP40 assembly/budding, amino acids 212KLR214 were targeted for mutagenesis based on the published crystal structure of VP40. These residues are part of a loop connecting two beta sheets in the C-terminal region and thus are potentially important for overall structure and/or oligomerization of VP40. A series of alanine substitutions were generated in the KLR region of VP40, and these mutants were examined for VLP budding, intracellular localization, and oligomerization. Our results indicated that (i) 212KLR214 residues of VP40 are important for efficient release of VP40 VLPs, with Leu213 being the most critical; (ii) VP40 KLR mutants displayed altered patterns of cellular localization compared to that of wild-type VP40 (VP40-WT); and (iii) self-assembly of VP40 KLR mutants into oligomers was altered compared to that of VP40-WT. These results suggest that 12KLR214 residues of VP40 are important for proper assembly/oligomerization of VP40 which subsequently leads to efficient budding of VLPs.
doi:10.1128/JVI.00853-07
PMCID: PMC2045517  PMID: 17699576
22.  Ebola Virus VP35-VP40 Interaction Is Sufficient for Packaging 3E-5E Minigenome RNA into Virus-Like Particles 
Journal of Virology  2006;80(11):5135-5144.
The packaging of viral genomic RNA into nucleocapsids and subsequently into virions is not completely understood. Phosphoprotein (P) and nucleoprotein (NP) interactions link NP-RNA complexes with P-L (polymerase) complexes to form viral nucleocapsids. The nucleocapsid then interacts with the viral matrix protein, leading to specific packaging of the nucleocapsid into the virion. A mammalian two-hybrid assay and confocal microscopy were used to demonstrate that Ebola virus VP35 and VP40 interact and colocalize in transfected cells. VP35 was packaged into budding virus-like particles (VLPs) as observed by protease protection assays. Moreover, VP40 and VP35 were sufficient for packaging an Ebola virus minignome RNA into VLPs. Results from immunoprecipitation-reverse transcriptase PCR experiments suggest that VP35 confers specificity of the nucleocapsid for viral genomic RNA by direct VP35-RNA interactions.
doi:10.1128/JVI.01857-05
PMCID: PMC1472164  PMID: 16698994
23.  Effect of Ebola virus proteins GP, NP and VP35 on VP40 VLP morphology 
Virology Journal  2006;3:31.
Recently we described a role for Ebola virus proteins, NP, GP, and VP35 in enhancement of VP40 VLP budding. To explore the possibility that VLP structure was altered by co-expression of EBOV proteins leading to the observed enhancement of VP40 VLP budding, we performed density gradient analysis as well as electron microscopy studies. Our data suggest that VP40 is the major determinant of VLP morphology, as co-expression of NP, GP and VP35 did not significantly change VLP density, length, and diameter. Ultra-structural changes were noted in the core of the VLPs when NP was co-expressed with VP40. Overall, these findings indicate that major changes in morphology of VP40 VLPs were likely not responsible for enhanced budding of VP40 VLPs in the presence of GP, NP and/or VP35.
doi:10.1186/1743-422X-3-31
PMCID: PMC1502131  PMID: 16719918
24.  Packaging of actin into Ebola virus VLPs 
Virology Journal  2005;2:92.
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.
doi:10.1186/1743-422X-2-92
PMCID: PMC1334228  PMID: 16367999
25.  L-Domain Flanking Sequences Are Important for Host Interactions and Efficient Budding of Vesicular Stomatitis Virus Recombinants 
Journal of Virology  2005;79(20):12617-12622.
Vesicular stomatitis virus (VSV) possesses a PPPY and a PSAP motif within the matrix (M) protein. The PPPY motif has significant L-domain activity in BHK-21 cells, whereas the PSAP motif does not. Since the core PSAP motif alone is insufficient to provide L-domain activity, we modified upstream or downstream amino acids flanking the PSAP core motif to determine their effect on L-domain activity. VSV recombinants were recovered that contained single or multiple amino acid mutations in upstream or downstream sequences flanking the PSAP core. Recombinant viruses were examined for growth kinetics, budding efficiency, and functional interactions with host proteins. We demonstrate that the composition of amino acids surrounding the L-domain core motifs are critical for efficient L-domain activity and for interactions with host proteins in the context of a VSV infection.
doi:10.1128/JVI.79.20.12617-12622.2005
PMCID: PMC1235845  PMID: 16188963

Results 1-25 (35)