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Search Results: 1 - 10 of 201672 matches for " Ronald N Harty "
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Packaging of actin into Ebola virus VLPs
Ziying Han, Ronald N Harty
Virology Journal , 2005, DOI: 10.1186/1743-422x-2-92
Abstract: 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.First, we sought to detect actin in budding VP40 VLPs. Human 293T cells were mock-transfected, or transfected with VP40 alone, VP40 + GP, VP40 + a mucin domain deletion mutant (GPΔM), or VP40 + secreted GP (sGP) (Fig. 1A). VP40 synthesis in all cell extracts is shown as an expression control (Fig. 1A, cells). As expected, VP40 alone was readily detected in budding VLPs; however, actin was weakly detectable in VLPs containing VP40 alone (Fig. 1A, VLPs, lane 2). Co-expression of either full-length wild type GP (lane 3), or GPΔM (lane 4) resulted in enhanced release of VP40. Similarly, release of cellular actin was also enhanced in VP40 VLPs containing full-length GP (lane 3), or GPΔM (lanes 4). In contrast, co-expression of sGP (lane 5) did not enhance release of either VP40 or actin (compare lanes 2 and 5). Both VP40 and actin were enhanced 5–6 fold (determined by phosphoimager analysis) in VLPs when GP or GPΔM were co-expressed along with VP40 compared to that when VP40 was expressed alone (data not shown). These results suggest that actin can be packaged in budding VP40 VLPs, and that co-expression of a membrane-anchored form of GP equally enhances release of both VP40 and actin. In addition, GP-mediated enhancement of VP40 VLP budding and actin packaging into VLPs is independent of the mucin-like domain of GP.To confirm that actin was indeed incorporated into VP40/GP VLPs and does not represent a cellular contaminant, protease protection (Fig. 1B) and
Effect of Ebola virus proteins GP, NP and VP35 on VP40 VLP morphology
Reed F Johnson, Peter Bell, Ronald N Harty
Virology Journal , 2006, DOI: 10.1186/1743-422x-3-31
Abstract: Ebola and Marburg viruses are members of the Filoviridae family of the order Mononegavirales. Both viruses are associated with recurrent outbreaks of deadly hemorrhagic fevers with mortality rates as high as 90% [1,2]. Currently, there are no approved vaccines, nor treatments for Ebola virus (EBOV) infection. A better understanding of the molecular aspects of EBOV replication will be necessary for successful development of specific treatments for EBOV infection.Ebola virus matrix protein, VP40, is the major virion protein and plays an essential role in virus assembly and budding [3,4]. VP40 buds from the cell surface forming virus-like particles (VLPs). VLP budding is mediated by viral L-domains present in the N-terminus of the protein, which interact with host factors such as Nedd4 and TSG101, leading to VLP release [3-7]. It is hoped that investigations into the mechanisms of VP40 VLP budding will lead to possible vaccines and therapeutics that will block late stages of the virus life-cycle.Recent evidence suggests that co-expression of other EBOV proteins will enhance VP40 VLP budding [8,9]. For example, co-expression of VP40+GP+NP enhanced VP40 release approximately 40-fold over that observed for VP40 alone [9]. We have also demonstrated that VP35 interacts with VP40, is enclosed within VP40 VLPs, and functions to specifically package the EBOV 3E-5E minigenome into VLPs [10]. Currently, the mechanism by which EBOV proteins enhance VP40 budding is unclear, as is their affect on VLP morphology. Thus, we are interested in examining VLPs that contain combinations of VP40, GP, NP, and VP35 to determine whether co-expression of different EBOV proteins affects density, length, diameter, and overall morphology. Investigating the morphology of EBOV VLPs may give us insight into the mechanism by which EBOV proteins contribute to the observed enhancement of VLP budding.Early EBOV reports suggest the virus particle is 970 nm in length and 80 nm in diameter with a density of
Bimolecular Complementation to Visualize Filovirus VP40-Host Complexes in Live Mammalian Cells: Toward the Identification of Budding Inhibitors
Yuliang Liu,Michael S. Lee,Mark A. Olson,Ronald N. Harty
Advances in Virology , 2011, DOI: 10.1155/2011/341816
Abstract: 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. 1. Introduction 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 [2]. 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) [3]. 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
In Vivo Replication and Pathogenesis of Vesicular Stomatitis Virus Recombinant M40 Containing Ebola Virus L-Domain Sequences
Takashi Irie, Elena Carnero, Adolfo García-Sastre and Ronald N. Harty
Infectious Diseases: Research and Treatment , 2012, DOI: 10.4137/IDRT.S10652
Abstract: 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.
Virus Budding/Host Interactions
Ronald N. Harty,Anthony P. Schmitt,Fadila Bouamr,Carolina B. Lopez
Advances in Virology , 2011, DOI: 10.1155/2011/963192
Abstract:
In Vivo Replication and Pathogenesis of Vesicular Stomatitis Virus Recombinant M40 Containing Ebola Virus L-Domain Sequences
Takashi Irie,Elena Carnero,Adolfo García-Sastre,Ronald N. Harty
Infectious Diseases: Research and Treatment , 2012,
Abstract:
Virus Budding/Host Interactions
Ronald N. Harty,Anthony P. Schmitt,Fadila Bouamr,Carolina B. Lopez,Claude Krummenacher
Advances in Virology , 2011, DOI: 10.1155/2011/963192
Abstract:
Cytopathogenesis of Vesicular Stomatitis Virus Is Regulated by the PSAP Motif of M Protein in a Species-Dependent Manner
Takashi Irie,Yuliang Liu,Barbara S. Drolet,Elena Carnero,Adolfo García-Sastre,Ronald N. Harty
Viruses , 2012, DOI: 10.3390/v4091605
Abstract: 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.
The YPLGVG sequence of the Nipah virus matrix protein is required for budding
Jared R Patch, Ziying Han, Sarah E McCarthy, Lianying Yan, Lin-Fa Wang, Ronald N Harty, Christopher C Broder
Virology Journal , 2008, DOI: 10.1186/1743-422x-5-137
Abstract: 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.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.Nipah virus (NiV) and Hendra virus (HeV) are emerging members of the family Paramyxoviridae that are distinguished by their ability to cause fatal disease in both animal and human hosts, and comprise the genus Henipavirus [1,2]. HeV was recognized as a novel paramyxovirus in 1994 during an outbreak in eastern Australia that resulted in the death of one human as a consequence of virus transmission from infected horses. Another person later died from relapsed encephalitis as a result of HeV infection that was identified retrospectively [3]. Repeated HeV spillover events have since occurred five times, all involving horses, with the most recent occurrence in July 2008 which also involved two human cases, one of which was fatal [4,5]. NiV was iden
High-fidelity two-qubit quantum logic gates using trapped calcium-43 ions
C. J. Ballance,T. P. Harty,N. M. Linke,D. M. Lucas
Physics , 2014,
Abstract: We study the speed/fidelity trade-off for a two-qubit phase gate implemented in $^{43}$Ca$^+$ hyperfine trapped-ion qubits. We characterize various error sources contributing to the measured fidelity, allowing us to account for errors due to single-qubit state preparation, rotation and measurement (each at the $\sim0.1\%$ level), and to identify the leading sources of error in the two-qubit entangling operation. We achieve gate fidelities ranging between $97.1(2)\%$ (for a gate time $t_g=3.8\mu$s) and $99.9(1)\%$ (for $t_g=100\mu$s), representing respectively the fastest and lowest-error two-qubit gates reported between trapped-ion qubits by nearly an order of magnitude in each case.
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