Gregory W Moseley

Monash University (Australia), Melbourne, Victoria, Australia

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Publications (27)124.47 Total impact

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    ABSTRACT: Background. Rabies virus (RABV) causes rabies disease resulting in >55,000 human deaths/year. The multifunctional RABV P-protein has essential roles in genome replication, and forms interactions with cellular STAT proteins that are thought to underlie viral antagonism of interferon-dependent immunity. However, the molecular details of P-protein-STAT interaction, and its importance to disease are unresolved.Methods. Studies were performed using sequence/structure analysis, mutagenesis, immunoprecipitation, luciferase and qRT-PCR-based signaling assays, confocal microscopy and reverse genetics/in vivo infection.Results. We identified a hydrophobic pocket of the P-protein C-terminal domain as critical to STAT-binding/antagonism. This interface was found to be functionally and spatially independent of the region responsible for N-protein interaction, which is critical to genome replication. Based on these findings, we generated the first mutant RABV lacking STAT-association. Growth of the virus in vitro was unimpaired, but it lacked STAT-antagonist function and was highly sensitive to interferon. Importantly, growth of the virus was strongly attenuated in brains of infected mice, producing no major neurological symptoms, compared with the invariably lethal wild-type virus.Conclusions. These data represent direct evidence that P-protein-STAT interaction is critical to rabies, and provide novel insights into the mechanism by which RABV coordinates distinct functions in interferon antagonism and replication.
    The Journal of Infectious Diseases 12/2013; · 5.85 Impact Factor
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    ABSTRACT: Bovine ephemeral fever virus (BEFV) is an arthropod-borne rhabdovirus that is classified as the type species of the genus Ephemerovirus. In addition to the five common rhabdovirus structural proteins (N, P, M, G and L), the large and complex BEFV genome contains several ORFs between the G and L genes (α1, α2/α3, β and γ) encoding proteins of unknown function. We show that the 10.5 kDa BEFV α1 protein is expressed in infected cells and, consistent with previous predictions based on its structure, has the properties of a viroporin. Expression of a BEFVα1-MBP-fusion protein in E. coli was observed to inhibit cell growth and increase membrane permeability to hygromycin-B. Increased membrane permeability was also observed in BEFV-infected mammalian cells (but not cells infected with an α1-defficient BEFV strain) and in cells expressing a BEFV α1-GFP fusion protein which was shown by confocal microscopy to localize to the Golgi complex. Furthermore, the predicted C-terminal cytoplasmic domain of α1, which contains a strong nuclear localization signal (NLS), was translocated to the nucleus when expressed independently and, in an affinity chromatography assay employing a GFP-trap, the full-length α1 was observed to interact specifically with importin β1 and importin 7, but not with importin α3. These data suggest that, in addition to its function as a viroporin, BEFV α1 may modulate components of nuclear trafficking pathways, but the specific role thereof remains unclear.
    Journal of Virology 11/2013; · 5.08 Impact Factor
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    ABSTRACT: Immune evasion by rabies virus depends on targeting of STAT1 and 2 proteins by the viral interferon-antagonist P-protein, but targeting of other STATs has not been investigated. Here, we find that P-protein associates with activated STAT3 and inhibits STAT3 nuclear accumulation and Gp130-dependent signaling. This is the first report of STAT3 targeting by the interferon-antagonist of a virus other than a paramyxovirus, indicating that STAT3 antagonism is important to a range of human pathogenic viruses.
    Journal of Virology 05/2013; · 5.08 Impact Factor
  • Michelle D Audsley, Gregory W Moseley
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    ABSTRACT: The paramyxoviruses are a family of > 30 viruses that variously infect humans, other mammals and fish to cause diverse outcomes, ranging from asymptomatic to lethal disease, with the zoonotic paramyxoviruses Nipah and Hendra showing up to 70% case-fatality rate in humans. The capacity to evade host immunity is central to viral infection, and paramyxoviruses have evolved multiple strategies to overcome the host interferon (IFN)-mediated innate immune response through the activity of their IFN-antagonist proteins. Although paramyxovirus IFN antagonists generally target common factors of the IFN system, including melanoma differentiation associated factor 5, retinoic acid-inducible gene-I, signal transducers and activators of transcription (STAT)1 and STAT2, and IFN regulatory factor 3, the mechanisms of antagonism show remarkable diversity between different genera and even individual members of the same genus; the reasons for this diversity, however, are not currently understood. Here, we review the IFN antagonism strategies of paramyxoviruses, highlighting mechanistic differences observed between individual species and genera. We also discuss potential sources of this diversity, including biological differences in the host and/or tissue specificity of different paramyxoviruses, and potential effects of experimental approaches that have largely relied on in vitro systems. Importantly, recent studies using recombinant virus systems and animal infection models are beginning to clarify the importance of certain mechanisms of IFN antagonism to in vivo infections, providing important indications not only of their critical importance to virulence, but also of their potential targeting for new therapeutic/vaccine approaches.
    World journal of virology. 05/2013; 2(2):57-70.
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    ABSTRACT: The evasion of host innate immunity by Rabies virus, the prototype of the genus Lyssavirus, depends on a unique mechanism of selective targeting of interferon-activated STAT proteins by the viral phosphoprotein (P-protein). However, the immune evasion strategies of other lyssaviruses, including several lethal human pathogens, are unresolved. Here, we show that this mechanism is conserved between the most distantly related members of the genus, providing important insights into the pathogenesis and potential therapeutic targeting of lyssaviruses.
    Journal of Virology 06/2012; 86(18):10194-9. · 5.08 Impact Factor
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    ABSTRACT: Regulated nucleocytoplasmic transport of proteins is central to cellular function and dysfunction during processes such as viral infection. Active protein trafficking into and out of the nucleus is dependent on the presence within cargo proteins of intrinsic specific modular signals for nuclear import (nuclear localization signals, NLSs) and export (nuclear export signals, NESs). Rabies virus (RabV) phospho (P) protein, which is largely responsible for antagonising the host anti-viral response, is expressed as five isoforms (P1-P5). The subcellular trafficking of these isoforms is thought to depend on a balance between the activities of a dominant N-terminal NES (N-NES) and a distinct C-terminal NLS (C-NLS). Specifically, the N-NES-containing isoforms P1 and P2 are cytoplasmic, whereas the shorter P3-P5 isoforms, which lack the N-NES, are believed to be nuclear through the activity of the C-NLS. Here, we show for the first time that RabV P contains an additional strong NLS in the N-terminal region (N-NLS), which, intriguingly, overlaps with the N-NES. This arrangement represents a novel nuclear trafficking module where the N-NLS is inactive in P1 but becomes activated in P3, concomitant with truncation of the N-NES, to become the principal targeting signal conferring nuclear accumulation. Understanding this unique switch arrangement of overlapping, co-regulated NES/NLS sequences is vital to delineating the critical role of RabV P protein in viral infection.
    Journal of Biological Chemistry 06/2012; 287(33):28112-21. · 4.65 Impact Factor
  • Gregory W Moseley, David A Jans
    Infectious disorders drug targets. 10/2011; 12(1):2-3.
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    ABSTRACT: Although the microtubule (MT) cytoskeleton has been shown to facilitate nuclear import of specific cancer-regulatory proteins including p53, retinoblastoma protein, and parathyroid hormone-related protein (PTHrP), the MT association sequences (MTASs) responsible and the nature of the interplay between MT-dependent and conventional importin (IMP)-dependent nuclear translocation are unknown. Here we used site-directed mutagenesis, live cell imaging, and direct IMP and MT binding assays to map the MTAS of PTHrP for the first time, finding that it is within a short modular region (residues 82-108) that overlaps with the IMPβ1-recognized nuclear localization signal (residues 66-108) of PTHrP. Importantly, fluorescence recovery after photobleaching experiments indicated that disruption of the MT network or mutation of the MTAS of PTHrP decreases the rate of nuclear import by 2-fold. Moreover, MTAS functions depend on mutual exclusivity of binding of PTHrP to MTs and IMPβ1 such that, following MT-dependent trafficking toward the nucleus, perinuclear PTHrP can be displaced from MTs by IMPβ1 prior to import into the nucleus. This is the first molecular definition of an MTAS that facilitates protein nuclear import as well as the first delineation of the mechanism whereby cargo is transferred directly from the cytoskeleton to the cellular nuclear import apparatus. The results have broad significance with respect to fundamental processes regulating cell physiology/transformation.
    Journal of Biological Chemistry 02/2011; 286(16):14335-51. · 4.65 Impact Factor
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    ABSTRACT: The fixed rabies virus (RV) strain Nishigahara kills adult mice after intracerebral inoculation, whereas the chicken embryo fibroblast cell-adapted strain Ni-CE causes nonlethal infection in adult mice. We previously reported that the chimeric CE(NiP) strain, which has the phosphoprotein (P protein) gene from the Nishigahara strain in the genetic background of the Ni-CE strain, causes lethal infection in adult mice, indicating that the P gene is responsible for the different pathogenicities of the Nishigahara and Ni-CE strains. Previous studies demonstrated that RV P protein binds to the interferon (IFN)-activated transcription factor STAT1 and blocks IFN signaling by preventing its translocation to the nucleus. In this study, we examine the molecular mechanism by which RV P protein determines viral pathogenicity by comparing the IFN antagonist activities of the Nishigahara and Ni-CE P proteins. The results, obtained from both RV-infected cells and cells transfected to express P protein only, show that Ni-CE P protein is significantly impaired for its capacity to block IFN-activated STAT1 nuclear translocation and, consequently, inhibits IFN signaling less efficiently than Nishigahara P protein. Further, it was demonstrated that a defect in the nuclear export of Ni-CE P protein correlates with a defect in its ability to cause the mislocalization of STAT1. These data provide the first evidence that the capacity of the RV P protein to inhibit STAT1 nuclear translocation and IFN signaling correlates with the viral pathogenicity.
    Journal of Virology 07/2010; 84(13):6699-710. · 5.08 Impact Factor
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    ABSTRACT: The nuclear envelope that encloses the nucleus is a significant barrier to non-viral vectors and shrouds the relationship between the trafficking of plasmid DNA to the nucleus and expression of an encoded transgene. Here, we use a novel single cell approach to quantify nuclear import of plasmid DNA following non-viral transfection and correlate this with reporter gene expression. Through the fractionation of intact nuclei from HeLa cells, the intranuclear copy number of plasmid DNA was quantified after transfection with either polyethylenimine (PEI) or LipofectAMINE2000 (LFA). Importantly, the use of a reporter protein that is incorporated into chromatin and retained in isolated nuclei permits analysis of gene expression by flow cytometry to be compared with nuclear plasmid delivery. PEI was found to mediate a greater and more rapid nuclear accumulation of plasmid DNA compared to LFA, but reporter gene expression was shown to be higher for LFA than PEI when an equivalent number of plasmids were in the nucleus. Sorting of the extracted nuclei according to the level of reporter expression demonstrated that reporter expression was dependent upon the number of plasmids delivered into the nucleus, with both threshold and saturation in expression evident with few or many nuclear plasmids. Our findings demonstrate formally that although the efficiency of plasmid nuclear delivery is a critical determinant of the level of transgene expression, intranuclear events also influence the transcriptional activity of the transgene, and must be taken into consideration when attempting to maximize the efficiency of non-viral vectors.
    The Journal of Gene Medicine 11/2009; 12(1):77-85. · 2.16 Impact Factor
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    ABSTRACT: Nucleocytoplasmic trafficking is a major consideration for the design of vehicles for the delivery of drug/DNA cargo to cell nuclei for cancer and gene therapies. Recent data indicate that efficient nuclear import can involve the microtubule (MT)/dynein network, such that nuclear delivery of exogenous cargo could be enhanced by attachment to peptide modules mediating association with dynein components, but this has not been investigated. Here, we report that the nuclear delivery of an exogenous cargo that enters the cell by protein transduction can be enhanced by attachment to a dynein-association sequence, with dependence on the MT network. This indicates that dynein/MT-association modules may provide useful modules for DNA/drug delivery approaches.
    Journal of Biotechnology 11/2009; 145(3):222-5. · 3.18 Impact Factor
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    ABSTRACT: Conventional nuclear import is independent of the cytoskeleton, but recent data have shown that the import of specific proteins can be either facilitated or inhibited by microtubules (MTs). Nuclear import of the P-protein from rabies virus involves a MT-facilitated mechanism, but here, we show that P-protein is unique in that it also undergoes MT-inhibited import, with the mode of MT-interaction being regulated by the oligomeric state of the P-protein. This is the first demonstration that a protein can utilise both MT-inhibited and MT-facilitated import mechanisms, and can switch between these different modes of MT interaction to regulate its nuclear trafficking. Importantly, we show that the P-protein exploits MT-dependent mechanisms to manipulate host cell processes by switching the import of the interferon-activated transcription factor STAT1 from a conventional to a MT-inhibited mechanism. This prevents STAT1 nuclear import and signalling in response to interferon, which is vital to the host innate antiviral response. This is the first report of MT involvement in the viral subversion of interferon signalling that is central to virus pathogenicity, and identifies novel targets for the development of antiviral drugs or attenuated viruses for vaccine applications.
    Journal of Cell Science 09/2009; 122(Pt 20):3652-62. · 5.88 Impact Factor
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    ABSTRACT: Members of the tetraspanin superfamily of proteins are implicated in a variety of complex cell processes including cell fusion. However, the contribution of individual tetraspanins to these processes has proved difficult to define. Here we report the use of recombinant extracellular regions of tetraspanins to investigate the role of specific members of this family in the fusion of monocytes to form multinucleated giant cells (MGC). In contrast to their positive requirement in sperm-egg fusion, previous studies using antibodies and knockout mice have indicated a negative regulatory role for tetraspanins CD9 and CD81 in this process. In an in vitro model of fusion using human monocytes, we have confirmed observations that antibodies to CD9 and CD81 enhance MGC formation; however, in contrast to previous investigations, we found that all members of a panel of antibodies to CD63 inhibited fusion. Moreover, recombinant proteins corresponding to the large extracellular domains (EC2s) of CD63 and CD9 inhibited MGC formation, whereas the EC2s of CD81 and CD151 had no effect. The potent inhibition of fusion and binding of labelled CD63 EC2 to monocytes under fusogenic conditions suggest a direct interaction with a membrane component required for fusion. Our findings indicate that the tetraspanins CD9, CD63 and CD81 are all involved in MGC formation, but play distinct roles.
    Immunology 07/2009; 127(2):237-48. · 3.71 Impact Factor
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    ABSTRACT: The identification of novel targets and strategies for therapy of microbial infections is an area of intensive research due to the failure of conventional vaccines or antibiotics to combat both newly emerging diseases (e.g. viruses such as severe acute respiratory syndrome (SARS) and new influenza strains, and antibiotic-resistant bacteria) and entrenched, pandemic diseases exemplified by HIV. One clear approach to this problem is to target processes of the host organism rather than the microbe. Recent data have indicated that members of the tetraspanin superfamily, proteins with a widespread distribution in eukaryotic organisms and 33 members in humans, may provide such an approach. Tetraspanins traverse the membrane four times, but are distinguished from other four-pass membrane proteins by the presence of conserved charged residues in the transmembrane domains and a defining 'signature' motif in the larger of the two extracellular domains (the EC2). They characteristically form promiscuous associations with one another and with other membrane proteins and lipids to generate a specialized type of microdomain: the tetraspanin-enriched microdomain (TEM). TEMs are integral to the main role of tetraspanins as 'molecular organizers' involved in functions such as membrane trafficking, cell-cell fusion, motility, and signaling. Increasing evidence demonstrates that tetraspanins are used by intracellular pathogens as a means of entering and replicating within human cells. Although previous investigations focused mainly on viruses such as hepatitis C and HIV, it is now becoming clear that other microbes associate with tetraspanins, using TEMs as a 'gateway' to infection. In this article we review the properties and functions of tetraspanins/TEMs that are relevant to infective processes and discuss the accumulating evidence that shows how different pathogens exploit these properties in infection and in the pathogenesis of disease. We then investigate the novel and exciting possibilities of targeting tetraspanins for the treatment of infectious disease, using specific antibodies, recombinant EC2 domains, small-molecule mimetics, and small interfering RNA. Such therapies, directed at host-cell molecules, may provide alternative options for combating fast-mutating or newly emerging pathogens, where conventional approaches face difficulties.
    BioDrugs 01/2009; 23(6):341-59. · 2.12 Impact Factor
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    ABSTRACT: Nucleocytoplasmic distribution of the rabies virus phosphoprotein is implicated in the evasion of cellular antiviral mechanisms by rabies virus and has been reported to depend on an N-terminal nuclear export sequence and a C-terminal nuclear localization sequence. This paper identifies a second nuclear export sequence that is located between key residues of the nuclear localization sequence in the phosphoprotein C-terminal domain. The C-terminal domain confers predominantly nuclear localization in unstimulated transfected cells, indicating that the nuclear localization sequence is the dominant signal at steady state. However, protein kinase-C activation or mutagenesis to mimic protein kinase-C phosphorylation at a site proximal to the C-terminal nuclear localization/export sequences shifts the targeting activity of the C-terminal domain toward nuclear exclusion, indicating that the nuclear export sequence becomes the dominant signal in activated cells. Mapping of these sequences within the three-dimensional structure of the C-terminal domain indicates that their activities may be coregulated by phosphorylation and/or conformational changes in the domain. The data are consistent with a model in which intimate positioning of the nuclear localization sequence, export sequence, and phosphorylation site within a single domain provides a switch mechanism to rapidly and efficiently balance the reciprocal import and export signals in response to cellular stimuli.
    Biochemistry 11/2007; 46(43):12053-61. · 3.38 Impact Factor
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    ABSTRACT: Macromolecules and supramolecular complexes are frequently required to enter and exit the nucleus during normal cell function, but access is restricted and exchange to and from the nucleus is tightly controlled. We describe the mechanisms which regulate nuclear import of endogenous molecules and indicate how viruses exploit these mechanisms during their life cycle. Opportunities exist to make use of natural pathways for delivery of therapeutic entities, in particular to develop safe and effective methods for gene therapy, although past attempts to design non-viral nuclear delivery systems have met with limited success. To increase the likelihood of success scientists will need an appreciation of the mechanisms by which viruses deliver their genomes to the nucleus, and will need a commitment to control the architecture of non-viral delivery systems at the molecular level. Effective delivery systems will require several attributes to facilitate endosomal escape, microtubular transport and uptake through the nuclear pore complex. The published literature provides a strong foundation for design of nuclear targeting systems. The challenge faced by delivery scientists is to assemble a system which is as effective as, for example, the adenovirus but which lacks its immunogenicity. This article reviews the relevant literature and indicates key areas for future research.
    Advanced Drug Delivery Reviews 09/2007; 59(8):698-717. · 12.89 Impact Factor
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    ABSTRACT: Nuclear localization sequence (NLS)-dependent nuclear protein import is not conventionally held to require interaction with microtubules (MTs) or components of the MT motor, dynein. Here we report for the first time the role of sequences conferring association with dynein light chains (DLCs) in NLS-dependent nuclear accumulation of the rabies virus P-protein. We find that P-protein nuclear accumulation is significantly enhanced by its dynein light chain association sequence (DLC-AS), dependent on MT integrity and association with DLCs, and that P-protein-DLC complexes can associate with MT cytoskeletal structures. We also find that P-protein DLC-AS, as well as analogous sequences from other proteins, acts as an independent module that can confer enhancement of nuclear accumulation to proteins carrying the P-protein NLS, as well as several heterologous NLSs. Photobleaching experiments in live cells demonstrate that the MT-dependent enhancement of NLS-mediated nuclear accumulation by the P-protein DLC-AS involves an increased rate of nuclear import. This is the first report of DLC-AS enhancement of NLS function, identifying a novel mechanism regulating nuclear transport with relevance to viral and cellular protein biology. Importantly, this data indicates that DLC-ASs represent versatile modules to enhance nuclear delivery with potential therapeutic application.
    Molecular Biology of the Cell 09/2007; 18(8):3204-13. · 4.60 Impact Factor
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    ABSTRACT: Nuclear protein import is dependent on specific targeting signals within cargo proteins recognized by importins (IMPs) that mediate translocation through the nuclear pore. Recent evidence, however, implicates a role for the microtubule (MT) network in facilitating nuclear import of the cancer regulatory proteins parathyroid hormone-related protein (PTHrP) and p53 tumor suppressor. Here we assess the extent to which MT and actin integrity may be generally required for nuclear protein import for the first time. We examine 10 nuclear-localizing proteins with diverse IMP-dependent nuclear import pathways, our results indicating that the cytoskeleton does not have a general mechanistic role in nuclear localization sequence-dependent nuclear protein import. Of the proteins examined, only the p110(Rb) tumor suppressor protein Rb, together with p53 and PTHrP, was found to require MT integrity for optimal nuclear import. Fluorescence recovery after photobleaching experiments indicated that the MT-dependent nuclear transport pathway increases both the rate and extent of Rb nuclear import but does not affect Rb nuclear export. Dynamitin overexpression experiments implicate the MT motor dynein in the import process. The results indicate that, additional to IMP/diffusion-dependent processes, certain cancer regulatory proteins utilize an MT-enhanced pathway for accelerated nuclear import that is presumably required for their nuclear functions.
    Traffic 07/2007; 8(6):673-86. · 4.65 Impact Factor
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    ABSTRACT: Human immunodeficiency virus type 1 (HIV-1) infection of human macrophages can be inhibited by antibodies which bind to the tetraspanin protein CD63, but not by antibodies that bind to other members of the tetraspanin family. This inhibitory response was limited to CCR5 (R5)-tropic virus and was only observed using macrophages, but not T cells. Here, we show that recombinant soluble forms of the large extracellular domain (EC2) of human tetraspanins CD9, CD63, CD81, and CD151 produced as fusion proteins with glutathione S-transferase (GST) can all potently and completely inhibit R5 HIV-1 infection of macrophages with 50% inhibitory concentration values of 0.11 to 1.2 nM. Infection of peripheral blood mononuclear cells could also be partly inhibited, although higher concentrations of EC2 proteins were required. Inhibition was largely coreceptor independent, as macrophage infections by virions pseudotyped with CXCR4 (X4)-tropic HIV-1 or vesicular stomatitis virus (VSV)-G glycoproteins were also inhibited, but was time dependent, since addition prior to or during, but not after, virus inoculation resulted in potent inhibition. Incubation with tetraspanins did not decrease CD4 or HIV-1 coreceptor expression but did block virion uptake. Colocalization of fluorescently labeled tetraspanin EC2 proteins and HIV-1 virions within, and with CD4 and CXCR4 at the cell surfaces of, macrophages could be detected, and internalized tetraspanin EC2 proteins were directed to vesicular compartments that contained internalized dextran and transferrin. Collectively, the data suggest that the mechanism of inhibition of HIV-1 infection by tetraspanins is at the step of virus entry, perhaps via interference with binding and/or the formation of CD4-coreceptor complexes within microdomains that are required for membrane fusion events.
    Journal of Virology 08/2006; 80(13):6487-96. · 5.08 Impact Factor
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    Journal of Virology 10/2005; 79(17):10839-51. · 5.08 Impact Factor

Publication Stats

606 Citations
124.47 Total Impact Points

Institutions

  • 2004–2013
    • Monash University (Australia)
      • Department of Biochemistry and Molecular Biology
      Melbourne, Victoria, Australia
  • 2005
    • The University of Sheffield
      • Academic Urology Unit
      Sheffield, ENG, United Kingdom
  • 2003
    • University of Melbourne
      Melbourne, Victoria, Australia