NEUROTROPIC VIRUS TRANSPORT

Abstract: A method of preventing or reducing transport of a neurotropic virus within a neuron, the method comprising altering or preventing interaction between a structural tegument protein of the virus and a motor protein in the neuron such that virus transport in the neuron is prevented or reduced. An antiviral composition comprising a compound capable of altering or preventing interaction between a structural tegument protein of a neurotropic virus and a motor protein in a neuron or cell.

Description: NEUROTROPIC VIRUS TRANSPORT Technical Field The present invention relates to viral infections, particularly cellular and tissue interactions during infection. Background Art Little is known about the mechanisms of transport of neurotropic viruses, such as Herpes simplex virus (HSV). varicella-zoster virus and rabies, within neurones. For these viruses, which replicate in the nucleus, anterograde transport from the cell body of dorsal root human ganglionic (DRG) neurons to the axon terminus occurs over long distances. In the case of HSV. there is increasing evidence for separate, fast anterograde axonal transport of viral nucleocapsids coated with tegument along microtubules (MTs) and of glycoproteins in axonal transport vesicles. An understanding of the mechanisms of transport of these viruses would be of benefit. Once this information is obtained, means to interrupt or prevent this transport would be a good candidate for antiviral therapy. HSV consists of an outer lipid envelope containing glycoproteins which surrounds the tegument. The inner capsid encloses the viral DNA to form the nucleocapsid. Early work has shown that HSV retrograde axonal transport in rat neurons utilizes microtubules (K. Kristensson. E. Lycke, J. Sjostrand. Ada Neuropathol. 17. 44. 1971). Previously, the present inventors have used a two chamber system to examine the mechanism of anterograde transport of HSV from infected dorsal root ganglionic (DRG) neurones in the central chamber along axonal fascicles to epidermal explants in the outer chamber. The velocity of viral transport (0.6 μm/s) indicated a mechanism based on fast anterograde transport (M. E. Penfold. P. Armati. A. L. Cunningham. Proc. Natl. Acad. Sci. USA 91. 6529. 1994). Using freeze- substitution transmission immuno-electron microscopy (TIEM) the virus was shown to be transported as unenveloped nucleocapsids. coated with tegument, separately from glycoproteins which are transported in vesicles (D. J. Holland. M. Miranda-Saksena, R. A. Boadle, P. J. Armati. A. L. Cunningham. /. Virol. 73. 8476. 1999). These findings were supported by experiments with nocodazole. an inhibitor of microtubule polymerization, and brefeldin A which inhibits transport through the Golgi. The transport of all three viral components, nucleocapsid. tegument and glycoproteins was inhibited by nocodazole. indicating their dependence on microtubule- associated transport. Brefeldin A, however, inhibited glycoprotein transport but not nucleocapsids confirming that the two are transported along separate pathways and indicating the close association of HSV glycoproteins with Golgi/intermediate compartment membranes. The present inventors have now obtained useful evidence for the direct interaction between a viral structural tegument protein and an ubiquitous cellular protein. This new information has been used to develop methods and compositions to alter viral transport and movement in cells. Disclosure of Invention In a first aspect, the present invention provides a method of preventing or reducing transport of a neurotropic virus within a neuron or cell, the method comprising altering or preventing interaction between a structural tegument protein of the virus and a motor protein in the neuron or cell such that virus transport in the neuron is prevented or reduced. In a preferred embodiment, the neurotropic virus is Herpes simplex virus (HSV). varicella-zoster virus, or rabies virus. More preferably, the virus is HSV. the structural tegument protein is USll, and the motor protein is kinesin. The method according to the invention can be carried out by providing a compound to a neuron or cell capable of altering or preventing interaction between a structural tegument protein of a neurotropic virus and a motor protein in a neuron or cell. In one preferred form, the compound is a motor protein-like molecule which binds the structural tegument protein thereby preventing the normal interaction of the virus with cellular motor protein. The motor protein-like molecule can be any suitable molecule and would include mimics of the motor protein or a parts of the motor protein to which the structural tegument protein binds or specifically interacts. When the motor protein is kinesin in the case of HSV, the kinesin-like molecule can be any suitable molecule and would include mimics of kinesin or a parts of kinesin to which the structural tegument protein USll of HSV binds or specifically interacts. In another preferred form, the compound is a structural tegument-like molecule which binds to a motor protein of a neuron or cell thereby preventing the normal interaction between the neuron or cell with the virus. The structural tegument-like molecule can be a mimic of a viral tegument protein or a part of the tegument protein to which a cellular motor protein binds or specifically interacts. Preferably, the virus is Herpes simplex virus, the structural tegument protein is USll. motor protein is kinesin and the structural tegument-like molecule is a mimic of USll or a part of USll to which the motor protein kinesin binds or specifically interacts. Knowing how these types of viruses move in neurons to infect neurons other cells and ultimately form lesions is an important advance and is useful for the development of antiviral strategies. Usually, during the latent state these viruses do not cause any real problems to an individual. It is during the active phase of the virus where clinical manifestations occur and suffering is caused. Preventing the actual transport of the virus down infected neurons should at least combat or prevent the clinical symptoms of re-infection. Similarly, preventing movement up the neuron may prevent the formation of latent state of the virus in ganglia or at least reduce the incidence of re-occurrence of manifestations of the disease in infected individuals. In a second aspect, the present invention provides a modified neurotropic virus which has lost or has reduced ability to be transported in neurons, the virvis comprising a mutation in a tegument protein such that the mutated tegument protein has abnormal interaction with a cellular motor protein. In a third aspect, the present invention provides use of a compound to prevent or reduce the interaction between a neurotropic viral tegument protein with a cellular motor protein to prevent or reduce viral transport in a neuron. In a preferred embodiment of the second and third aspects of the present invention, the neurotropic virus is Herpes simplex virus (HSV), varicella-zoster virus, or rabies virus. More preferably, the virus is HSV. the structural tegument protein is USll. and the motor protein is kinesin. In a fourth aspect, the present invention provides an antiviral composition comprising a compound capable of altering or preventing interaction between a structural tegument protein of a neurotropic virus and a motor protein in a neuron. Preferably, the neurotropic virus is selected from the group consisting of Herpes simplex virus, varicella-zoster virus, and rabies virus. Preferablv. the virus is Herpes simplex virus, the structural tegument protein is USll. and the motor protein is kinesin. In one preferred form, the compound is a motor protein-like molecule which binds to the structural tegument protein of the virus thereby preventing normal interaction of the virus with cellular motor protein of a neuron. Preferably, the motor protein-like molecule comprises a mimic of a cellular motor protein or a part of the motor protein to which a structural tegument protein of a virus binds or specifically interacts. In a further preferred form, the virus is Herpes simplex virus, the motor protein is kinesin and the motor protein-like molecule is a mimic of kinesin or a part of kinesin to which the structural tegument protein USll of Herpes simplex virus binds or specifically interacts. In another preferred form, the compound is a structural tegument-like molecule which binds to a motor protein of a neuron or cell thereby preventing the normal interaction between the neuron or cell with the virus. The structural tegument-like molecule can be a mimic of a viral tegument protein or a part of the tegument protein to which a cellular motor protein binds or specifically interacts. Preferably, the virus is Herpes simplex virus, the structural tegument protein is USll. motor protein is kinesin and the structural tegument-like molecule is a mimic of USll or a part of USll to which the motor protein kinesin binds or specifically interacts. The composition can further include additives, diluents, excipients and the like which can be used in clinical and health situations. This study raises many questions about the mechanisms of tegument formation, location of USll in the tegument of HSV. the potential role of motor proteins other than kinesin(s) and homologous interactions mediating the transport of other neurotropic viruses. Studies to map minimal binding regions of USll and uKHC and identification of other cellular factors which facilitate or contribute to the interaction will be useful. Inhibition of this interaction by peptides and/or peptidomimetic analogues could provide a new strategy for antiviral treatment for this and other neurotropic viruses. Incorporation of deletions into specifically attenuated live HSV (or other attenuated live neurotropic viruses) vaccine candidates could also be useful to prevent clinical recrudescence. Throughout this specification, unless the context requires otherwise, the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps. In order that the present invention may be more clearly understood, preferred forms will be described with reference to the following examples and drawings. Brief Description of Drawings Figure 1. Transmission immuno-electron micrographs of human fetal axons labelled with 5 and 10 nra immunogold particles recognising HSV major capsid protein VP5 and the stalk of human uKHC respectively. Arrows = 5 n gold particles. Arrowheads = 10 nm gold particles. Bars = 200 nm (A and C) or 100 nm (B). A Clustering of immunogold label for uKHC around axonal vesicles and diffuse labelling of other axons. Labelling with preimmune sera produced only sparse background label. B Colocalization of the two immunogold labels against uKHC and VP5 over a dense unenveloped viral particle in an axon. Arrows = VP5 (5 nm gold particle). C Extracellullar virus labelled only with anti-VP5. In the upper right corner is an axon with diffuse 10 nm immunolabel against uKHC. There is no immunolabel for uKHC over extracellular virus with only sparse background label outside axons. Figure 2. Identification of kinesin-binding HSVl proteins. Mock or virus-infected Hep-2 cell lysates were incubated, as indicated ( + ), with His- KHC fragments bound to nickel-activated beads. Eluted kinesin-viral protein complexes were analysed by immunoblotting with appropriate antibodies. A Polyclonal antibody to HSVl identified major viral bands at 65 and 20 kDa bound preferentially to His-KHCstalk/tail. B-C The viral proteins were identified with monoclonal antibodies as VP16 (B) and USll (C). respectively. In each immunoblot, the first two lanes are controls for antibody specificity. Figure 3. Direct binding of USll to uKHC and VP16. Shown are immunoblots of protein complexes eluted from nickel-activated beads. A Lysates of bacteria expressing untagged KLC or USll were incubated, as indicated ( + ). with His-KHC fragments. USll bound only to His- KHCstalk/tail in the absence of other HSVl proteins (lane 2, bottom panel). Overnight preincubation of the His-KHC fragments with KLC, which binds only to His-KHCstalk/tail (lane 4, top panel), prior to addition of USll. also showed binding of USll but this was slightly reduced (lane 4. bottom panel). B In a similar experiment, recombinant untagged VP16 did not bind to either His-KHC fragment (lanes 2 and 3). Expression of VP16 in bacterial lysates was confirmed in lane 1. Native VP16 from HSVl-infected Hep-2 cells (lanes 4 and 5) or recombinant untagged VP16 (lanes 6 and 7) was incubated with His-USll or His-KHCstalk. Both forms of VP16 interacted with His-USll. Figure 4. Transport of structural HSV antigens into axons of infected dissociated rat neonatal neurons in vitro. Dissociated DRG neurons were infected with either wt HSVl (CW strain), a USll deletion mutant of HSVl (R6604) or a USll rescuant of R6604 (R6606) at 20 pfu/neuron. The appearance and transport into the principle axon of HSVl tegument (USll), nucleocapsid (VP5) and envelope glycoprotein (gC) antigens was followed by serial fixation over 6 to 36 h. and then immunofluorescence and confocal microscopy. (A-I) HSVl antigens in the axon at 24 h post infection (hpi). Bars = 10 μm (A and C) or 25 μm (B and D-I). USll was present in axons infected with wt HSVl (A) but was completely absent from neurons infected with R6604 (B). USll was expressed in neurons and present in axons infected with R6606 (C). VP5 was present in axons infected with wt HSVl (D) but was not detected in axons infected with R6604 (E). VP5 was present in axons infected with R6606 (F). gC was detected in axons whether infected with wt HSVl (G). R6604 (H) or R6606 (I). Figure 5. Ultrastructural distribution of enveloped and unenveloped nucleocapsids from an HSVl USll deletion mutant (R6604) in dissociated rat DRG neurons (26 hpi). A Unenveloped (arrow) and enveloped (arrow head) nucleocapsids adjacent to the Golgi (G) within the cytoplasm of the cell body. B Enveloped virions (arrow) in the extracellular space adjacent to the cell body and longitudinal section of an axonal process (arrowhead) containing microtubules. Modes for Carrying Out the Invention METHODS Antibody HisKHC555-772. a oligohistidine (His)-tagged fragment of human uKHC (R. J. Diefenbach. }. P. Mackay. P. }. Armati, A. L. Cunningham. Biochemistry 37. 16663. 1998). was used to generate chicken polyclonal anti- uKHC. The cell pellet from a 1 litre bacterial culture expressing HisKHC555- 772 was resuspended in 25 ml of column running buffer (20 mM sodium diphosphate. pH 7.4. 0.5 M NaCl). lysed by sonication. clarified by centrifugation. and applied to a POROS 20 MC column (Perseptive Biosystems: 4.6 x 10 mm) which had been charged with nickel according to the manufacturer ' s instructions. The His-tagged fusion protein was eluted at 5 ml/min with 15 column volumes of a gradient from 0 to 0.5 M imidazole in running buffer. Pooled fractions containing purified HisKHC555-772 were then dialysed against PBS. Protein concentrations were determined using a Bio-Rad protein assay. A total of 2 mg of protein was then used to raise a polyclonal antibody in chickens (a service provided by the Institute of Medical and Veterinary Science. Veterinary Services Division. Gilles Plains, South Australia). Pre- and post-immune serum from a single immunised chicken was subsequently used in this study. The antibody was shown to be specific in immunoblots for human uKHC by comparing reactivity with the immunising antigen against human neuronal (n) KHC (expressed as a His- tagged fragment spanning amino acids 409-1032). cDNAs for uKHC (F. Navone. et al. J. Cell Biol. 117. 1263. 1992) and nKHC (J. Niclas. F. Navone, N. Hom-Booher. R. D. Vale. Neuron 12. 1059. 1994)) were provided by R. Vale. HisKHC Proteins The HisKHC proteins were expressed, harvested, bound to. and eluted from nickel-activated beads as previously described (R. J. Diefenbach, }. P. Mackay. P. J. Armati. A. L. Cunningham, Biochemistry 37 , 16663. 1998). Generation of virus stocks and titrations of wt HSVl (CWl) and modified viruses were performed in Hep-2. For binding assays. Hep-2 cells were infected at a multiplicity of 5 pfu/cell and incubated for 24 h. Cell monolayers were washed twice with PBS. resuspended in PBS (1 x 10 6 cells/ml), freeze-thawed 3 times, sonicated for 15 s, before addition of 1% (v/v) NP-40 and incubation for 1 h at 4°C. The soluble fraction was harvested by centrifugation at 4°C (lOOOOg for 15 min) before addition (1 ml) to HisKHC fragments on nickel-activated beads. Beads were incubated overnight with rocking at 4°C. washed with 3 x 20 volumes of wash buffer (150 mM NaCl. 120 mM imidazole. 20 mM Tris-HCl . pH 7.9). prior to elution of bound protein complexes. Protein complexes were separated by SDS-PAGE and identified by immunoblotting as previously described (R. J. Diefenbach, J. P. Mackay. P. J. Armati. A. L. Cunningham, Biochemistry 37, 16663. 1998). Antibodies used included mouse monoclonal anti-US 11 (R. J. Roller. B. Roizman. /. Virol. 66. 3624. 1992) (provided by B. Roizman). mouse monoclonal anti-VPl6 (C. McLean, et al.. J. Gen. Virol. 63. 297, (1982) (LPl; provided by T. Minson) and rabbit polyclonal anti-HSVl from Dako. Rabbit polyclonal antibody to HSVl capsid protein VP5 was provided by G. Cohen and R. Eisenberg (G. H. Cohen, et al.. J. Virol. 34. 521. 1980). Mouse monoclonal antibody to HSVl gC and KLC (L2) was obtained from Chemicon International. Plasmids Plasmid pRB4766 (H. Tsiang. E. Lycke. P. E. Ceccaldi, A. Ermine, X. Hirardot. /. Gen. Virol. 70. 2075. 1989) containing HSVl USll genomic DNA in pGEX-KG was provided by B. Roizman. An untagged USll construct was generated by digestion of pRB4766 with Ncol and insertion into the Ncol site of pET-28a. This resulted in an additional five amino acids (MGRLE) at the N- terminus of USll. His-tagged USll was constructed by inserting an EcoRL/Fspl USll-containing fragment from pRB4766 into EcoRI/Hindlll (Klenow filled-in) digested pΕT-28c. Between the oligohistidine tag and USll sequence are inserted amino acids LDSMGRLE. Genomic DNA containing HSVl VP16 was provided by P. O'Hare (T. A. Hughes. S. La Boissiere. P. O'Hare. /. Biol. Chem. 274. 16437. 1999: D. O'Reilly. O. Hanscombe. P. O'Hare, EMBO J. 16. 2420. 1997). The plasmid pP054 consisted of the gene for VP16 inserted into the BαmHI site of pBS. An untagged VP16 construct was generated by firstly digesting pP054 with BαmHI and inserting into the BαmHI site of pET-28c. VP16 was subsequently released from pET-28c by digestion with BamHI/Xhol and inserted into the yeast vector pACT2 also cut with BamHI/Xhol. VP16 was then released with an Ncol/Xhol digest and reinserted into the Ncol/Xhol site of pET-28a to allow expression of untagged VP16. The resulting fusion protein had an additional nineteen amino acids (MEAPGIRDPRSSFPYQPHP) at the N-terminus of VP16. Bacteria expressing untagged USll. VP16 or KLC (R. J. Diefenbach, J. P. Mackay. P. J. Armati. A. L. Cunningham, Biochemistry 37, 16663, 1998) were lysed in normal salt binding buffer (150 mM NaCl. 5 mM imidazole, 20 mM Tris-HCl . pH 7.9) for use in binding assays as described above. Virus Mutants R6604 is a partial USll gene deletion mutant which does not express the USll protein and R6606 is USll rescuant of R6604. To generate the HSVl mutant R6604 (USll-). a plasmid carrying a deletion in the USll coding sequence was constructed (pRB5173) by deleting the sequences present between BspEl and Xlwl in the BαmHI Z fragment of HSVl(F) genome. The 132 nucleotides deleted from the fragment removed all three ATG codons of the USll ORF without affecting the adjacent US12 ORF. The plasmid was cotransfected with the R3630 recombinant virus DNA containing the thymidine kinase gene inserted into US12. The selected recombinant progeny (R6603) contained a USll gene lacking the 132 nucleotides but an intact US12. The thymidine kinase gene at its natural locus was then restored to yield R6604. The genotype and phenotype of all recombinants was verified by Southern analysis and immunoreactivity of its proteins. RESULTS The candidate motor proteins for viral transport should be those involved in microtubule-dependent fast axonal anterograde transport of organelles. most likely the conventional kinesins (ubiquitous and neuronal) or possibly the kinesin-related protein KIF3. The colocalization of kinesin and HSVl nucleocapsids was examined in fetal human neurons using the DRG neuron-epidermal explant two chamber model system as previously described (D. J. Holland. M. Miranda-Saksena. R. A. Boadle. P. }. Armati, A. L. Cunningham. /. Virol. 73. 8476. 1999). Anterograde axonal transport of HSVl (CW strain: D. J. Holland. M. Miranda-Saksena, R. A. Boadle. P. J. Armati. A. L. Cunningham, /. Virol. 73. 8476, 1999) was visualized using freeze-substitution TIEM. Dual immunogold labelling (5 and 10 nm gold particles) of axons was performed with chicken polyclonal antibody to ubiquitous kinesin heavy chain (uKHC) and rabbit polyclonal antibody to HSVl major capsid protein VP5 (G. H. Cohen, et al.. J. Virol. 34. 521. 1980). Anti-uKHC showed diffuse labelling throughout axons with clusters of immunolabel for uKHC observed around electron dense axonal vesicles (Figure lA). Only sparse background label was found extra-axonally. With pre-immune sera only sparse background immunolabel was present on sections and there was no labelling of axonal vesicles. Immunolabel for uKHC and VP5 was found in and around unenveloped HSVl nucleocapsids, respectively, within axons (Figure IB). The anti-VP5 antibody was specific with no labelling of axons in mock-infected cultures. Only VP5 and not kinesin immunolabel was present on extracellular virus (Figure 1C). The immunolabelling for uKHC over intra-axonal virus (but not extra-axonal virus) was as intense as that around axonal vesicles and provides strong direct evidence that kinesin is involved in the anterograde axonal transport of HSV. The tegument protein VP16 has been demonstrated to colocalize with VP5-labelled nucleocapsids during anterograde transport (D. J. Holland, M. Miranda-Saksena. R. A. Boadle. P. J. Armati. A. L. Cunningham, /. Virol. 73, 8476. 1999). Therefore, tegument or outer capsid proteins are candidates for binding to conventional ubiquitous kinesin. Conventional kinesin is a heterotetramer consisting of two identical heavy and two identical light chains. The heavy chain has a three domain structure consisting of a N-terminal motor domain, highly conserved among the kinesin superfamily. a central stalk domain containing heptad repeats and a C-terminal tail domain. The present inventors used fragments of human uKHC tagged at the N-terminus with an oligohistidine (His) sequence, which were generated and characterised previously, as bait to identify kinesin-binding HSVl proteins. In this work, the fragments were designated His-KHCstalk (amino acid residues 555-772) and His-KHCstalk/tail (residues 771-963). HisKHC fragments attached to nickel-activated beads were incubated with a lysate of mock or HSVl-infected Hep-2 cells. Analysis of eluted complexes by immunoblotting with anti-HSVl identified predominantly a 65 and 20 kDa viral protein preferentially bound to KHCstalk/tail (Figure 2A). These viral proteins were subsequently identified as the tegument proteins VP16 (Figure 2B) and USll (Figure 2C). To establish whether USll and/or VP16 bound directly to uKHC. untagged forms of both were expressed in Escherichia coli. Addition of untagged USll to HisKHC showed that USll binds specifically to KHCstalk/tail (Figure 3 A. bottom panel). This also showed that the binding of USll to uKHC can occur in the absence of other viral proteins although it may well be modulated in vivo by viral or cellular factors. A complex of uKHC and KLC was generated by preincubating HisKHCstalk/tail with untagged KLC (Figure 3 A. top panel). This kinesin heavy/light chain complex also binds USll. though at a slightly reduced level when compared with heavy chain alone (Figure 3 A. bottom panel lanes 2 and 4). The minor reduction in USll binding is probably due to steric effects and not competitive inhibition since KLC and USll bind to different regions of HisKHCstalk tail. Therefore. KLC appears not to be directly involved in binding USll as there was no increase in the level of USll bound to a heavy/light chain complex compared with heavy chain alone. A similar observation has been documented using a KHCstalk/tail fragment from sea urchin which can bind to vesicles in the absence of KLC. In contrast. VP16 does not bind directly to uKHC (Figure 3B). The presence of VP16 co-eluting with KHC after incubation with viral lysates can be explained by the observation that VP16 binds directly to USll (Figure 3C). This binding was observed with both native and recombinant VP16 (in the absence of other viral proteins). Therefore uKHC binds USll which in turn binds VP16. This VP16/US11 interaction may represent an important structural interaction for USll in the tegument of the virus or may play a regulatory role in infected cells. VP16 has also been shown to bind to other tegument proteins including vhs (C. A. Smibert, B. Popova, P. Xiao, J. P. Capone. J. R. Smiley. /. Virol. 68. 2339. 1994) and VP22 (G. Elliott, G. Mouzakitis. P. O'Hare. /. Virol. 69. 7932. 1995), as well as to glycoprotein (gD; Q. Zhu. R. ). Courtney. Virology 204. 590. 1994). Therefore USll. a structural viral protein proposed to be part of the tegument, may also interact with one or both of these other tegument proteins. USll appears to be a multi-functional protein. It is a basic phosphoprotein which has RNA-binding activity, stably associates with 60S ribosomal subunits. has a role in post-transcriptional regulation of gene expression and localizes to the nucleoli. The RNA-binding properties of USll are dependent on the 20-24 RXP-repeats in the carboxy-half of the protein while the amino-half of USll is required for transactivation of gene expression. It has been suggested that the N-terminal domain may be involved in transport and translation of mRNA from the nucleus to the cytoplasm. This amino domain may well also bind to kinesin. The USll protein is well conserved between HSVl and 2 (63% homology) with the greater variation at the N-terminus. There are no USll homologues in varicella-zoster virus or rabies suggesting this virus may use different proteins for anterograde axonal transport. However, there are precedents though within the herpesviruses for similar functions on different proteins (eg entry into cells via gD in HSV (J. Rajcani. A. Vojvodova. Acta Virol. 42, 103. 1998) and via gE/I and gB in varicella zoster virus C. Grose, Rev. Infect. Dis. Suppl. 11. S960. 1991). To test the hypothesis that the USll-uKHC interaction mediated anterograde transport of HSV into the axons of rat neonatal DRG neurons, the axonal transport of wild-type (wt) HSVl. a USll deletion mutant of HSVl and its rescuant were examined. The appearance and distribution of structural HSV protein antigens from the tegument (USll). nucleocapsid (VP5) and envelope (gC) and their anterograde transport into axons of dissociated rat neonatal DRG was followed by fixation at serial time points over 24 hours and immunofluorescence/confocal microscopy. Firstly. USll antigen from wt HSVl (CW clinical strain), was demonstrated to move down the axon (a criterion for its proposed role in anterograde axonal transport: Figure 4A). The protein was not expressed in the USll deletion mutant of HSVl. R6604. but was present in the USll rescuant of R6604. R6606 (Figure 4B and C). The kinetics of transport of USll into axons was similar for wt and R6606 (Figure 4A and C). In neurons infected with wt HSVl. VP5 antigen was diffusely distributed in the cytoplasm of the cell body and transported throughout the axon by 24 hours (Figure 4D). The kinetics of transport of USll and VP5 from wt HSVl into axons was similar, supporting colocalization of USll with nucleocapsids (Figure 4A and D). In contrast, deletion of USll in R6604 abrogated nucleocapsid transport into axons since no VP5 antigen could be detected in the axons of infected neurons (Figure 4E), although there was a similar diffuse cytoplasmic distribution of VP5 to wt HSVl (Figure 4D and E). Rescue of USll expression in R6606 restored HSVl nucleocapsid transport into and along axons at a rate similar to wt (Figure 4F). Anterograde transport of gC from either wt HSVl. R6604 or R6606 into axons was unaffected (Figure 4G-I). For R6604. transmission electron microscopy at late time points (26 hours) showed large numbers of enveloped virions in the extracellular space and in the cytoplasm of the cell body of neurons, concentrated around the Golgi (Figure 5 A and B). Unenveloped nucleocapsids were in variable abundance in the nuclei and sparsely scattered in the perinuclear cytoplasm, often adjacent to the Golgi (Figure 5A). Thus the absence of USll does not prevent transport across the nuclear membrane, normal envelopment and viral egress in the cell body. Thus, there is no observable functional disruption of the tegument. These findings suggest that there are two separate mechanisms of viral egress, one via the cell body and the other via axon termini. The absence of USll appears to disrupt the latter pathway between the cell body and the axon. The presence of USll in the tegument, the similar kinetics of anterograde transport of USll and nucleocapsid VP5. and the inability of a HSV mutant deleted in USll to be transported into axons is consistent with the hypothesis that the observed USll-uKHC interaction has a major role in mediating anterograde transport of nucleocapsids into and along axons. Knowing how these types of viruses move in neurons to infect neurons, other cells and ultimately form lesions is an important advance and is useful for the development of antiviral strategies. Usually, during the latent state these viruses do not cause any real problems to an individual. It is during the active phase of the virus where clinical manifestations occur and suffering is caused. Preventing the actual transport of the virus down infected neurons should at least combat or prevent the clinical symptoms of re-infection. Similarly, preventing movement up the neuron may prevent the formation of latent state of the virus in ganglia or at least reduce the incidence of re-occurrence of manifestations of the disease in infected individuals. This work raises many possibilities about the mechanisms of tegument formation, location of USll in the tegument of HSV. the potential role of motor proteins other than kinesin(s) and homologous interactions mediating the transport of other neurotropic viruses. Inhibition of this interaction by peptides and/or peptidomimetic analogues could provide a new strategy for antiviral treatment for this and other neurotropic viruses. Incorporation of deletions into specifically attenuated live HSV (or other attenuated live neurotropic viruses) vaccine candidates could also be useful to prevent clinical recrudescence It will be appreciated by persons skilled in the art that numerous variations and/or modifications may be made to the invention as shown in the specific embodiments without departing from the spirit or scope of the invention as broadly described The present embodiments are. therefore, to be considered in all respects as illustrative and not restrictive

Claims: CLAIMS: 1. A method of preventing or reducing transport of a neurotropic virus within a neuron or cell, the method comprising altering or preventing interaction between a structural tegument protein of the virus and a motor protein in the neuron or cell such that virus transport in the neuron or cell is prevented or reduced. 2. The method according to claim 1 wherein the neurotropic virus is selected from the group consisting of Herpes simplex virus, varicella-zoster virus, and rabies virus. 3. The method according to claim 2 wherein the virus is Herpes simplex virus, the structural tegument protein is USll. and the motor protein is kinesin. 4. The method according to any one of claims 1 to 3 comprising providing to a neuron or cell a compound capable of altering or preventing interaction between a structural tegument protein of a neurotropic virus and a motor protein in the neuron or cell. 5. The method according to claim 4 wherein the compound is a motor protein-like molecule which binds to a structural tegument protein of the virus thereby preventing the normal interaction of the virus and neuron or cell. 6. The method according to claim 5 wherein the motor protein-like molecule comprises a mimic of a cellular motor protein or a part of the motor protein to which the structural tegument protein binds or specifically interacts. 7. The method according to claim 6 wherein the virus is Herpes simplex virus, the motor protein is kinesin and the motor protein-like molecule is a mimic of kinesin or a part of kinesin to which the structural tegument protein USll of Herpes simplex virus binds or specifically interacts. 8. The method according to claim 4 wherein the compound is a structural tegument-like molecule which binds to a motor protein of a neuron or cell thereby preventing the normal interaction between the neuron or cell with the virus. 9. The method according to claim 8 wherein the structural tegument-like molecule comprises a mimic of a viral tegument protein or a part of the tegument protein to which a cellular motor protein binds or specifically interacts. 10. The method according to claim 9 wherein the virus is Herpes simplex virus, the structural tegument protein is USll. motor protein is kinesin and the structural tegument-like molecule is a mimic of USll or a part of USll to which the motor protein kinesin binds or specifically interacts. 11. An antiviral composition comprising a compound capable of altering or preventing interaction between a structural tegument protein of a neurotropic virus and a motor protein in a neuron or cell. 12. The antiviral composition according to claim 11 wherein the neurotropic virus is selected from the group consisting of Herpes simplex virus, varicella-zoster virus, and rabies virus. 13. The antiviral composition according to claim 12 wherein the virus is Herpes simplex virus, the structural tegument protein is USll. and the motor protein is kinesin. 14. The antiviral composition according to any one of claims 11 to 13 wherein the compound a motor protein-like molecule which binds to a structural tegument protein of the virus thereby preventing the normal interaction of the virus and neuron or cell. 15. The antiviral composition according to claim 14 wherein the motor protein-like molecule comprises a mimic of a cellular motor protein or a part of the motor protein to which a structural tegument protein of a virus binds or specifically interacts. 16. The antiviral composition according to claim 15 wherein the virus is Herpes simplex virus, the motor protein is kinesin and the motor protein-like molecule is a mimic of kinesin or a part of kinesin to which the structural tegument protein USll of Herpes simplex virus binds or specifically interacts. 17. The antiviral composition according to any one of claims 11 to 13 wherein the compound is a structural tegument-like molecule which binds to a motor protein of a neuron thereby preventing the normal interaction between the neuron or cell with the virus. 18. The antiviral composition according to claim 17 wherein the structural tegument-like molecule comprises a mimic of a viral tegument protein or a part of the tegument protein to which a cellular motor protein binds or specifically interacts. 19. The antiviral composition according to claim 18 wherein the virus is Herpes simplex virus, the structural tegument protein is USll, motor protein is kinesin and the structural tegument-like molecule is a mimic of USll or a part of USll to which the motor protein kinesin binds or specifically interacts.

Inventors: ARMATI PATRICIA JOAN, CUNNINGHAM ANTHONY LAWRENCE, DIEFENBACH EVE MARGARET, DIEFENBACH RUSSELL JOHN, HOLLAND DAVID JAMES, MIRANDA-SAKSENA MONICA, PENFOLD MARK, ARMATI, PATRICIA, JOAN, CUNNINGHAM, ANTHONY, LAWRENCE, DIEFENBACH, EVE, MARGARET, DIEFENBACH, RUSSELL, JOHN, HOLLAND, DAVID, JAMES, MIRANDA-SAKSENA, MONICA, PENFOLD, MARK
Applicants: ARMATI PATRICIA JOAN, CUNNINGHAM ANTHONY LAWRENCE, DIEFENBACH EVE MARGARET, DIEFENBACH RUSSELL JOHN, HOLLAND DAVID JAMES, MIRANDA SAKSENA MONICA, PENFOLD MARK, UNIV SYDNEY, WESTMEAD HOSPITAL, ARMATI, PATRICIA, JOAN, CUNNINGHAM, ANTHONY, LAWRENCE, DIEFENBACH, EVE, MARGARET, DIEFENBACH, RUSSELL, JOHN, HOLLAND, DAVID, JAMES, MIRANDA-SAKSENA, MONICA, PENFOLD, MARK, THE UNIVERSITY OF SYDNEY
Application year: 2000
Main classification: A61K35
IPC codes: A61K38, A61K39, A61K45, A61P25, A61P31, C12N15