Dept. of Microbiology & Immunology

University of Tennessee, Memphis

J. Patrick Ryan, Ph.D.

Office: (901) 448-8764

Lab: (901) 448-8466

pryan@utmem.edu

Office: 201D M.S.B.

Lab: 210 M.S.B.

Laboratory web page

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Research Program:

Role of herpesvirus glycoproteins in virus entry and spread. My research program is centered on a genetic analysis of herpesvirus glycoproteins. In particular, we are interested in determining the roles of certain glycoproteins in the entry and spread of pseudorabies virus (PRV), a swine herpesvirus that is very similar to human herpes simplex virus. PRV is a 140 kilobase pair double-stranded DNA virus whose nucleocapsid is surrounded by a lipid bilayer (or envelope) that contains ten or more virus-encoded glycoproteins. Some of these glycoproteins mediate the entry of virions into the target cell while still others may serve to partially inactivate an immune response mounted against the infection.

Our approach to studying virus entry and spread has applied the principles of "reverse genetics." First, we introduce mutations into a plasmid-cloned copy of a PRV gene using either restriction enzymes to produce large deletions or site-specific mutagenesis to form point mutations. Next, the plasmids are propagated in Escherichia coli, and plasmid DNA containing the mutant allele is then introduced ("transfected") into cultured mammalian cells along with naked, infectious PRV genomic DNA that has been extracted from virus particles. Homologous recombination will occur in some of the cells between the wild-type copy of the gene in the genome and the mutant allele on the plasmid. This results in a virus that now contains only the mutant form of the gene, and we can detect this virus among the non-recombinant progeny of the transfection by an immuno-plaque assay. In this way, we have generated a number of PRV mutant virus.

Role of glycoprotein C (gC) in PRV entry of cells. The entry process among herpesviruses appears to be conserved. Initial attachment of virus is mediated by gC which binds to cell surface heparan sulfate proteoglycans (HSPGs), membrane-bound proteins that are heavily glycosylated. Although nonessential in cell culture, this binding facilitates a second binding event between viral gD and a family of cellular receptors referred to as Hve proteins. Subsequent to this binding, the viral envelope fuses with the target cell plasma membrane in a process involving several additional viral glycoproteins.

We have mapped the HSPG-binding domain in PRV gC by mutational analysis and demonstrated that it is composed of three discrete heparin-binding domains (HBDs), each consisting of six to eight amino acids. Within each HBD, three or four basic amino acids are arranged according to specific motifs that facilitate binding with the negatively-charged sulfate groups found on HSPGs. Interestingly, the HBDs perform a redundant function in cell culture: only one intact HBD is necessary to facilitate virus binding. Through a collaboration with Dr. Bergstrom’s lab at the University of Goteborg, Sweden, we have been able to show that each HBD preferentially binds a different form of heparan sulfate. This may indicate that the role of the three HBDs is to ensure virus attachment to a variety of cells or that the particular HSPG bound by the virus on a given cell type is important. Of course, synergistic interactions among the three HBDs remain a distinct possibility. Our work on the fine structure of the PRV gC HSPG-binding domain remains the most exact of any herpesvirus reported to date.

We have more recently turned our attention to the cellular aspect of PRV gC-HSPG interactions. We would like to identify the specific HSPGs bound by each of the HBDs found in PRV gC. To this end, we have begun characterizing fusion proteins composed of gC and a heterologous domain that will allow their immobilization on a matrix. This has allowed us to fractionate bound HSPGs from other cellular components and will facilitate the identification of the protein moiety of each relevant HSPG. In this way, we may be able to determine if the specific HSPGs bound by PRV are critical for efficient virus infection

Another cellular aspect of the gC-HSPG interaction that we are analyzing is the consequence for the cell of initial virus binding. It has long been known that wild-type strains of PRV enter cells more rapidly than those lacking gC. It is commonly held that gC-HSPG complexes serve only to facilitate the search for secondary receptors. That is to say, once the virus is bound to the cell surface, the search for secondary receptors can be limited to two dimensions rather than three. However, we have found that the initial gC-HSPG interaction is more directly influencing downstream events and that the actin cytoskeleton of the cell is required for efficient PRV entry. As visualized by immunofluorescence, the majority of wild-type particles lie over actin filaments after initial virus binding while a minority of gC null particles is coincident with actin. Moreover, cytochalasin D (cyto D), which depolymerizes filamentous actin, reduces both wild-type and gC null virus infectivity up to 40%. In the case of wild-type virus, cyto D treatment impairs the virus at two levels: movement from HSPG to secondary receptors and subsequent penetration of virions after binding these receptors. We currently favor a model in which gC-HSPG interactions promote the formation of stable complexes involving HSPGs and additional cellular proteins that interact with other viral glycoproteins. The actin cytoskeleton would act as the platform for receptor complex formation, and only when complex formation is achieved can rapid penetration of cells occur. In the absence of gC-HSPG interactions, receptor complexes can form, but by a relatively slow, perhaps random process. This work is of significance because while cellular receptors and numerous viral glycoproteins are emerging as components of virus entry, how these complexes are formed is not understood. Knowledge of these events could lead to novel approaches to limit herpesvirus spread.

Role of PRV UL43 protein in neuronal spread and neurovirulence. We have recently undertaken a collaboration with Dr. Mark LeDoux of the Dept. of Neurology, UT-Memphis, to look at a potential role for the UL43 gene product of PRV in neurovirulence. We discovered and characterized the UL43 locus of PRV a number of years ago. We determined the DNA sequence, identified the mRNA, and deleted the gene from the virus genome and demonstrated that no ill-effects were incurred by the virus in cell culture. The putative UL43 protein is predicted to span a membrane eight times, and appears to be a membrane/envelope protein.

The lack of a cell culture phenotype for UL43 deleted strains confounded our efforts to determine its function in the virus life cycle. However, recently we began inoculating rats with either wild-type or a UL43 deleted strain to ask whether the deletion strain (termed PRV528) is attenuated. The site of inoculation is the rat eyelid, which allows the wild-type virus to transport through motor, sensory, sympathetic, and parasympathetic neurons in both anterograde and retrograde directions. A robust inflammation is observed and encephalitis leads to a rapid decline to the rat’s health within 72 hours. In contrast, PRV528 infected rats show little overt signs of inflammation or encephalitis before being sacrificed at 72 hours postinfection. When brain sections are immunohistochemically stained for the presence of virus, results indicate that PRV528 spreads to a much less extent than wild type and transports only in a retrograde direction. We appear to be the only lab currently researching a herpesvirus UL43 homolog, and we have uncovered a novel phenotype with respect to neuronal spread. Our next efforts will focus on identifying the regions of UL43 that are important for spread and neurovirulence and to try and get a handle on the "function" of UL43 in the neurologic environment.

Selected Publications

Flynn, S. J., and P. Ryan. 1996. The receptor-binding domain of pseudorabies virus glycoprotein gC is composed of multiple discrete units that are functionally redundant. J. Virol. 70: 1355-1364.

Trybala, E., T. Bergstrom, D. Spillmann, B. Svennerholm, S. Olofsson, S. J. Flynn, and P. Ryan. 1996. Mode of interaction between pseudorabies virus and heparan sulfate/heparin. Virology 218: 35-42.

Ryan, P., and F. L. Shankly. 1996. A double-strand break in a herpesvirus genome stimulates targeted homologous recombination with exogenous, cloned viral sequences. J. Virol. Meth. 57: 95-107.

Trybala, E., T. Bergstrom, D. Spillmann, B. Svennerholm, S. J. Flynn, and P. Ryan. 1998. Interaction between pseudorabies virus and heparan sulfate/heparin. Pseudorabies virus mutants differ in their interaction with heparin/heparan sulfate when altered for specific glycoprotein C heparin-binding domain. J. Biol. Chem. 273: 5047-5052.

Rue, C.A., and P. Ryan. Pseudorabies virus glycoprotein C promotes efficient virus entry involving the actin cytoskeleton. Submitted for publication