Mary Elaine Whealy

Two α-herpesvirus strains are transported differentially in the rodent visual system

Abstract Uptake and transneuronal passage of wild-type and attenuated strains of a swine α-herpesvirus (pseudorabies [PRV]) were examined in rat visual projections. Both strains of virus infected subpopulations of retinal ganglion cells and passed transneuronally to infect retinorecipient neurons in the forebrain. However, the location of infected forebrain neurons varied with the strain of virus. Intravitreal injection of wild-type virus produced two temporally separated waves of infection that eventually reached all known retino-recipient regions of the central neuraxis. By contrast, the attenuated strain of PRV selectively infected a functionally distinct subset of retinal ganglion cells with restricted central projections. The data indicate that projection-specific groups of ganglion cells are differentially susceptible to the two strains of virus and suggest that this sensitivity may be receptor mediated.

Influence of infectious dose upon productive replication and transynaptic passage of pseudorabies virus in rat central nervous system.

Pseudorabies virus (PRV) is a neurotropic swine alpha herpesvirus that characteristically invades the nervous system and replicates within synaptically-linked populations of neurons. The invasive characteristics and ability of this family of viruses to replicate in neurons of the central nervous system (CNS) have been exploited to map functionally related populations of neurons in a variety of systems. In this report, we examined the effects of strain and concentration on the ability of PRV to infect retinal ganglion cells and pass transneuronally through central visual circuits. We find that the ability of virulent (PRV-Becker) and attenuated (PRV-Bartha) strains of PRV to produce a productive infection of visual circuitry is directly dependent upon the infectious of the injected virus. Injections of at least 10(5) total plaque forming units produce 100% infectivity, whereas lower infectious doses substantially reduce the percentage of animals exhibiting productive infection via this route of inoculation. Furthermore, the virulent strain of PRV consistently infects a higher percentage of animals across a broader range of titers than attenuated virus. These data demonstrate that viral titer and strain are important variables that should be considered in the design of studies and interpretation of data derived from investigations employing this pathogen for circuit analysis.

Construction of an infectious pseudorabies virus recombinant expressing a glycoprotein gIII―β-galactosidase fusion protein

An infectious herpesvirus mutant has been constructed in which a major structural envelope glycoprotein gene was replaced by a hybrid gene encoding a novel fusion protein consisting of the N-terminus of the viral glycoprotein joined to Escherichia coli beta-galactosidase (beta Gal). Specifically, we fused DNA encoding the first 157 amino acids of the structural glycoprotein gIII from pseudorabies virus strain Becker to the E. coli lacZ gene in a bacterial expression vector. The resulting hybrid gene was then used to replace the wild-type gIII gene in the virus by cotransfection of plasmid and viral DNA. The desired viral recombinants were identified by their inability to react with specific monoclonal antibodies that recognized only wild-type gIII protein. One such mutant virus, PRV-Z1, was chosen for further analysis. PRV-Z1 expressed a glycosylated gIII-beta Gal fusion protein after infection of PK15 cells. The fusion protein has no demonstrable beta Gal activity and, although glycosylated, remains sensitive to the enzyme endo-beta-N-acetylglucosaminidase H, unlike the mature gIII gene product, indicating that the fusion protein was incompletely processed.

Overall signal sequence hydrophobicity determines the in vivo translocation efficiency of a herpesvirus glycoprotein

We have described three mutant strains of Pseudorabies virus that contain mutations in the signal sequence coding region of a nonessential envelope glycoprotein, gIII. The alterations disrupt, truncate, or eliminate the hydrophobic core domain of the signal sequence. Each mutant was assayed for its ability to promote the translocation of gIII across the endoplasmic reticulum membrane and the subsequent localization of the mature form of the glycoprotein to the infected cell surface or the virus envelope. Our results confirm and extend findings in other systems that the overall hydrophobicity of the signal sequence core region is a major determinant of translocation efficiency. We were unable to correlate simply the length of the core or the average hydrophobicity of core residues with export efficiency. Because our work involved the use of infectious virus mutants, we were able to identify a virus defect associated with a complete block in gIII export. This defect will facilitate a pseudoreversion analysis of gIII signal sequence function.