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In trod u ction

DRG neurones lose excitability after 24 hours o f infection with wt HSV 17^. This is the result o f a dramatic decrease in sodium conductance as described in chapter 3. In this chapter an attempt has been made to determine which aspects o f w t HSV 17^ infection are responsible for the loss o f sodium conductance in DRG neurones. In classical virology, the function o f various components o f the life cycle o f HSV-1 have been studied using combinations o f viral mutants together with biochemical agents which block specific biological processes. The question is, what facet o f viral infection! elicits the dramatic decrease in sodium current amplitude?

The wt HSV 17^ virion contains many proteins, such as the capsomer proteins, tegument proteins and glycoproteins. It is possible that simply virion entry into DRG neurones may be sufficient to cause the loss o f sodium conductance. A virion protein, perhaps a glycoprotein, could act directly by blocking sodium channels, or indirectly by triggering a sodium channel down regulation mechanism.

The virion tegument protein, virus host shutoff protein (vhs), induces rapid degradation o f cellular mRNAs as well as the shutoff o f most host cell protein synthesis upon viral entry (Fenwick & McMenamin, 1984). Such general repression o f DRG neurone protein synthesis may indirectly cause the loss o f sodium conductance. A factor involved in sodium channel function or the sodium channels themselves may rapidly turnover. Depletion o f host proteins does occur during wt HSV 17^ infections, a specific example being the reduction o f the DNA-dependent protein kinase (DNA-PK) levels (Lees Miller et al. 1996). A perturbation in host cell protein synthesis may be sufficient to deplete levels o f a protein with a short half life involved in sodium channel function, hence the loss o f sodium current.

The loss o f sodium conductance in infected DRG neurones could also be the result o f viral proteins synthesised during the course o f infection. Upon entry o f viral DNA into the nucleus the first set o f genes to be expressed in the temporal cascade o f viral gene expression are the immediate early genes (Honess & Roizman, 1974). There are five immediate early genes encoding viral proteins, ICPO, ICP4, ICP27, ICP22 and ICP47 (Pereira et al. 1977). They are transcribed and translated by cellular machinery and do not require prior viral protein synthesis (Honess & Roizman, 1974; Costanzo et al. 1977). ICP22 and ICP47 do not affect viral gene expression (Everett, 1984). In contrast ICPO, ICP4 and ICP27 are involved in regulating immediate early, early and late gene expression (0-H are & Hayward, 1985). A summary the o f immediate early genes ICPO, ICP4 and ICP27 are listed below.

ICPO is a llO kD a nuclear phosphoprotein, also known as V m w l 10 and is the product o f immediate early gene 1 (Pereira et al. 1977). Transient expression assays show ICPO is a promiscuous transactivator o f viral and non viral promoters (Everett, 1984). In the absence o f ICPO, virus replication is inefficient or sluggish, with a lower probability o f initiating a productive infection. These defects can be overcome by increasing the multiplicity o f infection (Stow & Stow, 1986). Mutant viruses with deletions in the gene encoding ICPO are unable to reactivate from explanted sensory ganglia harboring latent virus, which suggests that ICPO plays a role in the switch between lytic and latent state (Leib et a l 1989). The phenotype o f cells infected with mutant HSV, with deletions in both genes encoding ICPO and 1CP4, can be rescued by transfection with plasmids encoding either ICPO or 1CP4, as both can activate immediate early, early and late gene expression. The levels o f expression, however, are much greater when both plasmids encoding ICPO and 1CP4 are transfected together. This suggests that the activity o f ICPO is synergistic with 1CP4 (Everett, 1984). Although ICPO is non-essential for viral growth, it does play a role in efficient initiation o f viral gene expression probably by stabilising viral proteins by binding to HAUSP (herpesvirus associated ubiqutin specific protease) (Everett et al. 1997).

1CP4 (expressed from immediate early gene 3) has an apparent molecular weight o f 175 kDa as estimated by SDS-PAGE, is a phosphoprotein and is located in the nucleus o f infected cells (Courtney & Benyesh Melnick, 1974; Pereira et al. 1977). 1CP4 is packaged into the tegument o f virons (Yao & Courtney, 1989). 1CP4 binds DNA as a homodimer, and is found in solution as a multimer or dimer, (Michael & Roizman, 1989; Metzler & Wilcox, 1985). O f the five immediate early genes, 1CP4 is perhaps the most important. Studies with temperature sensitive mutants which map to 1CP4, show an absence o f viral growth through failure to activate early and late promoters at non-permissive temperatures (Watson et al. 1979). In addition, experiments with temperature sensitive mutants (which map to 1CP4) showed that 1CP4 autoregulates its own synthesis and represses the expression o f other immediate early genes (DeLuca & Schaffer, 1988; Michael & Roizman, 1993). For this reason an overproduction of immediate early proteins, but no expression o f early or late genes, occurs during an infection with virus lacking functional 1CP4 (DeLuca et al. 1984). As described

above, transient expression assays show that ICPO and ICP4 have synergistic enhancing effects on early and late gene expression. In contrast, ICP27 can exert a positive or negative effect on ICP4 induced gene expression (Sekulovich et al. 1988).

ICP27 (gene product o f immediate early gene 2) is a nucleophosphoprotein, with an apparent molecular weight o f 63 kDa as estimated by SDS-PAGE (Wilcox et al. 1980; Knipe et al. 1987; Ackermann et al. 1984). Null ICP27 mutants do not replicate and show a decrease in viral DNA synthesis and late gene expression, whilst immediate early proteins accumulate (Rice & Knipe, 1990). ICP27 is involved in the switch between early and late gene expression and is absolutely essential for formation o f infectious progeny (Sacks et al. 1985; McCarthy et al. 1989). Experiments have shown that ICP27 can either activate or repress expression o f cotransfected reporter genes in uninfected cells (Rice et al. 1989; Everett, 1986). Activation o f reporter genes has been correlated with the presence o f particular polyadenylation signals, while repression has been correlated with the presence o f introns (Cai & Schaffer, 1989). The range o f regulatory feats performed by ICP27 includes stimulation of DNA synthesis, post transcriptional destabilisation o f immediate early gene RNA and inhibition o f RNA splicing (Smith et al. 1992; Leary et al. 1989). The inhibition o f host cell RNA splicing is thought to play a role in secondary shutoff o f host protein synthesis (Hardwicke & Sandri Goldin, 1994).

After the expression o f immediate early and early genes the virus begins to replicate its DNA and express late genes. Early genes encode for|proteins that are involved in DNA replication and the late proteins encode for virion structural proteins. Arresting this phase o f the cycle with a specific HSV DNA replication inhibitor, acyclovir, prevents the expression o f true late genes (Holland et al. 1980). Since early gene expression is inhibited by late genes, the presence o f acyclovir results in enhanced early gene expression.

The same tools which have been designed to dissect the life cycle o f HSV-1 can be used to determine the components responsible for loss o f sodium conductance in the host DRG neurones. The experiments described in this chapter use the chemical agents cycloheximide to investigate the role o f virion components, and acyclovir to investigate the role o f viral DNA synthesis in the loss o f sodium conductance. HSV-1 mutants lacking functional immediate early genes were used to study their effect on electrophysiological changes during an infection.

Finally, a cosmid library o f wt HSV \ Ÿ genome was used in an attempt to discover the gene(s) encoding the causative proteins involved in the loss o f sodium conductance. The wt HSV \ Ÿ genome is very large, consisting o f 152 Kb and encoding at least 70 distinct proteins (McGeoch et al. 1988). It is possible that a single protein could cause the loss o f sodium conductance in wt HSV 17^ infected DRG neurones. Five fragments together comprising the wt HSV 17^ genome were available in a cosmid library (Cunningham & Davison, 1993). Microinjection o f wt HSV 17^ genome fragments was used to locate possible gene products responsible for the loss o f sodium conductance.

In summary, the aim o f the studies reported in this chapter was to examine the possible features o f viral infection that cause the loss o f sodium conductance; virus entry, viral immediate early gene expression, and viral DNA replication. More specifically, the role o f expression o f specific viral genes was investigated using a cosmid library o f wt HSV 17^ genome.

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