Role of a cdk5-associated protein, p35, in herpes simplex virus type 1 replication in vivo

Abstract

Previous studies have shown that herpes simplex virus type 1 (HSV-1) replication is inhibited by the cyclin-dependent kinase (cdk) inhibitor roscovitine. One roscovitine-sensitive cdk that functions in neurons is cdk5, which is activated in part by its binding partner, p35. Because HSV establishes latent infections in sensory neurons, we sought to determine the role p35 plays in HSV-1 replication in vivo. For these studies, wild-type (wt) and p35−/− mice were infected with HSV-1 using the mouse ocular model of HSV latency and reactivation. The current results indicate that p35 is an important determinant of viral replication in vivo.

Figure 1

HSV-1 ocular shedding in p35-/- mice. C57BL/6 and p35-/- mice were ocularly infected with 1 × 10⁵ PFU/eye, and eye swabs were taken on days 1, 3, 5, and 7 post infection (p.i.). Viral titers of eye swabs (n = 8 per time point) were determined by standard plaque assays. Results displayed are logarithmic means, with error bars showing the standard error of the means (SEMs). The horizontal stippled line represents the lower limit of detection. Results from one experiment are shown; similar results were observed in a repeat experiment.

Figure 2

Acute viral replication in murine TG. TG from C57BL/6 and p35-/- infected mice were removed days 1, 3, 5, and 7 p.i., and TG homogenates were titered by standard plaque assays (n = 8 per time point). The results displayed are logarithmic means, with the error bars representing the SEMs. The horizontal stippled line represents the lower limit of detection. Results from one experiment are shown; similar results were observed in a repeat experiment.

Figure 3

Latent viral DNA loads in wt and p35-/- mice. TG were removed from C57BL/6 and p35-/- latently infected mice at 30 days p.i., and DNA was isolated from each TG. The relative establishment of HSV-1 latency for each mouse strain (n = 14 per group) was determined by real-time PCR using primers that amplified the HSV-1 UL50 gene. UL50 gene levels were normalized to a DNA loading control, the mouse adipsin gene. Data shown are logarithmic (base 2) means. Error bars represent SEMs. Mockinfected samples were below the limit of detection for the UL50 gene (data not shown).

Figure 4

HSV-1 reactivation in latently infected TG. On days 30 p.i., TG were removed from C57BL/6 and p35-/- latently infected mice and explanted on Vero cells. Medium from each sample was monitored daily for infectious virus over a period of 15 days (n = 8 for C57BL/6, n = 12 for p35-/-). The arrow at the top of the figure (day 10 post explant) indicates that samples were heat shocked at 43°C for 3 h to induce further reactivation. The results from one of two experiments are shown. The percent reactivation is the cumulative amount of reactivation for each set of samples analyzed.