Herpes simplex virus (HSV) causes diseases ranking in severity from annoying labialis and genital infections, to blinding keratitis, risk of developing encephalitis, risk of transmission to newborns, and increased risk of acquiring HIV-1 infection. Following lytic infection in epithelial cells at the portal of entry in the body, HSV establishes a latent infection in sensory neurons. Occasionally the virus is reactivated, generally as a result of a weakened immune system causing recurrent diseases. The current antiviral used to treat herpesvirus infections, acyclovir, although effective at blocking viral DNA synthesis, has limited bioavailability and acts late during the replication when many viral products are already present. Due to low lipophilicity it does not cross the blood brain barrier to prevent encephalitis. Drug resistance has been reported in immunocompromised individuals.
To infect and persist in the human body, HSV must overcome strong innate and adaptive immune responses. The infected cell protein 0 (ICP0), an immediate early protein of the virus, plays fundamental roles in this process. Its two most prominent functions are to render the infected cells resistant to the antiviral activity of interferons and to block the silencing of viral DNA and initiate transcription. ICP0 acts as an E3 ubiquitin ligase to degrade the innate immune components PML and SP100 that are constituents of the ND10 nuclear bodies where the viral genome is deposit and it is silenced. Following their degradation the ND10 bodies are dispersed and this is essential for viral gene expression. In tandem, ICP0 blocks the silencing of viral DNA through dissociation of repressor complexes. Subsequently, ICP0 recruits chromatin remodeling enzymes such as the histone acetyl transferase CLOCK (Circadian Locomotor Output Cycles Kaput), along with its partner BMAL-1, to activate viral gene expression. CLOCK is recruited to the viral genome via the direct interaction of ICP0 with BMAL-1. Failure of ICP0 to execute any of these functions impairs virus replication. ICP0 is essential in vivo and the ICP0 E3 ligase and the ICP0 null mutants fail to counteract IFN responses. This results in a failure to spread from the initial site of infection and less efficient reactivation. Given that ICP0 executes its functions immediately after the entry of the virus into the cell and before the onset of proteins synthesis, we hypothesize that small compounds interfering with these essential ICP0 functions will impede the viral infection and attenuate HSV reactivation.
To test our hypothesis we have formulated two Specific Aims: In Aim 1, we propose to identify compounds that block the HSV ICP0 E3 ligase activity in vitro. In Aim 2, we will identify compounds that block the interaction of ICP0 with BMAL-1 and thereby will block viral gene expression. The University of Kansas (KU)-High Throughput Screening collection (HTSC) of over 300,000 compounds will be utilized with the support of the KU High Throughput Screening Laboratory (HTSL). The results are expected to identify novel compounds with antiviral activity. Additionally, these compounds will serve as tools to characterize the ICP0 functions.