Granzyme B cleaves multiple HSV-1 and VZV gene products and VZV ORF4 inhibits Natural Killer cell dependent cytotoxicity.

Immune regulation of alphaherpesvirus latency and reactivation is critical for the control of virus pathogenesis. This is evident for herpes simplex virus type 1 (HSV-1), where cytotoxic T lymphocytes (CTLs) prevent viral reactivation, independent of apoptosis induction. This inhibition of HSV-1 reactivation has been attributed to granzyme B cleavage of HSV infected cell protein (ICP) 4, however it is unknown whether granzyme B cleavage of ICP4 can directly protect cells from CTL cytotoxicity. Varicella zoster virus (VZV) is closely related to HSV-1, however it is unknown whether VZV proteins contain granzyme B cleavage sites. Natural killer (NK) cells play a central role in VZV and HSV-1 pathogenesis and like CTLs utilize granzyme B to kill virally infected target cells. However whether alphaherpesvirus granzyme B cleavage sites could modulate NK cell mediated cytotoxicity has yet to be established. This study aimed to identify novel HSV-1 and VZV gene products with granzyme B cleavage sites and assess whether they could protect cells from NK cell mediated cytotoxicity. We have demonstrated that HSV ICP27, VZV ORF62 and VZV ORF4 are cleaved by granzyme B. However, in a NK cell cytotoxicity assay only VZV ORF4 conferred protection from NK cell mediated cytotoxicity. The granzyme B cleavage site in ORF4 was identified via site directed mutagenesis and surprisingly, the mutation of this cleavage site did not alter the ability of ORF4 to modulate NK cell cytotoxicity, suggesting that ORF4 has a novel immunoevasive function which is independent from the granzyme B cleavage site. IMPORTANCE HSV-1 causes oral and genital herpes and establishes life-long latency in sensory ganglia. HSV-1 reactivates multiple times in a person's life and can cause life-threatening disease in immunocompromised patients. VZV is closely related to HSV-1 and causes chickenpox during primary infection and establishes life-long latency in ganglia from where it can reactivate to cause herpes zoster (shingles). Unlike HSV-1, VZV only infects humans and there are limited model systems, thus little is known concerning how VZV maintains latency and why VZV reactivates. Through studying the link between immune cell cytotoxic functions, granzyme B and viral gene products an increased understanding of viral pathogenesis will be achieved.