Additional Evidence that HSV-1 LAT’s Anti-apoptosis Activity May be Involved in LAT’s Ability to Enhance the Spontaneous Reactivation Phenotype in Rabbits

Abstract

Abstract: : Purpose: To further demonstrate that a LAT (latency associated transcript) anti-apoptosis function is involved in LAT’s ability to enhance the reactivation phenotype. We previously substituted an alternate anti-apoptosis gene, the bovine herpes virus 1 (BHV-1) latency-related (LR) gene, for the HSV-1 LAT gene to produce a chimeric virus designated CJLAT. CJLAT, which contains the LR gene in place of LAT on an HSV-1 genomic background, has a high spontaneous reactivation rate (Perng, et al, 2002, J. Virol. 76:1224-1235) suggesting that an alternative anti-apoptosis gene can functionally replace LAT to restore the high spontaneous reactivation phenotype. A mutation was introduced into the LR gene resulting in loss of anti-apoptosis activity and examined the effect on the spontaneous reactivation phenotype. Methods: Three stop codons were inserted at the beginning of the LR open reading frame to prevent LR protein expression in all 3 reading frames. This alteration was previously shown to abrogate LR’s ability to block apoptosis in transient transfection assays by Ciacci-Zanella, et al.. The altered LR gene was then used to construct a virus, designated CJLATmut, that is identical to CJLAT, except that it does not express the LR protein and LR thus no longer blocks apoptosis. Rabbits were ocularly infected with 2x10⁵ PFU/eye of wt McKrae, CJLAT, CJLATmut, or dLAT2903. Relative spontaneous reactivation was determined by analysis of neutralizing serum antibody during latency. Results: CJLATmut and dLAT2903 had similar low spontaneous reactivation rates. In contrast wt McKrae and CJLAT had high spontaneous reactivation rates. All four viruses had similar in vivo replication and similar survival rates. Conclusion: The spontaneous reactivation phenotype of CJLATmut was similar to that of the LAT null mutant dLAT2903. Since the only difference between CJLATmut and CJLAT (which had a high spontaneous reactivation phenotype) is that CJLATmut does not express the LR-protein, this strongly suggests that in these chimeric viruses contain LR in place of LAT, expression of the LR-protein was required for the high spontaneous reactivation phenotype. These results also suggest that the LR anti-apoptosis activity was required for the high spontaneous reactivation phenotype. This in turn supports the hypothesis that LAT’s anti-apoptosis activity is involved in LAT’s ability to enhance the spontaneous reactivation phenotype.

Keywords: herpes simplex virus • molecular biology • microbial pathogenesis: experimental studies