Abstract
The herpes simplex virus type 1 (HSV-1) diploid gene γ134.5 encodes a neurovirulent factor, infected cell protein 34.5 (ICP34.5). The promoter to γ134.5 is located within the HSV-1 genome where there are repeated sequences. This region of the genome also contains important overlapping transcripts involved with the virus's ability to establish lytic and latent infections and reactivation. These transcripts include the latency-associated transcripts and regulator proteins ICP0 and ICP4. This study aimed to separate ICP34.5 from these overlapping transcripts and test if its expression from a single gene could restore wild-type HSV-1 strain 17+ virulence. To address these aims, different recombinant viruses were constructed using the Δ γ134.5 mutant 1716. Immunoblots probed with different ICP34.5 antisera demonstrated that one of the newly generated recombinant viruses, 1622, overexpresses ICP34.5 relative to a panel of wild-type viruses. Interestingly, the overexpression of ICP34.5 does not yield a more virulent virus. The onset of ICP34.5 expression from 1622-infected cells in vitro matched that of 17+, and its expression restored the function of maintaining protein synthesis in human neuroblastoma cells. Replication of 1622, however, was only partially restored to 17+ levels in vivo. Additionally, plaque morphology from 1622-infected cells indicates there is an additional defect. The authors report that the mutant virus 1622 can express ICP34.5 from a single γ134.5 gene and restore most (but not all) wild-type function. These findings are discussed with respect to the use of the γ134.5 deleted mutant, 1716, in oncolytic viral vector therapies and future studies for ICP34.5.
This work was funded, in part, by a scholarship from Glasgow University. The authors would like to express gratitude to the late Prof. Barklie Clements for generously providing several reagents and the bench space in which most of this work was carried out. The authors thank to Prof. Brown for communication and helpful comments during the course of this work. The in vivo studies indicated in text by an asterisk* were performed under the direction of Dr. N. M. Sawtell in her laboratory at Cincinnati Children's Hospital Medical Center, Cincinnati, Ohio, and supported by NIH R01 AI32121 (NMS). The authors thank Dr. Sawtell and David Nadziejka for the critical review and suggestions on the manuscript.