Mutations within the pathogenic region of HSV-1 gK signal sequences alter cell surface expression and neurovirulence

Abstract

Purpose: Glycoprotein K (gK) is a highly conserved and essential HSV protein. We previously demonstrated that immunization of mice with gK exacerbates eye disease following ocular HSV-1 infection independent of mice or virus strain. We also have reported that a recombinant HSV-1 expressing two extra copies of gK exacerbated eye disease in both mice and rabbit, further suggesting that gK is pathogenic. In addition, we had shown that the pathogenic region of the gK is located within the 8mer signal sequence of gK. In this study we investigated the role of the gK 8mer in corneal scarring (CS) and neurovirulence by constructing recombinant viruses with and without mutations within the 8mer region of the gK.

Methods: We constructed two recombinant viruses expressing two additional copies of the mutated (MgK) or native (NgK) form of the gK gene with a myc epitope tag, to facilitate detection, at their 3’ end. The replication of MgK, NgK, and revertant (RgK) viruses were determined in vitro and in vivo. In addition, the levels of gB and gK in the corneas, trigeminal ganglia (TG), and brains of infected mice on days 3, 5, and 28 post infection (PI) were determined. Finally, LD₅₀, CS and extent of explant reactivation in infected mice were determined.

Results: The replication of MgK virus was similar to that of NgK virus both in vitro and in vivo as well as in the TG of latently-infected mice. The levels of gB and gK transcripts in the corneas, TG, and brains of infected mice on days 3 and 5 PI were markedly virus- and time-dependent as well as tissue-specific. Point mutations in the 8mer region of gK in MgK virus blocked cell surface expression of gK-myc in rabbit skin (RS) cells and reduced LD₅₀ and CS in ocularly infected mice as compared with the NgK or RgK virus. MgK and NgK viruses and not the RgK virus had reduced extent of explant reactivation at the lower dose of ocular infection but not at the higher dose of infection. However, time of reactivation was not affected by overexpression of different forms of gK.

Conclusions: Taken together, these results strongly suggest that the 8mer peptide (ITAYGLVL) within the signal sequence of gK promotes cell surface expression of gK in infected cells and ocular pathogenesis in infected mice. Thus, these studies point to a key role for the 8mer within the signal sequence of gK in HSV-1-induced pathogenicity.