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Sariah Allen, Kevin Mott, Yoshiharu Matsuura, Kohji Moriishi, Konstantin Kousoulas, Homayon Ghiasi; Signal peptide peptidase (SPP) is required for HSV-1 infectivity through interaction with glycoprotein K (gK). Invest. Ophthalmol. Vis. Sci. 2013;54(15):1731.
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We previously demonstrated that vaccinating mice with expressed glycoprotein K (gK), but not any other of the known HSV 1 glycoproteins results in severely exacerbated corneal scarring and dermatitis following ocular infection with both virulent and avirulent HSV-1 strains. Although gK is not involved in the processes of virus attachment or penetration, it is involved in virus replication and egress. Thus, the goal of this study was to determine if binding of gK to any cellular factor(s) may contribute to HSV-1 infectivity and pathogenesis.
Bacterial two-hybrid assay using gK as bait and a mouse neuronal cDNA library was used to identify binding partners to gK. Binding was confirmed by co-immunoprecipitation and immunohistochemistry (IHC) in HeLa, Vero and RS cell lines. Blocking SPP-gK interaction was assayed using SPP dominant negative mutants, shRNA directed against SPP, and SPP chemical inhibitors (i.e., DAPT, L685,485, (Z-LL)2 ketone, Aspirin or Ibuprofen). The effect of blocking SPP on viral replication was monitored using standard plaque assay, real-time PCR and IHC. The effect of ocular administration of SPP inhibitor (Z-LL)2 ketone on ocular HSV-1 replication was evaluated in BALB/c and C57BL/6 mice by titering virus from tears.
Using a two-hybrid system we identified signal peptide peptidase (SPP) as a cellular protein which interacts with gK. Binding of gK to SPP was confirmed by pull-down assay and fluorescent colocalization. Blocking SPP binding to gK using SPP dominant negative mutants, shRNA construct against SPP or inclusion of chemical SPP inhibitors significantly reduced HSV-1 replication in vitro. Finally, HSV-1 replication in the eyes of mice that received (Z-LL)2 ketone as an eye drop before and during infection was significantly reduced compared to sham-treated animals.
We have demonstrated for the first time that HSV-1 gK interacts with cellular SPP and this interaction is required for virus infectivity as blocking SPP reduces virus replication in vitro and in vivo. As there is currently no effective treatment for HSV-1 recurrences, blocking SPP-gK interaction may represent a clinically effective and expedient target in developing HSV-1 therapeutics.
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