Expression of the enhanced green fluorescent protein by herpes simplex virus type 1 (HSV-1) as an in vitro or in vivo marker for virus entry and replication

Glycoprotein K (gK) is involved in membrane fusion phenomena during infecti ous virus production and egress and is an important determinant for neurovi rulence. To assess better the in vitro and in vivo roles of gK in virus rep lication, a recombinant virus was constructed expressing an engineered enha nced green fluorescent protein (EGFP) under the control of the human cytome galovirus immediate early gene promoter (HCMV-IEP) inserted in place of the gK gene. The EGFP gene insertion was confirmed by diagnostic polymerase ch ain reaction (PCR), and the presence of the EGFP protein was detected by we stern immunoblot analysis using anti-GFP monoclonal antibody. Fluorescence microscopy revealed that virus infected cells emitted bright fluorescence w hen examined using filters for fluorescein. Fluorescence emission was detec ted as early as 4 h post-infection. Fluorescence intensity increased over t ime and was stable at late times after infection at which point viral plaqu es continued to emit bright green fluorescence. The amount of fluorescence emitted by virus infected Vero cells was monitored by fluorescence cytometr y using a FACS cytometer. At an MOI of 3, all infected cells emitted strong green fluorescence as quantified by cytometry at 48 h post-infection. The Delta gK-EGFP expressing recombinant virus will enable the determination of the role of gK in virus entry and egress as well as the role of gK in the molecular pathogenesis of herpes simplex virus type 1 (HSV-1). (C) 1998 Els evier Science B.V. All rights reserved.