Construction and Characterization of Recombinant HSV-1 Expressing Early Growth Response-1

Early Growth response-1 (Egr-1) is a transcription factor that possesses a variety of biological functions. It has been shown to regulate HSV-1 gene expression and replication in different cellular environments through the recruitment of distinct cofactor complexes. Previous studies demonstrated that Egr-1 can be induced by HSV-1 infection in corneal cells but the level was lower compared to other cell types. The primary goal of this report is to generate a recombinant HSV-1 constitutively expressing Egr-1 and to investigate the regulation of viral replication in different cell types or in animals with Egr-1 overexpression. The approach utilized was to introduce Egr-1 into the BAC system containing complete HSV-1 (F) genome. To assist in the insertion of Egr-1, a gene cassette was constructed that contains the Egr-1 gene flanked by loxP sites. In this clone Egr-1 is expressed under control of CMV immediate-early promoter followed by another gene cassette expressing the enhanced green fluorescent protein (EGFP) under the control of the elongation factor 1α (EF-1 α) promoter. The constructed recombinant viruses were completed containing the Egr-1 gene within the viral genome and the expression was characterized by qRT-PCR and Western blot analyses. Our results showed that Egr-1 transcript and protein can be generated and accumulated upon infection of recombinant virus in Vero and rabbit corneal cells SIRC. This unique virus therefore is useful for studying the effects of Egr-1 during HSV-1 replication and gene regulation in epithelial cells and neurons. 1. Introduction Herpes simplex virus type-1 (HSV-1) has a large genome that can accommodate up to 30？kb of foreign sequences after deletion of a number of nonessential-for-replication genes [1]. This feature makes HSV-1-based vectors appropriate for a range of purposes in the development of gene therapy protocols. A number of strategies have been developed to manipulate the HSV-1 genome. The accessibility of a bacterial artificial chromosome (BAC) molecular clone of HSV-1 allowed efficient and rapid mutagenesis of large DNA sequences cloned into BACs, which were previously restricted to relatively small plasmids in E. coli [2]. In addition, Red recombination using PCR-amplified selectable markers as well as en passant mutagenesis utilizing markerless DNA strategy facilitates the insertions, deletions, and single point substitutions on a BAC-cloned DNA sequence [3]. In most circumstances, specific insertions that do not interfere with virus functions, while allowing stable expression of foreign genes, are