Herpes simplex virus type-1 (HSV-1) lytic infection causes diseases ranging from simple cold sores to dangerous keratitis and lethal encephalitis. The interaction between virus and host cells is being investigated extensively by many laboratories. Our data demonstrated that HSV-1 can rapidly induce the expression of multi-functional transcriptional factor early growth response-1 (Egr-1) during lytic infection in corneal cells. Egr-1 functions as a convergence point for many signaling cascades and is known to play an important role in regulating inflammation, cell proliferation and apoptosis. Western blot analyses showed that Egr-1 was absent in Vero and rabbit corneal cell line SIRC but the protein was detected starting from 24 hours post infection (hpi) and was directly proportional to the amount of virus used for infection. Infection with recombinant virus expressing EGFP followed by immunofluorescent studies revealed that Egr1 was expressed only within the infected cells. Reverse transcriptase polymerase chain reaction (RT-PCR) analyses indicated that Egr-1 mRNA was transcribed as early as 1 hpi and did not require de novo viral gene expression since UV inactivated virus initiated the Egr1 transcription but failed to produce protein. Chromatin Immunoprecipitation (ChIP) assays demonstrated that NFkB and cAMP response element binding protein (CREB) was induced by HSV-1 infection and recruited to the Egr-1 promoter upon infection. Collectively, these results suggest that Egr-1 is efficiently induced upon HSV-1 lytic infection and may play a key role in the viral replication and the disease progression in eyes. In this project, we will further identify the mechanisms that induced the Egr1 expression by HSV-1 infection and investigate the roles of Egr1 on HSV-1 replication through the following Specific Aims.
Specific Aim 1. To understand how viral infection induced Egr1 expression. In this Aim, we will examine if viral binding of the cells, viral gene expression, viral replication, or all of the above were required to initiate Egr1 transcription/translation.
Specific Aim 2. To determine the mechanism that controlled the transcription of Egr1. In this Aim, we will investigate the pathways that regulated Egr1 transcription.
Specific Aim 3. To evaluate the effect of Egr1 on HSV-1 gene expression and replication. Our published data showed that overexpression of Egr1 bound to ICP22 intron and inhibited transcription of ICP4 and ICP22. In this aim, we will use DNAzyme to eliminate Egr1 expression by infection and check if the elimination can affect HSV-1 gene expression and replication. Our immediate goal is to establish an active research program at the University of Louisiana Monroe (ULM) so the students can study Virology since our laboratory is the only research program at ULM and Northeast Louisiana dedicating to virus research. The long-term mission is to identify the regulatory mechanisms controlling viral gene expression and replication to develop novel therapeutic protocols for treatment of this devastating and severe viral disease.
Herpes Simplex Virus -1 (HSV-1) primary and recurrent infection may result in scarring of the cornea and is the leading cause of blindness in US and the developed world. We showed that HSV-1 infection rapidly induced the expression of an important multifunctional protein Egr-1 in corneal cells. The completion of this proposal will shed light on the molecular mechanism of HSV-1 mediated Egr1 expression and assist to develop new strategy to prevent viral replication and blindness caused by HSV-1.
