Virus vectors as tools to study herpes simplex virus type-1 in neurons

Abstract

Herpes simplex viruses are ubiquitous pathogens of humans. The hallmark of these viruses is their ability to establish and maintain a latent infection in the neurons of the host throughout its lifetime, creating a reservoir from which reactivation and transmission can occur. What neuronal or viral component(s) allow the virus to establish, maintain, or reactivate from a latent infection is not known. Potentially, the viral immediate early proteins ICP0. ICP4, and ICP27, which regulate gene expression during the productive infection, are important during the latent stages of the virus lifecycle as well. Virus vectors remain the best way to deliver and express genes in the nervous system. This is, in part, due to the unique characteristics of neurons to be in a post mitotic state and recalcitrant to other methods of introducing genes. Recombinant adenoviruses were used to deliver and express herpes simplex virus (HSV-1) immediate early proteins ICP0, ICP4, and ICP27 fused to the green fluorescent protein in cell lines or neurons. ICP4 or ICP27 fusion proteins showed fluorescence in the nucleus of cell lines or neurons, while the ICP0 fusion protein showed punctate cytoplasmic fluorescence in cell lines and was not detectable in neurons. These studies showed there might be differential expression of an HSV-1 immediate early protein in neurons. Virus vectors have also been used to ablate gene expression by expressing the complement of a gene's coding region, or anti-sense RNA. Anti-sense RNA produced from the complementary strand can then block the expression of its targeted protein by binding to the mRNA, which normally would be translated to produce a functional protein. We used an RNA virus vector and a DNA virus vector to produce anti-sense RNA targeted to ablate gene expression of two essential HSV-1 immediate early genes ICP4 or ICP27, and of one nonessential HSV-1 immediate early gene ICP0. A Sindbis (RNA) virus vector and an adenovirus (DNA) virus vector were able to express anti-sense RNA targeted against the 5' end of the ICP4 coding region. As ICP4 is essential for viral replication, we used the anti-sense expressing virus vectors to attempt to inhibit HSV-1 growth in cell lines or neurons. The Sindbis virus or adenovirus vectors, expressing anti-sense ICP4, did not inhibit HSV-1 growth in a specific manner. Control virus vectors, expressing RNA not specific for ICP4, inhibited HSV-1 growth to the same or greater extent. Control adenovirus vectors, interestingly, were able to inhibit 4-5 logs of HSV-1 growth. These studies suggest that using virus vectors to express anti-sense RNA to interfere with HSV-1 growth may be more difficult than expected.