In vitro Interaction and Colocalization of HSV-1 ORF P with a Cellular Splicing Factor (SC35) Using Pulldown Assay

Herpes simplex virus type-1 (HSV-1) causes a variety of diseases in human. This virus is a neurotropic pathogen of human that establishes latent infection in the sensory ganglia innervating the site of primary infection. A number of genes including ICP34.5 control HSV-1 pathogenicity and ICP34.5 has been identified as HSV-1 virulence gene. Open reading frame P (ORF P) is also a HSV-1 gene that might have a role in latency. A complication in the analysis of the role of ICP34.5 and ORF P in the HSV-1 life cycle is that these two are overlapping antisense genes. ORF P is also deleted in ICP34.5 negative mutants and to date, no definite function is attributed to it. To attribute characteristics which were originally attributed solely to ICP34.5 to each of these two genes (ORF P or ICP34.5), an approach is to construct a number of HSV-1 recombinant viruses that express ICP34.5 and ORF P independently. An alternative way is to determine if ORF P interacts with any of the cellular and viral proteins both in vitro and in vivo. Using Glutathione-S-transferase (GST) pulldown assay and Western-blotting, we showed that ORF P interacts with a cellular splicing factor (SC35) in vitro. To investigate the colocalization of ORF P and SC35, nuclear and cytoplasmic fractionation of ORF P/SC35 was also carried out. Our results showed that both SC35 and ORF P are located in the nucleus of HSV-1 infected cells. Conclusively, because ORF P interacts and colocalizes with SC35, it might have a role in splicing