Herpes simplex virus 1 (HSV-1) is important human pathogen widely known as the causative agent of cold sores, and it is the leading infectious cause of blindness, and the most common cause of sporadic encephalitis. Although intensively studied, many fundamental aspects of the HSV-1 biology are poorly understood, including the control of latency and regulation of gene expression. We have recently discovered numerous miRNAs, a class of small regulatory non-coding RNAs, expressed by HSV-1 and closely related virus, HSV-2, revealing a new paradigm for the HSV gene regulation. Some of these miRNAs are conserved between these two viruses and expressed differentially during different phases of infection; however function of most HSV miRNAs is unknown. We hypothesize that miRNAs have a central role in modulating the host-pathogen interaction that leads to efficient virus replication and establishment of latency. Also, we hypothesize that miRNA have a central role in establishing the threshold for the reactivation upon different stimuli, i.e. control of the lytic-latent switch. Here we seek to understand the roles of microRNAs (miRNAs), encoded by the virus and host, in the regulation of gene expression during lytic and latent HSV-1 infection. We primarily focus on functions of miR-H1 and miR-H6, HSV-1 miRNAs abundantly expressed during lytic infection, and lytic and latent infection, respectively, and miRNAs almost entirely complementary to each other. In our preliminary data, we have generated the HSV-1 mutant impaired for the expression of these miRNAs and characterize the mutant in vitro and in a mouse model. In addition, to enable high-throughput technologies, we have tested several in vitro latency models and characterized the expression of HSV-1 and host miRNAs in these models. At the conclusions of these studies we will have better understanding of the HSV-1 biology including the virus pathogenesis, and it might lead to novel approaches to cure HSV infections.