Protein Phosphoribosylation: Characterization of a novel protein modification - Abstract Herpesviruses are ubiquitous pathogens, and their infections are implicated in diverse diseases in human. Their infections can lead to life-threatening encephalitis under immune-deficient conditions. As obligate intracellular pathogens, viruses rely on cellular machinery to synthesize macromolecules for vial progeny production. Viruses are known to activate metabolic enzymes to fuel viral replication, however their functions beyond metabolism remain largely unchartered. By examining NAD+ metabolism in herpes simplex virus 1 (HSV- 1) infection, we discovered that the rate-limiting enzyme of the salvage NAD+ synthesis pathway, nicotinamide phosphoribosyltransferase (NAMPT), restricts HSV-1 lytic replication via dephosphoribosylating key structural proteins. Importantly, phosphoribosylation of these viral structural proteins facilitate their incorporation into virion progeny and subsequent infection. Among these structural proteins, VP22 is one of the most abundantly phosphoribosylated proteins of HSV-1. Protein phosphoribosylation is generally believed to be the degradation byproduct of ADP-ribosylation, a dynamic post-translational modification implicated in several diseased states, such as cancer, infection, chronic inflammation, and neurodegeneration. How phosphoribosylated proteins are generated from ADP-ribosylated proteins and its biological functions thereof have not been investigated in metazoans, particularly in the context of viral infection. For the first time, we have developed a system that enables the interrogation of the biochemistry and biological significance of protein phosphoribosylation in metazoans. Specifically, we have identified HSV-1 VP22 as a highly phosphoribosylated tegument protein and phosphoribosyl moieties of VP22 enhance its virion incorporation and subsequent infection of HSV-1 virions. This transition grant will characterize the biogenesis of phosphoribosylation of proteins using VP22 as a model substrate. Specifically, we will identify and characterize the enzyme(s) that catalyzes VP22 ADP-ribosylation (Aim 1) and subsequently process ADP-ribose to produce phosphoribose (Aim 2). In future, we will probe the roles of protein phosphoribosylation in HSV-1 infection in vivo. The reagents and system we develop here will constitute the toolkit for the study of protein post-translational modification to investigate viral protein phosphoribosylation in the subsequent project. Findings from this study will establish the first example of the biogenesis of protein phosphoribosylation in metazoan, pushing the boundary of our knowledge on protein post-translational modification in HSV-1 infection.
