The role of the herpes simplex virus type 1 UL28 protein in terminase complex assembly and function

Herpes simplex virus type I (HSV-1) is the causative agent of several pathologies ranging in severity from the common cold sore to life-threatening encephalitic infection. During productive lytic infection, over 80 viral proteins are expressed in a highly regulated manner, resulting in the replication of viral genomes and assembly of progeny virions. Cleavage and packaging of replicated, concatemeric viral DNA into newly assembled capsids is critical to virus proliferation and requires seven viral genes: UL6, UL15, UL17, UL25, UL28, UL32, and UL33. Analogy with the well-characterized cleavage and packaging systems of double-stranded DNA bacteriophage suggests that HSV-1 encodes for a viral terminase complex to perform these essential functions, and several studies have indicated that this complex consists of the viral UL15, UL28, and UL33 proteins. However, the inability to purify the terminase proteins has hampered biochemical analysis of these proteins. The goal of the following studies was to isolate a functional terminase complex from HSV-1-infected cells by affinity chromatography using a virus expressing a UL28-TAP fusion protein. The tandem affinity purification (TAP) procedure resulted in the isolation of soluble UL28 complexes containing the UL15 and UL33 proteins. Biochemical studies were performed to determine the protein composition and stoichiometry of the purified complex, and the associated nuclease activity was examined. Mass spectrometry was utilized to identify viral and cellular proteins that associate with the complex during infection. Finally, mutations or deletions within the nuclease domain of UL15 or the metal-binding domain of UL28 were introduced into the genome of the NTAP-UL28 fusion virus. Characterization of these viruses followed by the isolation of terminase complexes revealed that the domain mutations did not preclude complex formation but each virus was deficient in viral DNA cleavage, further demonstrating the importance of these domains during DNA encapsidation. The ability to purify the endogenous terminase complex is novel to the field and we view these studies as a critical step in understanding how the terminase complex functions in the context of productive HSV-1 infection.