Development of a synchronized assembly assay and investigation of herpes simplex virus-1 maturation
Church, Geoffrey Allen
MetadataShow full item record
To investigate late events in Herpes Simplex Virus (HSV) assembly, we have developed an assay system which allows HSV assembly to be synchronized in infected cells. This synchronous assay makes use of the reversible temperature sensitive HSV mutant, tsProt.A. The defect in this virus prevents cleavage of the capsid scaffold protein, ICP35, when infected cells are maintained at a non-permissive temperature. Under these conditions immature capsids form but cannot mature, thus blocking viral DNA packaging and all subsequent steps in HSV assembly. Upon downshift to the permissive temperature the block in assembly is removed, scaffolds are cleaved, and the capsid population matures in a single, synchronous wave, completing all the necessary steps of virion maturation until a burst of infectious virus is produced. Utilizing tsProt.A, we have determined the kinetics of capsid scaffold cleavage, concatameric viral DNA genome cleavage and packaging, and the acquisition of infectivity. This system was further utilized to determine the relationship between ongoing viral DNA synthesis and DNA packaging. We have found that, after treatment with a viral DNA synthesis inhibitor which completely blocked new DNA synthesis, a pre-existing population of viral DNA can be cleaved and packaged into maturing capsids. These studies have shown that, while HSV DNA synthesis may occur during packaging into capsids, these two events are not linked in an obligate manner. This work has also led to the development of a more rapid and quantitative assay to measure HSV DNA packaging. It is expected that this data will allow the establishment of an in vitro system to monitor viral DNA packaging and, perhaps, later events in HSV assembly.;This thesis work also encompassed studies which elucidated the formation and nature of tegument bodies. Tegument bodies are juxtanuclear structures which contain proteins that are normally incorporated into virions. They are believed to be involved in the envelopment of maturing virus. We present evidence to demonstrate that tegument bodies behave in a manner very similar to misfolded protein aggregates which form in cells when the proteasomal machinery is inhibited or overwhelmed. We therefore suggest that these structures may not lie on a pathway leading towards the production of infectious virus.