Identifier

etd-06122014-154723

Degree

Doctor of Philosophy (PhD)

Department

Veterinary Medical Sciences - Pathobiological Sciences

Document Type

Dissertation

Abstract

Herpes simplex virus type 1 (HSV-1) is a ubiquitous neurotropic alphaherpesvirus transmitted by contact with mucocutaneous surfaces of infected individuals. HSV-1 enters the host by fusion of the viral envelope with the host cell plasma membrane, followed by translocation of the viral capsids to the nucleus where viral DNA is injected into the host cell nucleus to initiate viral replication. To generate infectious virions, newly assembled capsids travel to the cytoplasm and undergo a process called secondary envelopment by budding into cytoplasmic vesicles derived from the trans-Golgi network. Cytoplasmic envelopment is a complex process involving interactions between a multitude of viral membrane and tegument proteins. To investigate the relative importance of a subset of viral membrane and tegument proteins in secondary envelopment, a number of recombinant viruses were constructed in the HSV-1(F) genetic background. A mutant virus unable to express gE, gM and the C-terminus of gD was characterized and compared to additional mutants unable to express both gE and gM or gE and the C-terminus of gD and to mutants lacking expression of just one of these glycoproteins, in addition to mutants lacking expression of both pUL11 and gM, and pUL20 alone. Characterization of all mutant viruses by plaque morphology, viral replication kinetics, electron microscopy and particle-to-PFU ratios revealed a hierarchy of defects in cytoplasmic envelopment and infectious virus production, with deletion of pUL20 having the greatest effect, followed by the deletion of pUL11 and gM. Characterization of additional mutants containing multiple mutations revealed that gE, gM and gD do not function in a redundant manner in cytoplasmic envelopment supporting a preeminent role for the pUL20/gK protein complex in cytoplasmic envelopment and egress. An epitope tag insertion adjacent to the pUL37 Y480 (DC480) exhibited a severe defect in cytoplasmic envelopment similar to gK and pUL20-null viruses. Importantly, this mutant virus was partially complemented when grown on cells expressing pUL20, suggesting an interaction with the pUL20/gK protein complex. This pUL37 interaction with pUL20/gK was verified by co-immunoprecipitation and proximity ligation assays suggesting that it facilitates cytoplasmic virion envelopment.

Date

2014

Document Availability at the Time of Submission

Student has submitted appropriate documentation to restrict access to LSU for 365 days after which the document will be released for worldwide access.

Committee Chair

Kousoulas, Konstantin

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