Date of Award

5-2015

Degree Name

Doctor of Philosophy

Department

Molecular Biology, Microbiology and Biochemistry

First Advisor

Halford, William P.

Second Advisor

Torry, Donald S.

Third Advisor

Trammell, Rita

Abstract

Both herpes simplex virus type-1 (HSV-1) and herpes simplex virus type-2 (HSV-2) are human pathogens that are capable of causing significant disease. More than 600 million people are infected with HSV-2 genital herpes and every day >40,000 new HSV-2 infections are transmitted to naïve recipients. The public health community has long agreed that a preventative vaccine is necessary to reduce the prevalence of HSV-2, but we continue to lack a vaccine to prevent the spread of this sexually transmitted disease. Herpes simplex viruses (HSV) establish a life-long infection in their host where they alternate between a state of latency and productive replication. One of principle determining factors of whether HSV reactivates from a latent state to cause disease is the accumulation of the immediate early viral protein, infected cell protein 0 (ICP0). ICP0 is a potent transactivator of HSV mRNA synthesis. However, in addition to the nuclear transactivating function of ICP0, our lab demonstrated that at later times during infection, ICP0 translocates from the nucleus to the cytoplasm where it reorganizes the host microtubule network (PLoS ONE 5(6): e10975). In the first part of my dissertation, we used the anti-neoplastic drug paclitaxel to determine if ICP0’s microtubule-reorganizing activity is required for efficient replication of HSV- 1. Surprisingly, we observed that paclitaxel prevented ICP0 from dispersing cellular microtubules and inhibited the spread of HSV-1 and HSV-2. Paclitaxel reduced HSV-1 plaque size by up to 100-fold, and inhibition of viral spread was proportional to the efficiency with which doses of paclitaxel stabilized microtubules against ICP0-induced dispersal. Analysis of infected Vero cells confirmed that paclitaxel treatment did not reduce the efficiency of HSV-1 mRNA, protein, DNA, or virion synthesis by more than 3-fold. In contrast, paclitaxel treatment caused an ~20-fold decrease in infectious virus release from HSV-1 infected cells. Comparison of ICP0+ versus ICP0- viruses yielded genetic evidence that ICP0 promotes infectious virus release from HSV-1-infected cells. These results provide the first evidence suggesting that ICP0’s microtubule-reorganizing activity may promote the release of infectious virus from HSVinfected cells. In the second part of my dissertation, I focus less on the basic-science quest to elucidate the function of ICP0 but instead focus on characterizing the antibody response elicited by a highly protective HSV-2 ICP0- vaccine that was developed in our lab. Specifically, despite the belief that virion glycoproteins such as glycoprotein D (gD-2) are the dominant antigens of herpes simplex virus 2 (HSV-2) we have demonstrated mice immunized with a live HSV-2 ICP0- mutant virus, HSV-2 0ΔNLS, are 10 to 100 times better protected against genital herpes than mice immunized with a HSV-2 gD subunit vaccine (PLoS ONE 6:e17748). In light of these results, we sought to determine which viral proteins were the dominant antibody-generators (antigens) of the live HSV-2 0ΔNLS vaccine. Western blot analyses indicated the live HSV-2 0ΔNLS vaccine elicited an IgG antibody response against 9 or more viral proteins. Many antibodies were directed against infected-cell proteins of >100 kDa in size, and only 10 ± 5% of antibodies were directed against gD. Immunoprecipitation (IP) of total HSV-2 antigen with 0ΔNLS antiserum pulled down 19 viral proteins. Mass spectrometry suggested 44% of immunoprecipitated viral peptides were derived from two HSV-2 infected cells proteins, RR-1 and ICP8, whereas only 14% of immunoprecipitated peptides were derived from HSV-2's thirteen glycoproteins. Collectively, the results suggest the immune response to the live HSV-2 0ΔNLS vaccine includes antibodies specific for infected cell proteins, capsid proteins, tegument proteins, and glycoproteins. This increased breadth of antibody-generating proteins may contribute to the live HSV-2 vaccine's capacity to elicit superior protection against genital herpes relative to a gD subunit vaccine. In the final section of my dissertation, I focus on the construction of stable Vero cell lines using a system that relies on Sleeping Beauty transposase to introduce two genetic elements into the chromosomes of target cells: 1. a Tet-regulatable gene-of-interest driven by the VP16- inducible HSV-1 ICP0 promoter and 2. a highly expressed bicistronic gene that expresses the Tet-repressor protein and a puromycin selection marker. The modified bidirectional HSV-1 ICP0 promoter used in this novel expression system 1. drives constitutive expression of a cell surface marker (tNGFR) from the reverse side of the ICP0 promoter, while 2. the Tetregulatable, forward side of the ICP0 promoter allows the expression of a downstream gene-ofinterest to be regulated over an ~100-fold range of gene expression through doxycyclineinduced derepression and induction with VP16. Using this system, stable and regulatable Vero cell lines are reproducibly obtained within one month of initial transfection by using puromycin selection followed by fluorescence-activated cell sorting (FACS) to isolate a pure population of tNGFR+ cells that contain the target gene-of-interest. Vero cell lines that carry a toxic gene, such as HSV-1 ICP0, can be routinely passaged and maintained in cell culture because basal expression of the gene-of-interest can be reduced to low to undetectable levels. However, when the same cell lines are de-repressed, they express adequate levels of HSV-1 ICP0 or HSV-1 origin-binding protein (OBP) to complement an HSV-1 ICP0- mutant . These data suggest this novel gene expression system may have broad uses in biological research because it allows for the efficient generation of stable cell lines that express a gene-of-interest in a highly regulatable manner.

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