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*Thesis Defense Announcement To: The George Mason University Community* *Candidate: Clinton W. Enos Program: Master of Science in Biology * *Date: Friday July 15, 2011 Time: 9:00 a.m. Place: George Mason University, Prince William campus Discovery Hall, Room 153 *Thesis Chair: Dr. Yuntao Wu Title: "Point Mutation H89A of the HIV-1 Nef Protein Renders a Moderate Impact on HIV-1 Replication and Infectivity: a Look into the Putative Nef/Pak2 Interaction"* * A copy of the thesis is on reserve in the Johnson Center Library, Fairfax campus. The thesis will not be read at the meeting, but should be read in advance. All members of the George Mason University community are invited to attend. *Abstract:* The human immunodeficiency virus type 1 (HIV-1) Nef protein is a multifunctional accessory protein believed to be necessary for HIV-1 pathogenesis and AIDS progression. Among the putative roles of Nef in HIV-1 infection is the association with an active cellular serine/threonine kinase, suggested as being the p21-activated kinase 2 (PAK2). The PAKs are serine/threonine kinases that are downstream effectors of Rac and Cdc-42 GTPases. The PAKs have been shown to play critical roles in many different host-cell functions including proliferation, survival, motility and cytoskeleton dynamics. The function of the Nef/PAK2 interaction in HIV-1 infection is not well understood. Proposed roles for Nef/PAK2 have been, for the most part, based on data employing the over expression of Nef or through the use of Nef mutants that, after subsequent studies, proved to have pleiotropic effects. However, recent studies have established the importance of a hydrophobic binding surface proposed to be specific for the association of Nef with PAK2. It was in our interest to uncover and further define a biological role of the Nef/PAK2 interaction in HIV-1 infection by designing a system that reflects physiological parameters and utilizes Nef mutants directed at the hydrophobic binding surface. To approach this problem we first designed a plasmid that exclusively produces the HIV-1 Nef protein in a physiologically relevant amount. The Nef-expression plasmid, pNLDYSE-Nef, is a pNL4-3 derived, Rev-independent expression vector that allows for exclusive expression of Nef under the control of the HIV-1 promoter, the LTR. pNLDYSE-Nef contains the necessary splicing sites for Nef expression (A1-D5, A4-D7) and lacks the HIV-1 packaging signal (Y). This results in physiological basal expression of Nef protein that, when constructing a virus, will be packaged into budding virion particles. pNLDYSE-Nef can also be used in transfection experiments to observe protein-protein interactions that are influenced by Nef alone. Additionally, we designed Nef/PAK2 mutants (F191R, H89A, and V85S) and cloned them into the pNL4-3 proviral vector as well as pNLDYSE-Nef. The resulting pNL4-3 Nef-mutant plasmids were used to generate virus. Infection of resting and pre-stimulated primary CD4 T lymphocytes with Nef/PAK2 mutant HIV-1 viruses demonstrated a mild phenotype as compared to /wild type /virus. Of note, point mutation H89A of HIV-1 Nef impacted multi-cycle replication in multiple donors. Infection of the Rev-dependent reporter cell line, G11, with /wild type/ virus, D/nef/ virus, and NL4-3 Nef-mutant virus indicates that the Nef/PAK2 interaction is influential on Nef's ability to increase infectivity of HIV-1 virion particles. Virions lacking a /nef /gene were observed to be merely half as infectious as /wild type/. A similar phenotype existed for the H89A Nef/PAK2 mutant. However, it appears that the Nef/PAK2 phenotype, exhibited by mutation H89A, is not sustained as long as D/nef. /Therefore, there may be an advantage for Nef/PAK2 at low levels of replication, however the phenotype is lost as multiple rounds of replication and infection have occurred. The /nef/ phenotype for infectivity enhancement appears to last through multiple rounds of infection and is most apparent at lower levels of replication. However, in a more active cellular state (spinoculation) replication levels are increased and a gradual disappearance of the /nef /phenotype was observed. Since Nef has been described as modulating the actin cytoskeleton in a PAK2-dependent manner, we investigated a role in the regulation of the actin binding protein, cofilin. Infection of the T cell line, CEM-SS, with /wild type /and /nef/ mutant viruses demonstrated no increase in phosphorylation levels of cofilin. Collectively, these data indicate a moderate phenotype for Nef/PAK2 in HIV-1 infection /in vitro/. However, ultimately the proposed association does not appear to be a requirement for HIV-1 replication. ###