*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.

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.