78132
Mapping the Essential Bipartite Nuclear Localization Signal Domain of UOL Protein
Presenter: Gwendolyn Le
Mentor: Guey-Chuen Perng
During the course of herpes simplex virus type 1 (HSV-1) infection, viral proteins shuttle in and out
of the cell organelles, such as the nucleus, playing a crucial regulatory role in infected cells. For a
protein to be localized in the nucleus, a nuclear localization signal sequence (NLS) is required to
fulfill the task. UOL, a newly identified gene from HSV-1 viral genome, contains two bipartite NLS.
My goal is to check whether these NLS in the UOL protein are functional. If functional, I will further
characterize which one of the two NLS' is (are) essential for the UOL protein in targeting the nucleus.
Preliminary results, using a red fluorescent protein (RFP) indicator gene fused to UOL, indicated that
the NLS in UOL protein is functional in transient transfected cells. I continued onto mapping the
NLS domain. In this process, five sets of overlapping PCR primers attached with universal restriction
enzyme sites, EcoRI and BamHI at 5' and 3' ends respectively, were designed to facilitate the
directional cloning. The amplified PCR fragments were then fused in frame to the RFP gene, which
will show a visible red signal. In my research so far, I have obtained three sets of fusion clones into
pDs-Red vector and am currently performing transient transfection assays to check for the organelle
localization of RFP signal. I have also cloned the remaining two sets into the pGEM-T vector and
am currently cloning them into the pDs-Red vector to further perform the transient transfection
assays.