Mason-Pfizer Monkey Virus Protein Trafficking

Anqi Jasmine Clark, Eric Hunter, Ph.D. 1 Emory College of Arts and Sciences
1 Yu ,

Introduction
Mason-Pfizer Monkey Virus (MPMV) is a simian retrovirus that
can be used to model human immunodeficiency virus (HIV) trafficking because of their similar genomes. gag viral matrix env viral envelope To observe these proteins in the host cells and to profile viral protein trafficking, gag and env are labeled with the fluorescent proteins constructs gagGFP and envCherry, respectively. Live-cell microscopy is then used to visualize the proteins as they move through the cell, which are hypothesized to employ motor proteins and the cytoskeleton for transport (Fig. 1):

Results

Conclusions
The viral proteins gag and env are colocalizing in CMMT cells as suggested by the nMDP and Lis ICQ. A positive nMDP number of 0.003 derived from the colocalization colormap (Fig. 5) indicates that there is a tendency to colocalize even though it is on a small scale. Lis ICQ (Figs. 6 and 7) confirms that the colocalization occurring is dependent colocalization. gag and env are also cotransporting in CMMT cells. This is determined by combining data from both Fig. 4 and Fig. 5 to distinguish truly colocalized particles. These particles trajectories were quantified and recorded using their x- and y- coordinates, and velocity and displacement data were gathered from these coordinates. The method of transportation is still uncertain. Viral proteins are known to use many modes of trafficking, and the results of this experiment does not conclude which mode was being utilized. Thus, more experiments are needed to determine the mode of transportation utilized by the viral proteins gag and env.

Figure 3. Raw screen capture of a co-transfected cell.

Figure 4. Trajectories from single-particle tracking.

Figure 1. A diagram of kinesis and dynein on a microtubule.

Videos are taken of the cells to record the movement of the proteins. Emphasis is placed on cells that are co-transfected with both fluorescent constructs ImageJ is used to process the videos and to obtain data. Data obtained include velocity, Lis intensity correlation analysis, colocalization color maps, and normalized mean deviation product (nMDP).
Figure 5. Colocalization colormap of the cell.

The data collected show that the bulk of the cotransportation (Fig. 4) and colocalization (Fig. 5) occurring in the cell are in the pericentriolar region and the periphery of the cell. The colocalization colormap in Fig. 5 yielded an nMDP of 0.003. A positive number indicates that the proteins are tending to colocalize, but the magnitude of the number indicates it is occurring on a small scale. When analyzing the live-cell videos, data from both Fig. 4 and Fig. 5 are used to determine which particles are truly colocalized gag and env proteins.

Future Directions
env is known to make use of multiple pathways; for example,
the endosomal pathways. Rab markers (Rab5, 7, 9, 11) can be used to track these endosomes:

Materials & Methods

Cocultivated monkey mammary tumor (CMMT) cells were used. They were allowed to grow until 80-90% confluence on a petri dish with Dulbeccos Modified Eagle Medium (DMEM) enriched with 10% fetal bovine serum (FBS). When ready for use in an experiment, the cells are transfected, following the protocol displayed in Fig. 2.

Data Analysis
Lis Intensity Correlation Analysis: Velocity:

Fig 3. Endosomal Pathways

OMEGA/OMERO program for data analysis. Other statistical methods to confirm data: Manders Coefficient, van Steensels Cross Correlation Coefficient (CCF).

Acknowledgements
Dr. Leah Roesch For serving as a wonderful temporary mentor and SIRE RPP coordinator. Becky Meyer and Dr. Stephanie Zlatic For being great mentors that I could bring up any questions to. Pranav Ramesh For helping with poster formatting. HHMI for funding the SIRE RPP Program.

References
Figure 2. Cell Transfection Protocol.
1. 2.

A DeltaVision Deconvolution Microscope was used to image the cells. Movies taken consisted of pictures taken by the microscope at five frames per second every five seconds for a total elapsed time of two minutes.

Figure 6 and 7. Lis ICA A (top) and ICA B (bottom) graphs. These yielded a positive intensity correlation quotient (ICQ) number of 0.357, indicating that most colocalization is dependent rather than segregated.

Figure 8 and 9. Trajectories are spatially quantified using the x- and y- coordinates (top). These coordinates are processed using Excel to get maximum velocities (bottom).

3. 4. 5. 6.

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