Real-time Visualization of the Viral and Host Cell Proteins Involved In The Intracellular Transport of Mason-Pfizer Monkey Virus Público
Clark, Jasmine Michelle (2013)
Abstract
Real-time Visualization of Viral and Host Cell Proteins Involved In The Intracellular Transport of Mason-Pfizer Monkey Virus
By Jasmine Clark
Mason-Pfizer Monkey Virus, the prototypic D-type retrovirus, co-translationally assembles immature capsids in the pericentriolar region of the cell. From here, immature capsids are transported to the plasma membrane for budding, release, and maturation into an infectious virions. Several mutational and biochemical studies have been employed to elucidate viral and host proteins important for this intracellular transport. Although these studies provided very important insight as to what proteins are involved in this portion of the viral life cycle, they have relied on population analysis of capsids. In this dissertation, we create a codon-optimized M-PMV Gag-eGFP fusion vector with an optimized Kozak consensus sequence and an alanine substitution at an internal methionine. This vector, co-expressed with untagged Gag, produces fluorescently-labeled capsids that are able to be visualized by real-time, live cell imaging. Characterization of the trafficking of these fluorescent capsids implicated a role for microtubules. This role was confirmed by employing the use of cytoskeletal inhibitors. Furthermore, using a mCherry-tagged M-PMV Env, co-transport of M-PMV Gag and Env was observed, and this co-transport as well as incorporation of Env into released virions required the presence of intact microtubules. This data provides strong support for the hypothesis that in order to produce infectious virus, M-PMV Env that has been transported to the plasma membrane from the trans Golgi network is endocytosed and recycled through the pericentriolar region, whereby these Env-containing recycling vesicles interact with assembled immature capsids, followed by co-transport, along microtubules, toward the plasma membrane for budding.Table of Contents
Table of Contents
Abstract
Dedication
Acknowledgements
Introduction....................................................................................... 1
Retroviruses.................................................................................... 1
Mason-Pfizer Monkey Virus............................................................... 5
Intracellular Vesicular Transport.................................................... 14
This Dissertation............................................................................ 20
A Mason-Pfizer Monkey Virus Gag-GFP Fusion Vector Allows Visualization of Capsid Transport in Live Cells and Demonstrates a Role for Microtubules......................................................................... 24
Abstract........................................................................................ 25
Introduction.................................................................................. 26
Materials and Methods.................................................................... 29
Results.......................................................................................... 38
Discussion...................................................................................... 57
Tables and Supporting Information.................................................. 63
Acknowledgements........................................................................ 72
Literature Cited............................................................................. 73
Direct evidence for intracellular anterograde co-transport of M-PMV Gag and Env on microtubules. 78
Abstract........................................................................................ 79
Materials and Methods.................................................................... 83
Results.......................................................................................... 89
Supporting Figures....................................................................... 115
Acknowledgements....................................................................... 121
Literature Cited........................................................................... 122
Discussion....................................................................................... 128
Summary.................................................................................... 136
Literature Cited (Introduction and Discussion)................................ 138
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Real-time Visualization of the Viral and Host Cell Proteins Involved In The Intracellular Transport of Mason-Pfizer Monkey Virus () | 2018-08-28 13:12:30 -0400 |
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Supplemental Files
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Dissertation_Chapter 3_Movie S2.wmv () | 2018-08-28 13:13:11 -0400 |
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Dissertation_Chapter 2_Movie S1.wmv () | 2018-08-28 13:13:45 -0400 |
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Dissertation_Chapter 3_Movie S4b.wmv () | 2018-08-28 13:13:48 -0400 |
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Dissertation_Chapter 3_Movie S5a.wmv () | 2018-08-28 13:13:59 -0400 |
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Dissertation_Chapter 2_Movie S2.wmv () | 2018-08-28 13:14:08 -0400 |
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Dissertation_Chapter 3_Movie S3b.wmv () | 2018-08-28 13:14:15 -0400 |
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Dissertation_Chapter 3_Movie S5b.wmv () | 2018-08-28 13:14:23 -0400 |
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Dissertation_Chapter 3_Movie S1.wmv () | 2018-08-28 13:14:30 -0400 |
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