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)

Permanent URL: https://etd.library.emory.edu/concern/etds/jd472w79n?locale=pt-BR
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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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