Study of the interaction of the matrix protein of the newcastle disease virus with lipid bilayers: implications for the mechanism of viral budding

Salamanca - Shnyrova , Anna

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Colecciones : TD. Ciencias biosanitariasDBBM. Tesis del Departamento de Bioquímica y Biología Molecular
Fecha de publicación : 11-abr-2008
La regulación dinámica de la forma de la membrana celular mediante interacciones proteolipídicas es importante para procesos intra- e inter-celulares. Se sabe que la gemación de muchos virus con envoltura se basa en la acción excusiva de una única proteína: la proteína matriz ó M. En el presente trabajo la gemación viral ha sido reconstituida usando la proteína M del virus de la enfermedad de Newcastle (NDV) y diferentes sistemas lipídicos modelo (membranas planas, vesículas unilamelares gigantes y grandes, y monocapas lipídicas). Así, la gemación por parte de la proteína M de NDV ha sido caracterizada mediante diferentes técnicas experimentales, tales como medidas de admitancia eléctrica, microscopía de fluorescencia, técnicas espectroscópicas, microscopía electrónica y medidas de presión superficial. Los datos experimentales obtenidos señalan que la interacción de la proteína matriz del NDV con los sistemas lipídicos modelo lleva a la gemación de membrana y producción de vesículas cuya distribución de tamaños es similar a la del NDV. De ésta forma, la proteína matriz del NDV es capaz de inducir la gemación de la membrana lipídica en ausencia de otros componentes de la maquinaria celular o viral. La adsorción y gemación por parte de la proteína matriz del NDV depende de la composición lipídica de la membrana, viendose ámbas favorecidas por la presencia de moleculas como el colesterol. Además, se observa que la proteína M del NDV organiza dominios fuidos sobre la membrana lipídica antes de que ocurra la gemación de la membrana. La unidireccionalidad de la gemación producida por la proteína M indica que el autoensamblaje de la proteína sobre la superficie de la membrana lipídica induce una curvatura negativa en la membrana. La formación de dominios proteolipídicos líquidos sobre la superfície de la membrana se propone como el mecanismo para la gemación de membrana celular por parte de la proteína M del NDV.Dynamic regulation of membrane shape by lipid-protein interactions is imperative for many intra and intercellular processes. Matrix proteins of many enveloped viruses have been suggested as the main responsible of the shape transformation of the cellular membrane into the viral vesicle. In order to investigate the molecular mechanism behind the matrix protein driven budding this process has been reconstituted with M protein purified from the NDV and different model lipid systems (planar lipid membranes, large and giant unilamellar vesicles and lipid monolayer). M protein budding activity has been characterized by different experimental approaches, including electric admittance measurements, fluorescence microscopy, spectroscopic approaches, electron microscopy and surface pressure measurements. The experimental data had shown that the interaction of the matrix protein from NDV with model lipid system results in membrane budding and production of membrane vesicles with a size distribution similar to that of the NDV. Thus, NDV matrix protein does not need the presence of any cellular component in order to activate its budding activity. NDV matrix protein binding and budding activities were dependent on the membrane lipid composition, being enhanced in the presence of molecules such as cholesterol in the membrane. Moreover, it was observed that the M protein of NDV organizes fluid-like domains on the lipid membrane prior to the membrane budding. The unidirectionality of budding produced by M protein indicated that the self-assembly of the protein on the membrane surface induced creation of a negative curvature. Fluid-like proteolipid domain creation had been proposed as the mechanism behind the cellular membrane budding induced by the matrix protein of the Newcastle Disease Virus.

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Publié par	Salamanca
Nombre de lectures	58
Licence :	En savoir + Paternité, pas d'utilisation commerciale, partage des conditions initiales à l'identique
Langue	Español
Poids de l'ouvrage	4 Mo

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UNIVERSIDAD DE SALAMANCA
FACULTAD DE BIOLOGÍA
Departamento de Bioquímica y Biología Molecular

STUDY OF THE INTERACTION OF THE MATRIX PROTEIN OF
THE NEWCASTLE DISEASE VIRUS WITH LIPID BILAYERS:
IMPLICATIONS FOR THE MECHANISM OF VIRAL BUDDING

ANNA SHNYROVA
2008

ENRIQUE VILLAR LEDESMA, CATEDRÁTICO DEL DEPARTAMENTO DE
BIOQUÍMICA Y BIOLOGÍA MOLECULAR DE LA UNIVERSIDAD DE
SALAMANCA

CERTIFICO:

Que la presente Tesis Doctoral titulada ¨Estudio de la interacción de la
proteína matriz del Virus de la enfermedad de Newcastle con bicapas lipídicas
implicaciones en el mecanismo de la gemación vírica¨, que para optar al grado de
Doctora en Biología presenta Dña. ANNA SHNYROVA, ha sido realizada bajo mi
dirección en el Departamento de Bioquímica y Biología Molecular de la Facultad de
Biología de la Universidad de Salamanca.

Considerando que dicha tesis se halla concluida, autorizo su presentación para
que pueda ser juzgada por el tribunal correspondiente.

Y para que así conste, firmo el presente certificado en Salamanca a 11 de
Abril de 2008.

Dr. Enrique Villar Ledesma

To my painter…

ACKNOWLEDGMENTS

I would like to deeply acknowledge my supervisor Dr. Enrique Villar and my co-
adviser Dr. Joshua Zimmerberg for their versatile support and inspiring discussions.
They opened my eyes toward new and exciting perspectives and provided a
reasonable degree of freedom in my work. Furthermore they skillfully revised this
thesis. I’m especially thankful to Dr. Zimmerberg for introducing me into the world of
membrane curvature that has adsorbed all of my attention.

I would like to thank Dr. Paul Blank for sharing his wisdom and experience with me.

I’m very thankful to Juan Ayllón, for being who purified the protein in the beginning
of this project and showed me how to do it latter and with whom we spent so many
hours of interesting discussions about science and life.

I would like to thank all the members of the Laboratory of Cellular and Molecular
Biology at NIH for their help and support. Special thanks to Drs. Elvira Rafikova and
Kamran Melikov, who were always there to help me.

I would like to thank the members of the Department of Biochemistry and Molecular
Biology in Salamanca, for their help and comprehensiveness in many moments of my
scientific life.

I have to specially acknowledge my parents, who gave me education and remain my
most wisdom teachers. They introduced me to science in my infancy and I’ve been
suffering from this disease since then. But my deepest gratitude to them is for their
unconditional support through all my good and bad moments.

This work is dedicated to my husband, the painter of my life.

PREFACE

This thesis project has been carried out in the collaborative framework
between the laboratory of Dr. Enrique Villar Ledesma from the University of
Salamanca in Spain and the Laboratory of Dr. Joshua Zimmerberg from the National
Institutes of Health in the USA.
This work has been supported by the intramural research program of the
Eunice Kennedy Shriver National Institute of Child Health and Human
Development and by the grant from Fondo de Investigaciones Sanitarias (FIS) #
PI05/1796 to Enrique Villar.

This thesis has been partially published in the Journal of Cell Biology (JCB
179 (4), 627-633, (2007)) . Also, part of the thesis results has been communicated at
the following International Meetings:

- Biophysical Society Meeting in 2006 and 2007, USA

- EMBO workshop on Cell Membrane Organization and Dynamics, 2006, Bilbao,
Spain

- Thirteenth International Conference on Negative Strand Viruses, 2006, Salamanca,
Spain

- International Conference “Membrane Biophysics of Fusion, Fission and Rafts in
Health and Disease”, 2007, Society of General Physiologists, Woods Hole, USA

ABBREVIATIONS

ANTS - 8-aminonaphthalene-1,3,6-trisulfonate
BLM – Bilayer Lipid Membrane
BODIPY - boron dipyrromethane difluoride
BSA – Bovine Serum Albumin
++Ca - Calcium ion
COP – Coat Protein Complex
Corp. - Corporation
DOPC - 1,2-Dioleoyl-sn-Glycero-3-phosphocholine
DOPE - 1,2-Dioleoyl-sn-Glycero-3-phosphoethanolamine
DOPG - 1,2-Dioleoyl-sn-Glycero-3[pospho-rac-(1-glycerol)] (sodium salt)
DPX - p-xylenebis(piridinium bromide)
EM - Electron Microscopy
ER – Endoplasmic Reticulum
ESCRT - Endosomal Sorting Complex Required for Transport
F protein – viral fusion protein
FITC - Fluorescein isothiocyanate
GUV – Giant Unilamellar Vesicles
HIV - Human Inmunodeficiency Virus
HN protein - hemagglutinin-neuraminidase protein
HPAIV - highly pathogenic avian influenza virus
ILV - Intralumenal vesicles
Inc. – Incorporated
Lab. - Laboratories
L protein – Viral RNA polymerase
LUV – Large Unilamellar Vesicles
M protein – Viral matrix protein
MVB – Multivesicular Body
ND - Newcastle Disease
NDV – Newcastle Disease Virus
NP protein – Viral nucleoprotein
PAGE - Polyarylamide Gel Electrophoresis
PI – Phosphatidylinositol
POPC - 1-Palmitoy-l,2-oleoyl-sn-Glycero-3-phosphocholine
PS – Phosphatidylserine
Rh – Rhodamine
RNA – Ribonucleic acid
SDS – Sodium dodecyl sulfate
TEM –Transmitted Electron Microscopy
VLP – Virus-Like Particle

CONTENTS

I. INTRODUCTION ....…………………………………………………….............. 1

1.1. Membrane budding: from complexity of intracellular trafficking
to the relative simplicity of viral particle creation ……………………….. 2
1.1.1. Classical coated vesicles and novel membrane microdomains .....……. 2
1.1.2. Budding to the opposite direction: multivesicular body
and viral budding .......……………………….………………………. 6
1.2. Physical-chemical principles behind lipid membrane budding …………. 10
1.2.1. Phospholipids and their polymorphism ................................................ 10
1.2.2. Energetics of membrane budding ......................................................... 13
1.2.2.1. Bending energy ...................................................................... 13
1.2.3. Domain structure of biological membranes
and its role in membrane budding ..................................................... 15
1.3. Newcastle Disease Virus and its matrix protein .......................................... 20
1.3.1. Newcastle Disease Virus: classification and characteristics ............... 20
1.3.2. Matrix protein of Newcastle Disease Virus: its known
and unknown properties ……………………………………………. 24
1.4. Objectives ........................................................................................................ 27

II. MATERIALS AND METHODS ....................................................................... 29
2.1. Apparatus and materials ……………………………………………..…… 30
2.2. Chemicals and lipids ……………………………...……………………….. 33
2.3. Biological samples ………………………………...………………………... 35
2.4. Analytical Software ………………………………...……………………… 35
2.5. Methods ……………………………………………...…………………...… 36
2.5.1. Virus and matrix protein purification …………...…………………... 36
2.5.2. SDS-polyacrylamide gel electrophoresis, gel staining and analysis ... 37
2.5.3. Preparation of large unilamellar vesicles .…………………...………. 38
2.5.4. Analysis of matrix protein binding to large unilamellar vesicles ...…. 40
2.5.5. Matrix protein interaction with large unilamellar vesicles:
fluorescence measurements …………………………………R