Characterization of factors and mechanisms of virulence of arenaviruses through mutational analysis Pubblico

Abstract

Arenaviruses are one of the most neglected tropical pathogens. The most prominent human pathogen belonging to this group is Lassa Fever virus (LASV), which is responsible for ~5,000 - 10,000 deaths and ~2 million infections annually. Mutational analysis of arenaviral proteins may provide insight into the pathological mechanisms of these viruses.

A related arenavirus, Pichinde virus (PICV), is capable of causing a disease in guinea pigs that mimics LASV infection in humans. We have sequenced two strains of PICV with different disease phenotypes in infected guinea pigs. The P2 strain causes a mild disease from which the animals quickly recover. The P18 strain results in a highly virulent disease, which results in death. While the phenotypes are vastly different, relatively few sequence changes exist between them.

We have developed reverse genetics systems for both the P2 and P18 strains of PICV, which have allowed us to generate recombinant viruses. Through mutational analysis, we have identified sequence changes in the L polymerase that contribute to the increased pathogenesis of P18. Three amino acid changes in the C terminus of L are required for increased virulence of the P18 strain by increasing the rate of viral genome replication, indicating that the rate of viral genome replication is an important factor in arenavirus pathogenesis.

We have also developed a LASV minireplicon system in order to perform mutational analysis of the LASV nucleoprotein (NP). We have shown that LASV NP contains a cap-binding domain in its N terminus that is required to initiate viral RNA transcription. We have also shown that LASV NP contains a 3'-5' exoribonuclease domain in its C terminus that is required to mediate type-I interferon inhibition. We therefore propose a novel mechanism of immune evasion in which the NP protein degrades viral RNAs that would otherwise be detected by innate immune sensors, therefore allowing these viruses to replicate to high levels in infected patients.

Collectively, this work characterizes two important viral factors (NP and L polymerase) required for arenaviral replication, transcription and immune evasion. This information will aid in the development of vaccines and therapeutics against arenaviral hemorrhagic fever.

Table of Contents

CHAPTER 1: INTRODUCTION
1.1 Targeting virulence mechanisms for the prevention and therapy of
arenaviral hemorrhagic fever…………………………………………........ 1
Abstract………………………………………………………………………………… 2
Introduction……………………………………………………………………………. 3
Arenavirus genome structure and replication cycle…………………………………… 6
The role of individual arenaviral proteins in hemorrhagic fever disease pathogenesis… 13
Antiviral compounds targeting different steps of the arenaviral life cycle…………….. 19
Host immune suppression and inflammatory responses………………………………. 22
Conclusion…………………………………………………………………………….. 26
Tables………………………………………………………………………………….. 28
Figures…………………………………………………………………………………. 30
Figure legends…………………………………………………………………………. 33
1.2 A comparative analysis of the pathology and molecular mechanisms of
New World and Old World Arenaviruses…………………………………. 35
Abstract………………………………………………………………………………… 36
Introduction……………………………………………………………………………. 37
Phylogeny and epidemiology………………………………………………………….. 39
Disease manifestations………………………………………………………………… 42
Pathology………………………………………………………………………………. 44
Coagulopathy…………………………………………………………………………... 45
Immune response………………………………………………………………………. 48
Molecular strategies……………………………………………………………………. 51
Current development in vaccines………………………………………………………. 57
Summary……………………………………………………………………………….. 59
Tables…………………………………………………………………………………... 62
Figures………………………………………………………………………………….. 64
Figure legends………………………………………………………………………….. 65
CHAPTER 2:
2.1 Genome comparison of virulent and avirulent strains of the Pichinde
Arenavirus…………………………………………………………………… 66
Abstract………………………………………………………………………………… 67
Introduction……………………………………………………………………………. 68
Materials and methods…………………………………………………………………. 70
Results and Discussion………………………………………………………………… 73
Acknowledgements……………………………………………………………………. 82
Tables…………………………………………………………………………………... 83
Figures…………………………………………………………………………………. 86
Figure legends………………………………………………………………………….. 90

2.2 Development of infectious clones for virulent and avirulent Pichinde
viruses: a model virus to study arenavirus-induced hemorrhagic fever…. 92
Abstract………………………………………………………………………………… 93
Introduction……………………………………………………………………………. 94
Materials and Methods………………………………………………………………… 96
Results and Discussion………………………………………………………………… 102
Acknowledgements……………………………………………………………………. 108
Tables………………………………………………………………………………….. 109
Figures…………………………………………………………………………………. 110
Figure legends…………………………………………………………………………. 115
2.3 Identification of virulence determinants within the L genomic segment
of the Pichinde arenavirus………………………………………………….. 117
Abstract………………………………………………………………………………... 118
Introduction……………………………………………………………………………. 119
Materials and Methods………………………………………………………………… 122
Results………………………………………………………………………………… 126
Discussion……………………………………………………………………………... 133
Acknowledgements……………………………………………………………………. 138
Tables………………………………………………………………………………….. 139
Figures…………………………………………………………………………………. 140
Figure legends…………………………………………………………………………. 145
CHAPTER 3:
Cap binding and immune evasion revealed by Lassa nucleoprotein
Structure……………………………………………………………………. 147
Abstract……………….………………………………………………………………. 148
Introduction…………………………………………………………………………… 149
Results…………………………………………………………………………………. 150
Conclusion…………………………………………………………………………….. 158
Methods………………………………………………………………………………... 159
Figures…………………………………………………………………………………. 168
Figure legends…………………………………………………………………………. 179
CHAPTER 4: Discussion…………………………………………………………….. 184
REFERENCES……………………………………………………………………….. 197

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School	Laney Graduate School
Department	Biological and Biomedical Sciences
Degree	PhD
Submission	Dissertation
Language	English
Research Field	Biology, Molecular Biology, Virology
Parola chiave	Pichinde Arenavirus Lassa Hemorrhagic
Committee Chair / Thesis Advisor	Ly, Hinh, University of Minnesota
Committee Members	Rota, Paul A, Emory University Hunter, Eric A, Emory University Liang, Yuying, Emory University Gewirtz, Andrew T, Emory University Ansari, Aftab A, Emory University

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