SPECIES SPECIFIC DIFFERENCES IN TRIM5alpha FUNCTION AND GENOMIC COMPOSITION Pubblico

Abstract

It has recently become apparent that the alpha splice variant of tripartite motif-containing protein 5 (TRIM5alpha ) can impose a major barrier to cross-species retroviral infection. This antiviral activity is most prominently exerted shortly after viral entry into a target cell and results in an abortive infection prior to viral genome integration into the host chromatin. Work from other groups has shown that TRIM5 alpha is capable of restricting infection by a variety of Gammaretroviruses, Lentiviruses, and Spumaviruses. In this dissertation molecular virological approaches were used to demonstrate that a prototype Betaretrovirus, Mason-Pfizer Monkey Virus (M-PMV), encounters a virus specific post-entry block to replication in cells from three New World monkey species. In two cases TRIM5 alpha was identified as being responsible for this block, thus expanding known TRIM5 alpha antiviral activity to Betaretroviruses.

In primate genomes, TRIM5 is found in conjunction with the related genes TRIM6, TRIM22 and TRIM34. Similar to TRIM5, TRIM22 has been shown to possess antiviral activity and both genes have been identified as having faced strong selective pressures during primate evolution. In contrast, the neighboring genes have been under purifying selection. Using genomics approaches it was discovered that elevated rates of transposable element fixation has occurred within the introns of TRIM5 and TRIM22, including the fixation of multiple endogenous retroviral (ERV) long terminal repeat (LTR) elements in the first intron of these genes. Using molecular biological techniques it was further shown that differential fixation of one such LTR element has resulted in a species-specific regulation of TRIM22 in response to p53 activation. As TRIM5 and TRIM22 are found adjacent each other in the genome and are situated such that they share a 5 kilobase (kb) promoter region, it is likely that the various LTRs fixed in these genes similarly modulate gene expression. These results are suggestive of a scenario where primate genomes have co-opted inserted endogenous retroviral elements as a mechanism for rapid alteration of gene expression, presumably to match the tissue tropism of a pathogenic virus challenge.

Table of Contents

TABLE OF CONTENTS

INTRODUCTION ................................................................................................................................................... 1
Retroviridae ......................................................................................................................................................... 1
Conserved Retroviral Genetics ................................................................................................................................ 3
Retroviral Accessory Genes .................................................................................................................................... 8
Retroviral Lifecycle ............................................................................................................................................... 13
Restriction Factors and Evasion Mechanisms ............................................................................................................ 26
TRIM5alpha ......................................................................................................................................................... 40

Chapter 1: IDENTIFICATION OF POST-ENTRY RESTRICTIONS TO MASON-PFIZER MONKEY VIRUS INFECTION IN

NEW WORLD MONKEY CELLS ................................................................................................................................. 53

Chapter 2: ALTERED TRANSCRIPTIONAL REGULATION OF MEMBERS OF THE TRIM5, TRIM22, TRIM34, TRIM6

LOCUS AS A RESULT OF DIFFERENTIAL FIXATION OF ENDOGENOUS RETROVIRAL ELEMENTS IN

THE NON-CODING REGIONS OF THESE GENES ......................................................................................................... 84
DISCUSSION ....................................................................................................................................................... 117
LIST OF ABBREVIATIONS ...................................................................................................................................... 124
REFERENCES ....................................................................................................................................................... 132

LIST OF FIGURES
INTRODUCTION
1. Retroviral phylogeny ........................................................................................................................................... 2
2. Schematic representation of the Mason-Pfizer Monkey Virus (M-PMV) genomic RNA and coding structure ....................... 4
3. Schematic of a mature particle from a hypothetical retrovirus ................................................................................... 6
4. Lifecycle of a typical retrovirus ............................................................................................................................. 15
5. Major steps during reverse transcription ................................................................................................................ 18
6. Model for the hexameric structure of HIV-1 capsid domains in the immature and mature lattices ................................... 26
7. Schematic representation of the proposed evolution and transfer of vpu and nef during zoonotic transfer of Lentiviruses

in Old World primates .............................................................................................................................................. 41
8. Representation of the TRIM5 coding structure and protein domain architecture ............................................................ 43
9. Stereo model of a mature HIV-1 capsid core ........................................................................................................... 48
10. Composition and structure of the of TRIM5 genomic locus ....................................................................................... 49

Chapter 1. IDENTIFICATION OF POST-ENTRY RESTRICTIONS TO MASON-PFIZER MONKEY VIRUS INFECTION

IN NEW WORLD MONKEY CELLS
1. M-PMV infectivity is reduced in New World monkey cell lines ...................................................................................... 65
2. Observed infectivity differences are specific to M-PMV ............................................................................................... 68
3. Quantitative PCR analysis of reverse transcriptase products in primate fibroblast cells ................................................... 69
4. Exogenous expression of tamarin and squirrel monkey TRIM5alpha inhibit M-PMV infection ............................................ 71
5. Quantitative PCR analysis of reverse transcriptase products in TRIM5alpha expressing CRFK cells ................................... 75

6. Truncated forms of TRIM5 have dominant-negative activity in TRIM5alpha expressing CRFK cells .................................... 77

7. Expression of dominant-negative spider monkey TRIM5 does not enhance M-PMV infectivity in spider monkey fibroblasts .. 79

Chapter 2. ALTERED TRANSCRIPTIONAL REGULATION OF MEMBERS OF THE TRIM5 , TRIM22 , TRIM34 , TRIM6

LOCUS AS A RESULT OF DIFFERENTIAL FIXATION OF ENDOGENOUS RETROVIRAL ELEMENTS IN THE

NON-CODING REGIONS OF THESE GENES
1. Primate TRIM6/34/5/22 gene locus ........................................................................................................................ 88
2. TRIM genes are variably conserved as a result of differential rates of indel turnover ...................................................... 96
3. TRIM5 and TRIM22 contain more transposable elements than TRIM6 or TRIM34 ........................................................... 98
4. Graphical depiction of the genomic structure and location of transposable elements in the analyzed TRIM genes ............... 99
5. Transcription of TRIM22 from different primate species is differentially regulated following p53 induction ....................... 104
6. Reduced binding of p53 to the LTR10D element within the first intron of rhesus macaque TRIM22 ................................. 107
S1. Amino acid alignment of human and rhesus macaque p53 ..................................................................................... 115
S2. Nucleic acid alignment of the LTR10D element found within the first intron of Old World primate TRIM22 ..................... 116

LIST OF TABLES
INTRODUCTION

1. Viral restriction by primate TRIM5alpha proteins ...................................................................................................... 46

Chapter 1. IDENTIFICATION OF POST-ENTRY RESTRICTIONS TO MASON-PFIZER MONKEY VIRUS INFECTION
IN NEW WORLD MONKEY CELLS

1. Cells lines used in this study ................................................................................................................................. 57

2. Correlation of viral infectivity in New World monkey fibroblasts and CRFK cells expressing TRIM5alpha from these

New World monkeys ................................................................................................................................................ 74

Chapter 2. ALTERED TRANSCRIPTIONAL REGULATION OF MEMBERS OF THE TRIM5 , TRIM22 , TRIM34 , TRIM6

LOCUS AS A RESULT OF DIFFERENTIAL FIXATION OF ENDOGENOUS RETROVIRAL ELEMENTS IN THE

NON-CODING REGIONS OF THESE GENES
1. Summary of demographic and genetic information for human and non-human subjects ............................................... 106

S1. Nucleotide differences seen in the LTR10D elements within rhesus macaque TRIM22 ................................................. 115

About this Dissertation

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School	Laney Graduate School
Department	Biological and Biomedical Sciences
Subfield / Discipline	Microbiology & Molecular Genetics
Degree	PhD
Submission	Dissertation
Language	English
Research Field	Biology, Genetics Biology, Molecular Biology, Virology
Parola chiave	evolution TRIM5 antiviral
Committee Chair / Thesis Advisor	Hunter, Eric, Emory University
Committee Members	Song, Byeongwoon, Emory University Silvestri, Guido, Emory University Spearman, Paul, Emory University Speck, Sam, Emory University Derdeyn, Cynthia, Emory University Parslow, Tristram G, Emory University

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