TRANS-ACTING FACTORS AFFECTING RETROVIRAL RECODING Lisa Green Submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Graduate School of Arts and Sciences COLUMBIA UNIVERSITY 2012 © 2012 Lisa Green All Rights Reserved Thesis Abstract Trans-Acting Factors Affecting Retroviral Recoding Lisa Green The production of retroviral enzymes requires a translational recoding event which subverts normal decoding, either by direct suppression of termination with the insertion of an amino acid at a stop codon (readthrough), or by an alteration of the reading frame of the mRNA (frameshift). It has been determined that retroviral readthrough and frameshift require cis-acting factors in the mRNA to stimulate recoding on the eukaryotic ribosome. Here we investigate the affects of trans-acting factors on recoding, primarily in the context of the MoMLV gag-pol junction. We report the effects of a host protein, Large Ribosomal Protein Four (RPL4), on the efficiency of recoding. Using a dual luciferase reporter assay, we show that transfection of cells with an RPL4 cDNA expression construct enhances recoding efficiency in a dose-dependent manner. The increase in the frequency of recoding can be more than 2-fold, adequate to disrupt normal viral production. This effect is cell line specific, and appears to be distinct to RPL4 among ribosomal proteins. The RPL4 increase occurs with both retroviral readthrough and frameshift sequences, and even at other viral readthrough regions that do not involve RNA secondary structures. We show that RPL4 effects are negated by release factor over-expression, and that RPL4 will increase readthrough above the levels of a hyperactive mutant and in addition to G418. When co- transfected with Moloney murine leukemia provirus, the RPL4-mediated increase in readthrough reduces the amount of virus released. We also examined the effects of aminoglycoside drugs and the small molecule PTC124 on readthrough of the MoMLV gag-pol junction. We show that G418, paromomycin and PTC124 increase readthrough of our MoMLV reporter in a dose dependent manner in 293A cells. These drugs reduce viral replication, as measured by a recombinant transducing virus assay. We further examine G418 and paromomycin in an in-vitro system; readthrough is increased to higher levels than those seen in vivo. G418 displays deleterious effects on cell viability and overall translation. Paromomycin does not appear as toxic, suggesting differences in interactions by which these drugs enhance readthrough. Table of Contents List of Figures and Tables iii Acknowledgements viii Chapter 1: Introduction 1 Retroviruses 2 Translation 15 Recoding 30 Chapter 2: Materials and Methods 49 Chapter 3: RPL4 effects on retroviral recoding 72 mRPL4 enhances readthrough at the MoMLV gag:gag-pol junction 76 RPL4 effects on readthrough are cell line dependent 78 Mouse and human RPL4 both enhance MoMLV/XMRV gag-pol readthrough 81 RPL4 enhancement of readthrough is distinctive among ribosomal proteins 83 RPL4 mutants do not increase readthrough 91 RPL4 Time Course 99 RPL4 enhances translation of other functional viral recoding sequences 101 RPL4 effect on readthrough of non-recoding premature stop codons is undetermined 104 PseudoKnot mutants and RPL4 112 RPL4 increases readthrough at all three stop codons 115 RPL4 does not enhance readthrough in in-vitro translation reactions 119 Release factor over-expression negates RPL4 enhanced readthrough 124 RPL4 and G418 act additively to increase MoMLV readthrough 127 i RPL4 enhancement of readthrough is not due to ribosome biogenesis 130 RPL4 increase in gag-pol readthrough has deleterious effects on viral replication 135 Chapter 4: Readthrough Promoting Drugs and Retroviral Recoding 141 Aminoglycosides increase MoMLV readthrough in vivo 146 Readthrough promoting drugs can affect retroviral replication 154 Aminoglycosides and PTC124 increase Sindbis/Leaky stop codon readthrough in vivo 160 G418 and PTC124 may enhance HIV-1 frameshift in vivo 162 Aminoglycosides do not increase readthrough of MoMLV in drug-resistant cells 164 Aminoglycosides increase MoMLV/PTC readthrough in vitro 164 Chapter 5: Discussion 177 RPL4 177 Readthrough promoting drugs 191 Final thoughts 195 References 197 Appendix 207 Functional Pseudoknot FACS screen 207 Cryo-EM structure: The pseudoknot on the ribosome 219 ii Figures Figure 1-1. Retroviral particle 3 Figure 1-2. Retroviral replication cycle 6 Figure 1-3. Simple and complex retroviral genome 7 Figure 1-4. Reverse Transcription 9 Figure 1-5. General Structure and Organization of the ribosome 14 Figure 1-6. A, P, and E Site occupancy during translation elongation 19 Figure 1-7. 80s ribosome 20 Figure 1-8. Cognate fit; normal elongation 23 Figure 1-9. Decoding center 26 Figure 1-10. eRF1 structure 28 Figure 1-11. Recoding: Frameshift 32 Figure 1-12. Recoding: Readthrough 33 Figure 1-13. Possible models of -1 programmed ribosomal frameshift 35 Figure 1-14. Proposed mechanics of frameshifting pseudoknot 38 Figure 1-15. Readthrough of the MoMLV gag-pol junction 41 Figure 1-16. Schematic of MoMLV pseudoknot 42 Figure 1-17. Functional effects on mutations to the MoMLV pseudoknot 44 Figure 1-18. Traditional and revised MoMLV pseudoknot structure 45 Figure 1-19. Inactive and active conformations of the MoMLV pseudoknot 47 iii Figure 3-1. 60S ribosome and RPL4 74 Figure 3-2. Schematic of MoMLV 13PK6 luciferase assay reporter constructs and calculation of readthrough 75 Figure 3-3. mRPL4 effects on readthrough of MoMLV gag-pol junction 77 Figure 3-4. RPL4 effects on MoMLV gag-pol readthrough: cell line survey 80 Figure 3-5. mRPL4 and hRPL4 induced increase of MoMLV gag-pol readthrough 82 Figure 3-6. Survey of effect of additional ribosomal proteins on MoMLV gag-pol readthrough. 84 Figure 3-7. Effect of myc-tagged ribosomal proteins on MoMLV gag-pol readthrough 85 Figure 3-8. Expression of myc-tagged ribosomal proteins 86 Figure 3-9. Expression of N-terminal flag-tagged RPL4 88 Figure 3-10. Expression of mRPL4 and hRLP4 89 Figure 3-11. qPCR analysis of RPL4 mRNA levels in 293 cells 90 Figure 3-12 RPL4-FS mutant 92 Figure 3-13. Effect of L4-FS on readthrough of the MoMLV gag-pol junction 93 Figure 3-14. C-terminal differences between mouse and human RPL4 and schematic of RPL4 c- terminal truncation mutants 95 Figure 3-15 RPL4 C-terminal truncation mutants effect on readthrough 96 Figure 3-16. Schematic of RPL4 helicase-binding domain mutants 97 Figure 3-17. Effect of RPL4 helicase-binding domain mutants on MoMLV gag-pol readthrough 98 Figure 3-18 Timecourse of RPL4 enhancement of readthrough and corresponding RPL4 expression 100 Figure 3-19. Effect of RPL4 on additional viral recoding sequences 103 Figure 3-20. Readthrough frequency of the 13ScrPK6 reporter 106 Figure 3-21. Linear range of the dual luciferase assay 107 iv Figure 3-22. UPF1 dominant negative mutant effects on readthrough of 13ScrPK6 109 Figure 3-23. Readthrough efficiency of 13ScrPK6_RSE reporter 111 Figure 3-24. Comparison of readthrough efficiency of reporter constructs 113 Figure 3-25. RPL4 effects on CollStop readthrough 114 Figure 3-26. Effects of RPL4 on MoMLV pseudoknot mutants 116 Figure 3-27. Effects of RPL4 on MoMLV hyperactive pseudoknot mutant 117 Figure 3-28. RPL4 enhancement of readthrough of alternate stop codons 118 Figure 3-29. RPL4 effect on readthrough in TnT IVT reaction 121 Figure 3-30. Effect of pre-translated RPL4 on MoMLV readthrough in TnT IVT reaction 122 Figure 3-31. Effect of RPL4‐GST tagged protein on readthrough in TnT IVT reaction 123 Figure 3-32. Release factor effect on MoMLV readthrough 125 Figure 3-33. Combined effects of Release Factors and RPL4 on MoMLV readthrough 126 Figure 3-34. Combined effects of RPL4 and RPL7 on MoMLV readthrough 128 Figure 3-35. eRF1 effects on RPL4 enhancement of readthrough 129 Figure 3-36. Combined effects of RPL4 and G418 on readthrough 131 Figure 3-37. Monoclonal antibody detection of endogenous mouse and human RPL4 133 Figure 3-38. Levels of endogenous RPL4 in responsive and non-responsive cells 134 Figure 3-39. RPL4 overexpression in responsive and non-responsive cell lines 136 Figure 3-40. RPL4 overexpression effects on L10a expression 137 Figure 3-41. Effects of RPL4 overexpression on 28S rRNA in responsive and non-responsive cell lines 138 Figure 3-42. Effects of RPL4 on viral replication 140 Figure 4-1. Aminoglycoside Structures 142 Figure 4-2. Substitutions and variations in aminoglycoside structure 143 Figure 4-3. Paromomycin bound to 16S rRNA 145 Figure 4-4. G418 effects on MoMLV readthrough in 293A cells 147 v Figure 4-5. G418 effects on viral production: transducing assay 149 Figure 4-6. G418 effects on cell viability and readthrough 150 Figure 4-7. Readthrough promoting drug effects on MoMLV readthrough 152 Figure 4-8. Readthrough promoting drug effects on MoMLV readthrough: fold change summary 153 Figure 4-9. Streptomycin effects on MoMLV readthrough in 293A cells 155 Figure 4-10. Drug effects on viral replication: transducing virus assay 156 Figure 4-11. Drug effects on viral replication:: detail, 1:4 virus dilution 157 Figure 4-12. Drug effects on viral replication: producer cells 158 Figure 4-13. Drug effects on Sindbis leaky stop codon 161 Figure 4-14: Drug effects on HIV-1 frameshift 163 Figure 4-15. Drug effects on MoMLV readthrough in 293T neo-resistant cells 165 Figure 4-16. G418 effects on MoMLV readthrough in vitro 167 Figure 4-17. G418 effects on MoMLV readthrough in vitro: fold change 168 Figure 4-18. G418 effects on CollStop readthrough in vitro 169 Figure 4-19. G418 effects on CollStop readthrough in vitro: fold change 170 Figure 4-20. Paromomycin effects on MoMLV readthrough in vitro 172 Figure 4-21. Paromomycin effects on MoMLV readthrough in vitro: fold change 173 Figure 4-22.