Foamy viruses (FVs), like other retroviruses, have gained interest due to their applications as viral vectors in gene therapy and various other fields. Despite their potential applications, FV stands out as a unique retrovirus, with distinct features in its replicative cycle that make it a compelling subject of study. However, it remains one of the least studied retroviruses. A crucial aspect of the retroviral replicative cycle, especially for its use as in gene transfer, is the selective packaging of its viral genomic RNA (vgRNA) out of the vast pool of RNAs found in virus producing host cells. This process, known as vgRNA enrichment, involves efficiently packaging vgRNA among the cellular and processed viral genetic material. Unlike other retroviruses, the selective packaging process of vgRNA in FV has not been explored, making it largely unknown. Previous studies on other retroviruses have indicated that vgRNA packaging selectivity is achieved through specific features within the vgRNA itself, such as specific sequences elements called packaging signals as well as dimerization of vgRNA resulting in the presence of two copies vgRNA in each retrovirus particle. Additionally, viral structural proteins, particularly Gag, play a significant role by interacting with vgRNA through specific protein regions. To shed light on the selective packaging process of FV vgRNA, the roles of structural proteins Env, Pol, and Gag were investigated, along with the vgRNA's dimerization capacity. To quantify the vgRNA selective packaging, an enrichment assay (E-assay) and a competitive assay (C-assay) were established. The E-assay allows to compare FV vgRNA packaging efficiency relative to non-viral RNAs, whereas the C-assay enables the determination of the preferential packaging of one vgRNA over another when both are present within a cell. The results obtained regarding the role of the structural proteins on the vgRNA selective packaging emphasized the delicate balance required between viral protein expression and vgRNA levels. Overexpression of Env and Gag severely disrupted selective packaging. Particularly the excess of Env protein amount led to an increased production of subviral particles that lack the capability to selectively package vgRNA. An interesting observation was the impact of the RNA template used for translating Gag on vgRNA enrichment. Expressing Gag from vgRNA (cis) enhanced vgRNA packaging selectivity, while expression from an RNA containing only expression-optimized gag ORF sequences (trans) reduced vgRNA enrichment. Nevertheless, the results from the C-assays suggest that non-Gag-translating vgRNA can still be selectively packaged over non-selectively packable dimerization deficient vgRNA. This indicates that while Gag might have a cis-acting mechanism in FV vgRNA selective packaging, this role appears to be non-essential. As mentioned earlier, vgRNA dimerization appears to be a crucial factor in the selective packaging process of most retroviruses. In the case of FV, the dimerization process was previously reported to be facilitated by three specific regions on the vgRNA known as dimerization sites one to three (DS-I to -III). Among these sites, DS-II stands out as being indispensable for vgRNA dimerization due to its 10 nt palindromic sequence, a determinant reported to be essential for the interaction between the two strands of vgRNA for most retroviruses. To investigate the significance of FV vgRNA dimerization in its selective packaging, we conducted E- and C-assays to assess the vgRNA packaging efficiency and specificity in FV DS-II mutants, previously identified as non-dimerizing (DS-II-M6 and -M7) or exhibiting a low dimerization rate (DS-II-M2). Intriguingly, FV vgRNA packaging was significantly negatively affected in the non-dimerizing FV mutants (DS-II-M6 and -M7), and to a lesser extent in the DS-II-M2 mutant, where dimerization was reported of occur at lower rates. This reveals a direct correlation between vgRNA packaging efficiency and the reported vgRNA dimerization potential of these DS-II mutants. These results suggest that, similar to other retroviruses, vgRNA dimerization plays a pivotal role in FV's selective packaging. Furthermore, it is well-documented that secondary structures within vgRNA in some retroviruses facilitate dimerization, thereby enhancing the selective packaging process. In-silico analyses of the FV vgRNA predicted the formation of a stem loop created by the palindromic sequence (SL-Pal). To gain insight into the role of these secondary structures within the DS-II region of FV vgRNA in its selective packaging, we designed a series of new dimerization mutants. These mutants were meticulously engineered to disrupt, modify, or restore the SL-Pal structure by introducing mutations inside or in proximity to the palindromic sequence based on computational secondary structure prediction. Notably, we observed that the palindrome's sequence could be mutated, as long as the SL-Pal structure and G-C proportion along all the stem-loop were preserved in a manner identical to the original structure. This preservation was crucial to ensuring the selective packaging of vgRNA and maintaining viral infectivity. Lastly, the evaluation encompassing protein analysis, vgDNA quantification, and infectivity assessment conducted on DS-I and DS-II mutants revealed a significant decrease not only on vgRNA selective packaging but also on viral infectivity, Pol packaging, cleaving, and the RTr process in non-dimerizing mutants. This underscores the intricate interrelation of these processes, emphasizing their collective importance for successful viral production. In summary, the findings presented in this project represent a significant advancement in understanding FV vgRNA selective packaging and dimerization. They offer valuable and novel insights that contribute to the expansion of our knowledge about FV molecular biology and its potential applications as a viral transfer vector.:I. ACKNOWLEDGEMENTS I
II. TABLE OF CONTENT III
III. INDEX OF FIGURES VI
IV. INDEX OF TABLES VIII
1 INTRODUCTION 1
1.1 Retroviruses 1
1.1.1 Taxonomy of retroviruses 2
1.1.2 General features of retroviruses 3
1.2 Foamy viruses 6
1.2.1 PFV virion structure and genome organization 8
1.2.2 Viral proteins 11
1.2.2.1 Gag 11
1.2.2.2 Pol 13
1.2.2.3 Env 15
1.2.3 Replication cycle 17
1.2.3.1 Early phase 18
1.2.3.2 Late phase 19
1.3 Selective packaging of retroviral vgRNA 20
1.3.1 selective vgRNA packaging in orthoretroviruses 20
1.3.1.1 Role of the capsid protein Gag on the selective vgRNA packaging 20
1.3.1.2 vgRNA packaging signals 22
1.3.1.3 vgRNA dimerization and selective packaging 23
1.3.1.4 Factors that determine the vgRNA fate 24
1.3.2 Selective packaging in Hepadnaviruses 26
1.3.3 Selective packaging in PFV 26
2 THESIS AIM 29
3 MATERIALS AND METHODS 30
3.1 Buffers and solutions 30
3.2 Enzymes 34
3.3 Commercial kits 34
3.4 Nucleic acids 35
3.4.1 Oligonucleotides 35
3.4.1.1 Oligonucleotides for cloning 35
3.4.1.2 Oligonucleotides for qPCR analysis 37
3.4.2 Plasmids 39
3.4.2.1 Plasmid constructs used in this project: 39
3.4.2.2 New plasmid constructs 42
3.5 Bacteria strains 52
3.6 Cell lines 52
3.7 Antibodies 53
3.8 Software and Devices 54
3.9 Consumables 56
3.10 Molecular Biology methods 56
3.10.1 Bacteria culture 56
3.10.2 Transformation of competent bacteria 57
3.10.3 Plasmid extraction 57
3.10.4 Molecular cloning 58
3.10.4.1 Polymerase Chain Reaction (PCR) 58
3.10.4.2 Plasmid digest 59
3.10.4.3 Fragment purification 59
3.10.4.4 Ligation 60
3.10.4.5 Transformation of ligated plasmid constructs 60
3.10.4.6 Plasmid preparation small-scale 61
3.10.4.7 Plasmid Sequencing 61
3.10.4.8 Plasmid quantification 61
3.10.4.8.1 Photometric quantification 61
3.10.4.8.2 Fluorometric quantification 62
3.1 Biochemistry methods 62
3.1.1 SDS-Polyacrylamide Gel Electrophoresis (SDS-PAGE) 62
3.1.2 Semi-Dry Western Blot 63
3.1.3 Immunodetection 63
3.1.4 Quantification of viral proteins by Western Blot 63
3.2 Cell Culture and Virological Methods 64
3.2.1 Cell lines 64
3.2.2 Cell passaging 64
3.2.3 Calcium phosphate transfection 65
3.2.4 Harvest of viral particles 65
3.2.5 Cell harvesting 66
3.2.6 Viral infectivity determination 66
3.3 Molecular virology methods 67
3.3.1 Viral RNA extraction 67
3.3.2 Cellular RNA extraction 67
3.3.3 DNase treatment 68
3.3.4 RNA quantification by RT-qPCR 68
3.3.5 DNA quantification by qPCR 69
3.3.6 Enrichment assay (E-assay) 70
3.3.7 Competitive assay 72
3.3.8 Secondary RNA structure prediction 74
4 RESULTS 75
4.1 Establishment of methodologies for quantification of selective vgRNA packaging efficiency 75
4.1.1 Establishment of the Enrichment (E)-assay 75
4.1.1.1 Reference mRNA 75
4.1.1.2 Background subtraction 77
4.1.2 Establishment of the Competition (C)-assay 80
4.1.2.1 Silent mutants characterization 81
4.1.2.2 Primer design 83
4.2 Selective packaging in foamy virus 88
4.2.1 Role of the viral structural proteins on the selective vgRNA packaging 92
4.2.1.1 Role of Env on the selective vgRNA packaging 92
4.2.1.2 Role of Pol on the selective vgRNA packaging 97
4.2.1.3 Role of Gag on the selective vgRNA packaging 100
4.2.1.3.1 Gag cis-acting mechanism on the selective vgRNA packaging 103
4.2.2 Role of dimerization on the selective packaging 109
4.2.2.1 Characterization of the dimerization mutants 111
4.2.2.2 Selective vgRNA packaging and dimerization 114
4.2.2.3 PFV vgRNA secondary structure (prediction) and dimerization potential 118
4.2.2.4 Dimerization and RTr 124
5 DISCUSSION 129
5.1 Establishment of methodologies for vgRNA selective packaging determination 129
5.1.1 E-assay 129
5.1.2 C-assay 130
5.2 Viral factors involved in the FV vgRNA selective packaging 132
5.2.1 Selective packaging in PFV 132
5.2.2 Role of the structural viral proteins on the vgRNA selective packaging 133
5.2.2.1 Env 133
5.2.2.2 Pol 136
5.2.2.3 Gag 136
5.2.3 Role of vgRNA dimerization on its selective packaging 139
5.2.3.1 vgRNA DS-II secondary structure and dimerization 141
5.2.3.2 vgRNA dimerization and RTr 142
6 CONCLUSION 145
7 REFERENCES 146
8 APPENDICES 162
8.1 Abbreviations list 162
8.2 Anlage 1 165
8.3 Anlage 2 166
| Identifer | oai:union.ndltd.org:DRESDEN/oai:qucosa:de:qucosa:90711 |
| Date | 04 June 2024 |
| Creators | Aguilar Hernández, Nayeli |
| Contributors | Lindemann, Dirk, Temme, Achim, Technische Universität Dresden |
| Source Sets | Hochschulschriftenserver (HSSS) der SLUB Dresden |
| Language | English |
| Detected Language | English |
| Type | info:eu-repo/semantics/publishedVersion, doc-type:doctoralThesis, info:eu-repo/semantics/doctoralThesis, doc-type:Text |
| Rights | info:eu-repo/semantics/openAccess |
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