Mammals are constantly challenged by numerous pathogens that pose a threat to their health. Upon infection, retroviruses quickly integrate their genome into that of their host thereby permanently modifying it. Protein members of the APOBEC3 (A3) family exhibit cytidine deaminase activity that specifically acts on single-stranded DNA to deaminate deoxycytidine bases into deoxyuridines. This process is potentially mutagenic because uracil directs the insertion of adenine on the opposite DNA strand. High levels of mutations induced by A3 proteins in the retroviral genome ultimately inactivate progeny viruses. However, under conditions where low levels of A3 proteins are present, sub-lethal mutagenesis can occur and is generally believed to be beneficial for the virus. Powerful and affordable techniques designed to detect rare deamination events generated by these deaminases along the full length of retroviral genomes are therefore essential. Through the course of my studies, I developed such a new tool that I called HyperHRM which was instrumental to my project’s success.
In addition to the antiretroviral affects of their catalytic activity, some members of the A3 family have the ability to hinder reverse transcription independently of their enzymatic properties. Yet, the details underlying the deamination-independent restriction by the proteins remain unclear. Through my work, I have advanced our current understanding of this elusive process by defining the essential role for RNA-binding in the inhibition of the early steps of infection by APOBEC3G (A3G). I also demonstrate that the ability to bind RNA is important for the selection of DNA dinucleotides targeted for deamination by A3 enzymes. Based on the premise that the DNA context for deamination may alter viral fitness in various ways, I then investigated the gene inactivation potency of different A3 based on their preferred DNA substrate. My experiments showed that mutations introduced in a 5'CC context by A3G are much more lethal for the virus because of the high frequency of termination codons that are generated. I therefore clearly established that deamination target specificity has a strong influence on the overall restriction potency of A3 proteins and demonstrated that such specificity was linked to the ability of A3 proteins to bind RNA.
Finally, in addition to retroviruses, mobile elements such as retrotransposons can also lead to genomic instability if not properly controlled. The A3 protein family has been shown to play a crucial role in the restriction of these elements through a mechanism that is not believed to require the enzymatic activity of the proteins, although the details of the restriction mechanism are not yet understood. Here, I provide molecular insights on the potential mechanism of retrotransposon restriction by showing that the RNA-binding properties of the enzymes are not involved in the restriction of L1 retrotransposition. A complete elucidation of the modes of restriction employed by the A3 could lead to the development of a new generation of antiretroviral drugs.
Overall, my research has led to the design of a new research tool to detect and quantify A3-induced mutations in retroviruses, but more importantly, it has enabled a better understanding of how the RNA-binding abilities of A3 proteins play an essential role in the overall restriction potency of retroviruses and retrotransposons.
| Identifer | oai:union.ndltd.org:uottawa.ca/oai:ruor.uottawa.ca:10393/34353 |
| Date | January 2016 |
| Creators | Bélanger, Kasandra |
| Contributors | Langlois, Marc-André |
| Publisher | Université d'Ottawa / University of Ottawa |
| Source Sets | Université d’Ottawa |
| Language | English |
| Detected Language | English |
| Type | Thesis |
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