One of the greatest barriers to curing HIV-1 is the ability of the virus to establish a state of latent infection within infected cells. During viral latency, the virus lies dormant in the form of an integrated, replication-competent provirus within the infected cell. In this state, the virus is undetectable by the host immune system and is unaffected by antiretroviral drugs due to extremely weak or null transcription. These viruses, however, can be induced to produce infectious virus later on and propagate the infection as well as reseed the latent reservoir. The factors that lead to the establishment and maintenance of HIV-1 latency are not all known. Current latency reversal methods are unable to effectively purge the latent reservoir in HIV-1-infected patients, which begs the question whether there are populations of latently-infected cells that are not being targeted by present therapeutics.
One of our first lines of defense against retroviral infection is the APOBEC3 family of cytidine deaminases. These enzymes restrict retroviral infection mainly through hypermutation of the proviral DNA, leading to inactivation of the virus. However, it has been shown that low levels of mutations do not necessarily inactivate the virus and can sometimes be beneficial for the virus by increasing genetic diversity and viral fitness. Sublethal mutagenesis has been studied on coding regions of the virus, however, their effects on regulatory regions of the virus such as the LTR promoter have been scarcely explored. My project aimed to examine the effects of APOBEC3-induced mutations on the activity of the HIV-1 LTR promoter and investigate whether these mutations could be generating a subset of latently-infected cells.
A library of HIV-1 clones with A3G- or A3F-mutated LTRs was generated. We discovered that the 5’ LTR is not mutated during the first round of HIV-1 infection in our system while the 3’ LTR accumulates mutations. A second round of infection is required for the mutations in the 3’ LTR to be copied over to the 5’ LTR and influence promoter activity. The mutations generated a range of effects on the promoter, with some clones being completely inactivated and no longer responsive to Tat or PMA/Ionomycin induction, while other clones were still highly infectious. The clones of interest were the ones in between this spectrum that were weakly-infectious (≤0.25%) prior to stimulation but were able to be induced above a given threshold (≥10-fold). We denoted these clones latency-prone viruses (LPVs) and were able to identify 10 of these clones within our library. We also explored the effects of individual mutations on the promoter and although these clones were highly infectious, we identified 8 positions of interest that led to weaker infectivity and fluorescence when mutated. The mutations in our library were found to hit important transcription factor binding sites and were also identified in a bioinformatics search of HIV-1-infected patient sequences, which confirms the relevance of our in vitro-identified mutations in a physiological setting.
This is the first investigation and evidence of the APOBEC3 proteins contributing to the generation of a subset of latent viruses and constitutes an important contribution to the understanding of HIV-1 latency and its reversal. This previously uncharacterized pool of latently-infected cells could explain why current therapies are ineffective as they do not target these molecular modifications but rather focus on reversing epigenetics and reactivation of the virus. These newly-discovered latency-prone viruses would be a useful tool in testing reactivation drugs and establishes the need to develop novel antiretrovirals that will target a broader spectrum of latently-infected cells. This project has effectively illustrated the dual role of this host-encoded restriction factor and provided further insight into HIV-1 latency, the major hurdle towards a cure for HIV-1.
| Identifer | oai:union.ndltd.org:uottawa.ca/oai:ruor.uottawa.ca:10393/39811 |
| Date | 06 November 2019 |
| Creators | Lam, Cindy |
| Contributors | Langlois, Marc-André |
| Publisher | Université d'Ottawa / University of Ottawa |
| Source Sets | Université d’Ottawa |
| Language | English |
| Detected Language | English |
| Type | Thesis |
| Format | application/pdf |
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