Biochemical characteristics of different subtypes of human immunodeficiency virus type 1 reverse transcriptase and its interactions with the host factor : apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like 3G

Dr. Stefan G. Sarafianos, Thesis Supervisor.Field of study: Veterinary pathobiology."December 2017."[ACCESS RESTRICTED TO THE UNIVERSITY OF MISSOURI AT REQUEST OF AUTHOR.] Human immunodeficiency virus (HIV) is divided into type 1 (HIV-1) and type 2 (HIV-2). Whereas HIV-2 accounts for 5% of global infections, HIV-1 is responsible for 95% of the global pandemic. HIV-1 is classified into four groups; M, N, O and P. Group M is the most prevalent, including subtypes or clades of A-D, F-H, J, K, over 100 Circulating Recombinant Forms (CRFs), and several Unique Recombinant Forms (URFs). In developed countries (i.e. countries in North America, Western Europe, and Australia), HIV-1B is the most prevalent strain. However, it accounts for only about 12% of worldwide infections. On the other hand, HIV-1C, which is most prevalent in Low- and Middle-Income Countries (LMICs) (e.g. India, Brazil, and many countries in Sub-Saharan Africa), accounts for [about]52% of all HIV infections. Due to its prevalence in developed countries, HIV-1B has been the major target of HIV studies and anti-HIV drug development. However, it has been reported that HIV-1non-B patients have higher rates of treatment failure than HIV-1B patients. To understand the mechanism behind this observation, we applied in vitro biochemical assays, which were performed with four patient-derived reverse transcriptase (RT) proteins isolated from HIV-1B, HIV-1C, CRF01_AE, and CRF02_AG viruses. RT is the HIV enzyme that generates viral DNA from genomic RNA during the early stages of infection, and it is the target for many anti-HIV drugs. RT inhibitors are divided into two types depending on their structure. First, Nucleoside/nucleotide Reverse Transcriptase Inhibitors (NRTIs), which bind the active site of the RT and act as chain terminators. In addition, a new type of NRTI, called 4'-ethynyl-2-fluoro-2'-deoxyadenosine (EFdA), works as a translocation inhibitor. Second, Non-Nucleoside Reverse Transcriptase Inhibitors (NNRTIs) inhibit RT by binding to an allosteric site. Nevirapine (NVP) belongs to the first generation of NNRTIs, however HIV-1 develops resistance mutations frequently during NVP treatment. Rilpivirine (RPV) has been developed to target NVP-resistant viruses but it is not frequently used in LMICs. In this study, we determined the biochemical characteristics of HIV-1 RTs from various subtypes and determined kinetic constants of inhibition by EFdA, NVP, and RPV. The results show that all of the tested HIV-1 RTs incorporate EFdA with better efficiency than their natural cognate substrate, dATP, which suggests that EFdA would be effective against all of the tested HIV-1 subtypes. Furthermore, generally NVP binds RTs with a lower affinity than RPV, implying that NVP would be a less effective inhibitor than RPV. However, in the case of CRF02_AG RT, NVP binds with similar affinity to RPV, suggesting CRF02_AG patients may respond better to NVP than patients infected with other subtypes. HIV-1C has less binding affinity to RPV than other subtypes, which is consistent with clinical reports showing that HIV-1C patients have a higher rate of treatment failure with RPV. Finally, we have studied the effect of a host factor, apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like 3G (APOBEC3G), on HIV-1 RT. APOBEC3G (A3G) has been reported to inhibit several retroviral infections. To date, there are two established mechanisms of this inhibition, which are hyper-mutagenesis and roadblock. In this study, we investigated a third mechanism, the direct inhibition of HIV-1 RT, using in vitro RT assays. Our results showed that A3G does inhibit the activity of HIV-1 RT by affecting the catalytic rate of dNTP incorporation (kcat), supporting the direct inhibition mechanism. Interestingly, A3G had similar inhibitory activity against a related viral RT (from Moloney Murine Leukemia Virus), but no activity against DNA polymerase I.Includes bibliographical references (pages 78-87)