ROLE OF APOBEC3 ENZYMES IN HIV-1 EVOLUTION

There are approximately 36.7 million people living with HIV while 1.8 million become newly infected every year. HIV belongs to the Retroviridae family and is an enveloped, positive sense (+) single stranded RNA virus. HIV has two subtypes, HIV-1 and HIV-2. HIV-1 is responsible for 95% of infections worldwide and is the focus of this thesis. There has not been any report on a conventional cure of this disease and the diagnosed HIV+ individuals need to receive lifelong treatment with antiretroviral drugs. Besides the antiretroviral drugs, there are host restriction factors that fight the infection in addition to the conventional immune barriers and responses. The APOBEC3 (A3) family of enzymes are part of an intrinsic immune system in humans and can act as host restriction factors to restrict the replication of HIV in CD4+ T cells by deaminating cytosine to uracil on the (-) DNA of HIV during reverse transcription. This promutagenic activity, if it occurs frequently enough, can cause viral hypermutation and inactivation. The A3 family contains seven members (A3A to A3H, excluding A3E) some of which developed gene variations that result in protein polymorphisms due to selective pressure over evolutionary time. These polymorphisms acquired enhanced antiretroviral activity to fight off HIV. To combat this, HIV has an accessory protein termed virion infectivity factor (Vif) that interacts with A3 enzymes and components of a Cullin5 E3 ligase complex. This complex causes ubiquitination and degradation of A3 enzymes in HIV infected host cells. HIV also has a high level of genetic variation that allows the virus to escape immunological and pharmacological barriers. Besides the lack of proofreading activity of reverse transcriptase, viral recombination and high rate of replication, the A3 enzymes have a potential role in increasing the genetic diversity of the HIV replicating pool if Vif mediated degradation is not complete. As a result, human A3 enzymes can contribute to this diversity directly by causing sublethal mutations and indirectly when the lethally mutated RNA genomes are “rescued” after virus recombination. While A3 enzymes may have this potential, and some research has found that A3 enzymes cause drug resistance mutations, other research groups have reported that in the presence of Vif the mutation rate of A3s is lower than the mutation rate of reverse transcriptase and their contribution to HIV evolution is not significant. To identify all the contributors to genetic variation of HIV and have a reliable answer for the current controversy in the field we aimed to determine the mutation rate of A3s in wild-type HIV-1 infection and investigate if A3-induced mutations can yield drug resistant HIV-1 variants. Moreover, we aimed to study the restriction abilities of A3F allele variants carrying a single nucleotide polymorphism (SNP). We chose the SNP that causes the A3F 231I/V polymorphism because most people carry the heterozygous alleles. To test if A3 enzymes could contribute to HIV-1 evolution, we established a system to isolate HIV-1 drug resistant variants using peripheral blood mononuclear cells (PBMCs). We then used this method for a continuous spreading infection on infected U87 CD4+ CXCR4+ cells with a HIV-1 that was or was not exposed to A3G and A3F in a single replication cycle to discern the contributions of A3-mediated mutagenesis. The viruses were grown in the presence of increasing amounts of antiretroviral drugs. Viruses were collected and tested for viability using a reverse transcriptase activity assay. The data from four independent experiments with different drugs showed that A3G and A3F can induce drug resistance more times than when the virus was grown in their absence. However, drug resistant viruses exposed to A3G and A3F had less replication capacity than drug resistant variants that arose from HIV reverse transcriptase errors alone. Integrated proviral DNA and HIV genomic RNA (converted to cDNA) were sequenced to analyze A3-induced mutations. Importantly, for the first time we have quantified the amount of A3-induced mutations that occurs in the presence of Vif and shown that A3-induced mutations are approximately 4-fold above the background mutation level of HIV-1 reverse transcriptase alone. We also conducted similar experiments in CEM CD4+ T cells where endogenous A3 enzymes are expressed and obtained similar results. It is known that A3s have numerous polymorphisms which can affect antiretroviral activity of these enzymes. Namely, the CEM cells used in our experiments and numerous other labs have heterozygous alleles for A3F. As a result, we wanted to examine if the resulting A3F polymorphisms can be an additional factor that effects A3 activity in the presence or absence of Vif. We found that the A3F 231V polymorphism, in comparison to the A3F 231I, resulted in higher expression and virus encapsidation in the presence and absence of Vif. Specifically, we observed approximately 4-fold more virus restriction and mutations in proviral DNA. Altogether, these data suggest that A3 enzymes can be a viable option as a therapy for HIV-1 if the interaction between Vif and A3s were disrupted. However, such a potential therapy would also depend on if the A3 genotype of the person contained highly active variants, such as A3F 231V