Thesis (Ph. D.)--University of Missouri-Columbia, 2007. / The entire dissertation/thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file (which also appears in the research.pdf); a non-technical general description, or public abstract, appears in the public.pdf file. Title from title screen of research.pdf file (viewed Oct. 16, 2007). Vita. Includes bibliographical references.

ABSTRACT APOBEC3G (Apo3G) is a host cell restriction factor of viruses that produce a singlestranded (ss) DNA replication intermediate (Sheehy et al., 2002; Suspene et al., 2004). Apo3G is studied primarily for its ability to restrict propagation of the retrovirus, HIV. In cell culture, Apo3G can only inhibit HIV if it lacks its virion infectivity factor (Vif). The host-pathogen interface between Apo3G and HIV has become a new target of study for the development of novel HIV therapeutics (Prochnow et al., 2009; Sheehy et al., 2003). Apo3G induces mutagenesis of the HIV proviral DNA (Mangeat et al., 2003; Zhang et al., 2003). Apo3G has the ability to induce transition mutations, i.e. cytosine to thymine, through deamination of cytosine to form uracil. Deamination activity induces numerous mutations that causes gene inactivation of the HIV provirus thus restricting the HIV lifecycle. Apo3G attenuates HIV virion infectivity in the absence of the virion infectivity factor (Vif) by inducing genome mutations through deamination of cytosine to uracil in HIV minus strand DNA. Independent from deaminase activity, Apo3G may also interfere with HIV reverse transcription by preventing full length cDNA from forming (Iwatani et al., 2007), nucleocapsid (NC) mediated strand annealing (Guo et al., 2007; Guo et al., 2009; Li et al., 2007), and RNaseH activity of the reverse transcriptase (Li et al., 2007). Whether Apo3G is able to restrict HIV by a deamination-independent mode remains controversial. In particular, the existence of the deamination independent mode was challenged since the Apo3G deamination null mutant E259Q was shown to have limited or no ability to inhibit HIV-1 replication (Schumacher et al., 2008). This research assesses the ability of Apo3G to inhibit reverse transcription of HIV genomic RNA. It is hypothesised that based on the ability of Apo3G to bind and oligomerize on single stranded nucleic acids (Chelico et al., 2008), and its high affinity for RNA (Chelico et al., 2010), that Apo3G can inhibit RT mediated primer extension as well as nucleocapsid mediated strand annealing. Additionally, it is hypothesized that Apo3G cannot inhibit RT RNAseH activity, as Apo3G has been shown to have a low affinity for DNA/RNA hybrids (Iwatani et al., 2006). We will test these hypotheses by using in vitro assays that mimic in vivo reverse transcription events. Here we have shown that Apo3G is able to decrease the efficiency with which HIV-1 reverse transcriptase synthesizes DNA from an RNA primer annealed to an RNA template. Apo3G had a minimal affect on primer initiation and primarily inhibits primer elongation. Using iii the monomeric mutant, F126A/W127A, we show that the deamination independent mode of inhibiting reverse transcriptase is impaired without oligomerization on template RNA. We also provide evidence that the Apo3G mutant E259Q should not be considered a deamination null proxy for native Apo3G since it exhibits decrease in RNA binding affinity compared to the native form. We did not find that Apo3G inhibited HIV NC-mediated strand annealing activity or RNaseH activity of HIV-1 reverse transcriptase. The data suggest a two-tiered mechanism for inhibition of reverse transcriptase-mediated DNA synthesis that is dependent upon 1) the ability of Apo3G to oligomerize on RNA substrates and 2) bind RNA with high affinity. Ascribing a mechanism to the deamination independent mode of HIV-1 restriction by Apo3G suggests that the enzyme may use this mechanism in vivo to delay completion of proviral DNA synthesis which, may negatively impact the HIV-1 lifecycle.

BACKGROUND: APOBEC3H (A3H) is a member the Apolipoprotein B mRNA-editing catalytic (APOBEC) polypeptide seven cytidine deaminases family. These APOBEC3 enzymes have the innate ability to deaminate a cytidine base converting it to a uridine base. Previous research has suggested that human A3H haplotypes have similar properties like those of A3G and A3DE, in which these APOBEC3 enzymes are known to entail anti-viral activity toward viruses such as human immunodeficiency virus. METHODOLOGY: Human A3H gene was amplified by polymerase chain reaction and inserted into E.Coli plasmids. Expression tests were conducted to verify that the clones were able to express protein. Ten liters of the cloned cells were grown, and A3H protein was purified using an established protocol in Dr. Chen's lab for deamination to test the enzyme activity. An inactive mutant of A3H was also cloned to use as a negative control for the wild type A3H. PRINCIPAL FINDINGS: The isolated A3H haplotype 1 (referred to as A3H hereafter) is indeed active, but the activity is very weak in comparison to other APOBEC3 proteins, in which A3F and A3A were used as controls. A3H deamination is most optimal with an acidic environment of pH 5.5 or 6.0, and concentrated protein will produce more deamination product (darker bands). The inactive A3H produced some unexpected visible smaller DNA bands, which may be the result of contaminated protein activities. In conclusion, the wild type A3H protein may have weak activity, but additional studies are required to confirm that the inactivated A3H mutant has no activity on the DNA substrates.

Thesis (Ph. D.)--University of Missouri-Columbia, 2001. / Typescript. Vita. Includes bibliographical references. Also available on the Internet.

Thesis (Ph. D.)--University of Missouri-Columbia, 2001. / Typescript. Vita. Includes bibliographical references. Also available on the Internet.

Thesis (Ph. D.)--University of Missouri-Columbia, 2003. / Accompany CD-ROM contains html files that illustrate in 3-D the structures discussed in Chapter 1. Typescript. Vita. Includes bibliographical references. Also available on the Internet but lacking CD-ROM contents.

Thesis (Ph. D.)--University of Missouri-Columbia, 2003. / Accompany CD-ROM contains html files that illustrate in 3-D the structures discussed in Chapter 1. Typescript. Vita. Includes bibliographical references. Also available on the Internet but lacking CD-ROM contents.

Thesis (Ph. D.)--University of Missouri-Columbia, 1996. / Typescript. Vita. Includes bibliographical references. Also available on the Internet.

Thesis (Ph. D.)--University of Missouri-Columbia, 1996. / Typescript. Vita. Includes bibliographical references. Also available on the Internet.

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.