Structural inferences for reverse transcribing viruses and drug resistance

Abstract

[ACCESS RESTRICTED TO THE UNIVERSITY OF MISSOURI AT REQUEST OF AUTHOR.] As we narrowly focus on the fundamental questions of our respective systems it is important to interact with the broader scientific community to gain greater perspective and further refine our research. When it comes to the roles of proteins in architecture and enzymology, 'form follows function', and this is an important recognition as much of modern biochemical and pharmaceutical insight has advanced through characterization of the precise relationship between protein structure and function. Engaging related but divergent fields and identifying suitable equivalent models can both provide unexpected insight and accelerate the identification of novel approaches/questions. My dissertation work has developed deeply towards identifying and characterizing enzymatic inferences grounded firmly in conserved protein structure with the specific applications in anti-viral drug resistance and enzyme kinetics. Rather than focusing on a single protein or system, the research presented here underlines two unique viral proteins: Integrase (IN) and Reverse Transcriptase (RT), while highlighting their divergent and conserved contributions to the life cycle of related 'high-profile' viruses. Chapter 1 will introduce the spectrum of viral replication mechanisms then further delve into the biology of the retrovirus Human Immunodeficiency Virus (HIV) with the objective of comparing its life cycle to related reverse transcribing viruses; Rice Tungro Bacilliform Virus (RTBV) and Hepatitis B Virus (HBV). Because of the conserved function but divergent form of RT, insights based on the thorough study of HIV can lend themselves to a much deeper understanding of these related viruses. Additionally, recognizing that in spite of the wealth of HIV enzyme knowledge, the development of the latest anti-HIV inhibitors targeting IN have relied heavily on the availability of Prototype Foamy Virus (PFV) IN crystal structures, often in the presence of small molecule inhibitors. Expanding on the inferences provided by the range of PFV IN structures and incorporating the recently characterized HIV-1 IN intasome structure, Chapter 2 focuses on the role of strain specific polymorphisms (PMs) and drug induced mutations on HIV-1 IN enzymology. Moving towards the development of personalized medicine, it is becoming increasingly more important to identify the genetic factors that determine drug susceptibility or resistance and the underlying mechanisms behind them. A major resource in this work has been access to clinical isolates from HIV-1 infected individuals representing the major infectious subtypes. The sequencing of these viral genomes, with emphasis on those patients that failed drug treatment, has opened the door for in-depth analysis of emerging resistance mutations. Here we expand on IN subtype PMs and acquired mutations identified when patients fail treatment and evaluate the biochemical processes affected by drug resistance. This work introduces the application of novel techniques to monitor IN assembly and the impact of drug resistant mutations on critical enzyme functions. In the future we expect to utilize these tools to enhance our understanding of the critical functions of this enzyme and the development of next-gen pharmaceuticals with a more targeted activity. Returning focus to the enzyme that ties all these viruses together, Chapter 3 will discuss the broad conservation among viral genomic coding regions, which allowed the identification of plant pararetroviral RT and the subsequent cloning and expression of the protein for kinetics studies. Drawing heavily from the wealth of research surrounding HIV-1 and related retroviral RT, the boundaries of plant pararetroviral RT structure and function within the viral replication cycle is explored. Emphasizing the economically significant RTBV as a model virus for this work we aim to expand the techniques developed here to inform the study of related reverse transcribing DNA plant pararetroviruses such as Cauliflower Mosaic Virus (CaMV) and the more distantly related HBV. The ability to isolate and express the RT from these viruses in their native conformation while maintaining their solubility has been a rate limiting step excluding the development of enzyme kinetics, crystallography, and ultimately drug discovery. Here I will outline the journey of RT coding region resolution as well as the application of common techniques predicted to help improve solubility while focusing on the critical outcome of maintaining predicted native enzymatic function. Chapter 4 will consolidate the protocols developed to solubilize the RTBV RT and build a firm understanding of the viral life cycle through determination of its characteristic enzyme functions with emphasis on divalent metal ion mediated catalysis. Drawing inferences from the HIV-1 structures to determine the enzyme polymerase and RNase H active site residues, we explore the structure function relationship through directed mutations of critical amino acids and monitoring of enzymatic activity. The identification of a non-canonical enzyme active site and its impact on divalent metal dependence is a major focus of this work and has the potential to change our understanding of the enzyme and its role in the viral life cycle. The conservation, and divergence, of the two functional domains of this enzyme allow for a range of activities and rates that directly feed into the viral genome replication and ultimately fitness of the virus. This is a delicate balance that has evolved to match the host organism environment, and as we've seen with extensive HIV-1 studies, can be thrown into disarray through targeted drug design. The implications of this work stand to benefit the study of plant pararetroviruses and could greatly inform the study of HBV RT, which to this day has not been expressed in a soluble form. Finally, in Chapter 5 the long-term implications of structure-based approach to enzyme determination and drug resistance is summarized along with future objectives for the presented projects. Included in Appendix is the manifestation of this work in a parallel project focused on the design of peptides targeting HIV-1 capsid (CA). In this work a 'conversation' with the available crystal structures utilizing techniques discussed in earlier chapters are applied to the study of HIV-1 CA and the demonstration of a novel inhibitory peptide with encouraging preliminary target binding. The design of this peptide inhibitor was based on the conservation of host factors and small molecule inhibitors interacting with the amino acid residue side chains at a high traffic binding pocket. This novel approach to drug design serves as a practical demonstration for the importance of structure-based approaches in targeted clinical drug discovery with broader applications in crop sciences and transgenic approaches to pararetrovirus resistance.