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theses

Retroviruses continue to attract public health attention due to the increasing disease burden these viruses continue to have in animals. An estimated 38 million people worldwide are infected with HIV which underscores the importance of these viruses to humans. In the absence of a cure or vaccine for HIV, therapeutic interventions that have culminated in the taming of the disease, which used to be a death sentence before the discovery of these drugs, has relied on detailed understanding of key processes which underline the replication of the virus in its hosts. It is not surprising that the great majority of the currently approved drugs for treating HIV infections target the various enzymatic proteins which carry out chemical reactions that enable the virus to infect new cells.
Increasing resistance to these drugs, and problems of compliance and toxicity, have necessitated the need to search for more potent and less toxic drugs to manage the disease. It is in this regard that understanding the structural and mechanistic details of polyprotein processing is important. Synthesis of polyprotein precursors which are subsequently processed into mature enzymes is unique to pathogenic viruses such as retroviruses and RNA viruses. Developing drugs selective to these polyproteins would reduce the potential for cross-reactivity with host proteins.
The crystal structure of the PFV protease-reverse transcriptase (PR-RT), solved to 2.9 Å resolution, has revealed a monomeric protein containing individually folded subdomains. In PFV, PR-RT is the mature entity following the proteolytic release of IN from the C-terminus of the PFV Pol polyprotein. The structure of the PR resembles a monomer of the homodimeric HIV-1 PR even though the N-terminal and C-terminal residues involved in the formation of anti-parallel β-sheets which mediate the dimer interface, remain unstructured. The RT also contains the canonical subdomains: fingers, palm, thumb and connection, as well as the RNase H domain, which characterize retroviral RTs. The relative orientations of these subdomains are however different, resembling more the compact p51 subunit of HIV RT. This is an inactive RT conformation since the nucleic acid binding cleft is occluded. It however offers insights into the possible subdomain arrangements of the monomeric precursor polyproteins.
In this work also, the Pol polyproteins from the prototype foamy virus (PFV), as well as Pol and Gag-Pol polyproteins from HIV-1 have been characterized using a combination of enzymatic assays and other biophysical methods including: gel filtration, dynamic light scattering (DLS), small angle X-ray scattering (SAXS), and single particle cryo-EM. The production of these proteins, which were isolated in yields and purity suitable for biophysical studies from bacteria for the first time, relied on a new media formulation for E. coli growth. Fundamentally, it was discovered that at non-physiologic Mg2+ concentration (~50 mM) or low pH (≤ 6.0), the proteolytic degradation of these polyproteins and by extension, heterologously expressed proteins in bacteria, are highly attenuated. This decreased proteolytic processing enabled the expression and purification of these polyprotein precursors.
Single particle cryo-EM analysis of the HIV-1 Pol polyprotein revealed a dimeric RT similar to the mature heterodimer in configuration. The formation of the heterodimer at a very early stage of the maturation process offers a plausible explanation as to why only one RNase H domain of RT is cleaved in the virus during maturation, since in this configuration, the RNase H cleavage site of the p66 subunit is sequestered. The dimeric RT brings the two N-terminal PR monomer close to each other, which enables them to dimerize and cleave their substrates.
Advanced three-dimensional classification analysis of the HIV-1 Pol structures further revealed that the dimerization tendency of the PR may be reduced compared to the mature enzyme with various classes showing density for only one PR at the N-terminus of either the p51-like subunit or the p66-like subunit. This structural and configurational heterogeity offers a plausible explanation to why the immature PR is less sensitive to drugs that target the active site of the mature PR. The IN domain in the structure of the Pol is disordered and not visible. However, it binds the integrase-binding domain (IBD) of lens epithelium-derived growth factor (LEDGF/ p75) fused to the C-terminus of the maltose-binding protein (MBP), and the MBP-IBD fusion pulls down the full-length HIV-1 Pol dimer even at high salt concentrations (1.0 M NaCl). This suggests that the IN is also in a dimeric organization since IBD binds dimeric IN and not monomers. By assembling the dimeric units of these enzymes, which are the functional units or the building blocks for the functional entity, in the case of IN, very early in the maturation process, the virus embarks on an irreversible voyage committed to infect new cells by ensuring that these enzymes required later in the life cycle, are available in active forms when needed. The structures of the PFV Pol and HIV-1 Gag-Pol will provide a more comprehensive understanding of the structural underpinnings of PR activation and how maturation is orchestrated in the retroviral family.

ETD_10394

Ph.D.

Includes bibliographical references

doi:10.7282/t3-81et-xe54

ETD doctoral

The author owns the copyright to this work.