HIV-1 PROJECTS

The acquired immunodeficiency syndrome (AIDS) continues to spread unchecked since its first documentation in 1981. Twenty-five years later, nearly 50 million individuals are living with HIV/AIDS world wide, according to the figures released by the Joint United Nations Program on HIV/AIDS (UNAIDS) and World Health Organization (WHO).

Given the importance and extent of the HIV epidemic around the world, studies to achieve a better understanding of underlying viral pathways that prevent us from finding a cure for this disease are of crucial importance. Extensive research and clinical trials have led to the discovery that certain enzymes such as protease (PR), reverse transcriptase (RT) and integrase (IN), are of crucial importance for viral infectivity. HIV protease is an aspartic protease involved in the processing of the viral Gag and Gag/Pol polyproteins; this step is essential for gaining viral infectivity. Without an active protease the virion is unable to maturate and infect other CD4+ cells. Currently, there are eight protease inhibitors (PIs) used to treat HIV-positive individuals. These PIs are administered in conjunction with a combination of RT inhibitors and/or integrase inhibitors and/or fusion inhibitors, and this cocktail, called HAART, has improved the prognosis of HIV-infected patients.
             But, even with this complex therapeutic approach, the development of drug resistance poses the greatest challenge for treating the HIV infection and the margin of success for achieving and maintaining virus suppression is narrow. At the molecular level, resistance to PIs predominantly takes the form of mutations within the PR molecule that preferentially lower the affinity of PIs with respect to protease substrates, while still maintaining viable catalytic activity. Basically, resistant mutations arise because they provide the virus with an advantage to survive in the presence of the drug. A number of resistance mutations have been characterized for all clinically used inhibitors, including atazanavir, the most recent one on the market. These difficulties only add to the significant genetic diversity that characterizes HIV. While HIV-1 subtype B has been the most widely studied, subtypes A, C, and D predominate worldwide.  Among the approximately 40 million people living with HIV/AIDS in 2003, more than 80% were infected with HIV-1 non-B subtypes. Substitutions at the positions designed as secondary resistance mutations in subtype B occur at high rates in certain non-subtype B viruses as naturally occurring or baseline polymorphisms. Whereas in treatment-naïve patients many of these baseline polymorphisms do not confer resistance to drugs per se among different clades, they may facilitate the development of drug resistance. Recent studies have shown that non-B isolates were statistically associated with more rapid progression to resistance after antiretroviral therapy and they had different mutational patterns as compared to B isolates. The combined effect of naturally existing polymorphisms and drug resistant mutations might have important consequences on the feasibility of continuing to use current HIV-1 protease inhibitors for non-subtype B infections.

Our HIV protease-related research develops into two main directions: (1) analysis of primary and secondary drug resistance mutations within HIV protease and (2) phylogenetic diversity of HIV-1 protease and its impact on response to current anti –HIV therapy. Our lab has performed extensive biochemical and structural studies to identify and characterize primary and secondary mutations.

 1. BIOCHEMICAL AND STRUCTURAL CHARACTERIZATION OF HIV-1 NON-B SUBTYPE PROTEASES including A₂, C, D, H, and F and recombinant forms A/G, A/C, and B/F, which we have obtained from the NIH AIDS Research & Reference Reagent Program. The worldwide spread of AIDS and the presence of a variety of different forms of the virus demand that we evaluate the properties of proteases from subtypes other than subtype B, which is the form that is predominant in the US and has been used in all studies of resistance development. The genes have been subcloned, and the proteins have been expressed, purified, and analyzed for kinetic properties and inhibitor binding using clinically-approved drugs, and three-dimensional structure in complex with a variety of the same inhibitors. This will be accomplished with the help of Dr. Robert McKenna.

A student working on this project will learn how to: 
-  express the HIV protease in E. coli expression system, to extract, refold to the native conformation, and purify the enzyme.
-  measure enzyme kinetic properties (K_m, k_cat, k_cat/K_m) and inhibition (K_i) with all clinically used inhibitors and for other inhibitors undergoing clinical trials. The student will also learn how to assay enzymatic activity using a chromogenic substrate that mimics the natural substrate of the enzyme. To further study each different protease, determination of kinetic constants would be employed: K_m, k_cat, and K_i.
-  crystallize and solve the three-dimensional structure of the different variants of HIV protease, alone or complexed with inhibitors.

 Working on this project: Roxana Coman – graduate student
                                                     Taylor Gilliland – undergraduate student
                                                     Marty Fernandez – undergraduate student
                                                     Kajia Zhu – undergraduate student

 

 

2. CHARACTERIZATION OF THE PROPERTIES OF THE GAG-POL PROTEINS OF NON-B SUBTYPES. Processing of these precursor polyproteins (Figure 1) is accomplished by the viral PR encoded within Gag/Pol without assistance from cellular proteases and occurs at the cell membrane during virion packing. Both the order and kinetics of cleavage as well as the extent of precursor processing appear to be critical steps in the generation of fully infectious, correctly assembled viral particles.

Figure 1. Gag and Gag/Pol Cleavage Sites. The HIV protease cleaves 5 sites within Gag, 11 sites within Gag/Pol polyproteins, and 1 cleavage site within accessory protein Nef (not shown).

We focus on the influence of mutations within the Gag/Pol fusion protein on the efficiency and order of PR processing. Our current work has identified several points within the region from the start of protein p2 through the end of the p6^Pol where mutations affect processing. We will employ this in vitro transcription/translation system to screen natural variants for altered processing patterns. We will pursue the analysis of the effects of antiviral inhibitors on the processing pathway and rate. This will begin with the clinically approved compounds now available from the NIH AIDS Research & Reference Reagent Program, but will also include new compounds that we develop or discover in our studies. All these goals will be accomplished in collaboration with Dr. Maureen Goodenow.

For a student to be able to work on this project, he/she must take the radioactivity safety class offered by UF.

Working on this project: Roxana Coman  – graduate student

 

3. EXPLORATION OF THE PROPERTIES AND THE STRUCTURE OF A VARIETY OF EXTENDED FORMS OF HIV-1 PROTEASE. These are constructs containing the protease itself and increasingly longer sequences upstream of the protease. Our studies focus on the N-terminal end of the Gag/Pol protein sequence, with protease located at the C-terminus of the constructs, as we know that the initial steps in processing occur in the region upstream of protease. We hypothesize that there is a structural pre-organization of Gag/Pol that brings the C cleavage site [p2/NC] near the active site cleft of the protease, resulting in the initial rapid cleavage of that junction (Figure 2). If this structure can be observed by crystallography, through the construction of an inactive protease-Gag fusion protein, then we will identify a new target for drug discovery, i.e., the interaction surface for formation of the pre-cleavage structure.

Figure 2. Model of the region from p2 (red) through protease (yellow), with NC (green) and transframe-p6^Pol intervening (orange). MA-CA is not shown in this model. This model was built using the structures of the protease (yellow tube, with catalytic aspartic residues in ball-and-stick representation, and the structure of NC (green tube) bound to DNA (shown in ball-and-stick representation) [1]. The p2 segment is represented as a helix (red) and the TR-p6^Pol is shown as a coil (orange). This structure brings the p2/NC cleavage junction near the active site cleft of the protease and would be a possible intermediate just before the start of Gag/Pol self-processing. The second p2/NC/TF segment could be disordered at this point, awaiting successful cleavage of the first segment, and is not shown in this figure. Red balls in the figure show points where mutations have been found to alter Gag-Pol processing. Two points in the p2 segment are represented by a black ball.

We will try to express, purify, refold and crystallize two variants: the active p2-PR construct and the inactive D25N variants.

Working on this project: Lashara Livingston – undergraduate student
                                                   Chriss Lazo – undergraduate student