Co-evolution of HIV-1 Protease and its Substrates: A Dissertation

Drug resistance is the most important factor that influences the successful treatment of individuals infected with the human immunodeficiency virus type 1 (HIV-1), the causative organism of the acquired immunodeficiency syndrome (AIDS). Tremendous advances in our understanding of HIV and AIDS have led to the development of Highly Active Antiretroviral Therapy (HAART), a combination of drugs that includes HIV-1 reverse transcriptase, protease, and more recently, integrase and entry inhibitors, to combat the virus. Though HAART has been successful in reducing AIDS-related morbidity and mortality, HIV rapidly evolves resistance leading to therapy failure. Thus, a better understanding of the mechanisms of resistance will lead to improved drugs and treatment regimens. Protease inhibitors (PIs) play an important role in anti-retroviral therapy. The development of resistance mutations within the active site of the protease greatly reduces its affinity for the protease inhibitors. Frequently, these mutations reduce catalytic efficiency of the protease leading to an overall reduction in viral fitness. In order to overcome this loss in fitness the virus evolves compensatory mutations within the protease cleavage sites that allow the protease to continue to recognize and cleave its substrates while lowering affinity for the PIs. Improved knowledge of this substrate co-evolution would help better understand how HIV-1 evolves resistance and thus, lead to improved therapeutic strategies. Sequence analyses and structural studies were performed to investigate co-evolution of HIV-1 protease and its cleavage sites. Though a few studies reported the co-evolution within Gag, including the protease cleavage sites, a more extensive study was lacking, especially as drug resistance was becoming increasingly severe. In Chapter II, a small set of viral sequences from infected individuals were analyzed for mutations within the Gag cleavage sites that co-occurred with primary drug resistance mutations within the protease. These studies revealed that mutations within the p1p6 cleavage site coevolved with the nelfinavir-resistant protease mutations. As a result of increasing number of infected individuals being treated with PIs leading to the accumulation of PI resistant protease mutations, and with increasing efforts at genotypic and phenotypic resistance testing, access to a larger database of resistance information has been made possible. Thus in Chapter III, over 39,000 sequences were analyzed for mutations within NC-p1, p1-6, Autoproteolysis, and PR-RT cleavage sites and several instances of substrate co-evolution were identified. Mutations in both the NC-p1 and the p1-p6 cleavage sites were associated with at least one, if not more, primary resistance mutations in the protease. Previous studies have demonstrated that mutations within the Gag cleavage sites enhance viral fitness and/or resistance when they occur in combination with primary drug resistance mutations within the protease. In Chapter III viral fitness in the presence and absence of cleavage site mutations in combination with primary drug resistant protease mutations was analyzed to investigate the impact of the observed co-evolution. These studies showed no significant changes in viral fitness. Additionally in Chapter III, the impact of these correlating mutations on phenotypic susceptibilities to various PIs was also analyzed. Phenotypic susceptibilities to various PIs were altered significantly when cleavage site mutations occurred in combination with primary protease mutations. In order to probe the underlying mechanisms for substrate co-evolution, in Chapter IV, X-ray crystallographic studies were performed to investigate structural changes in complexes of WT and D30N/N88D protease variants and the p1p6 peptide variants. Peptide variants corresponding to p1p6 cleavage site were designed, and included mutations observed in combination with the D30N/N88D protease mutation. Structural analyses of these complexes revealed several correlating changes in van der Waals contacts and hydrogen bonding as a result of the mutations. These changes in interactions suggest a mechanism for improving viral fitness as a result of co-evolution. This thesis research successfully identified several instance of co-evolution between primary drug resistant mutations in the protease and mutations within NC-p1 and p1p6 cleavage sites. Additionally, phenotypic susceptibilities to various PIs were significantly altered as a result of these correlated mutations. The structural studies also provided insights into the mechanism underlying substrate co-evolution. These data advance our understanding of substrate co-evolution and drug resistance, and will facilitate future studies to improve therapeutic strategies

Upper and Lower Gastrointestinal Endoscopic Findings in HIV-Infected Patients in the Era of Highly Active Antiretroviral Therapy.

Background: Endoscopic evaluation with biopsies are instrumental in the diagnosis and management of gastrointestinal (GI) disorders in the setting of human immunodeficiency virus (HIV) or acquired immunodeficiency syndrome (AIDS), especially in the era of highly active antiretroviral therapy (HAART). Methods: A retrospective chart review of 304 HIV-positive and 199 HIV-negative patients who had undergone upper and/or lower endoscopy in an urban community hospital from the years 2012 - 2017 was performed. Inclusion criteria included men and women between the ages of 45 to 75 years, which had undergone colonoscopies between within 2012 - 2017 and had tested positive for HIV. They were selected from that population if they had complete charts that included information regarding symptoms, viral load, cluster of differentiation 4 (CD4) count, prescribed HAART medication, findings from the upper and lower colonoscopy both from the gastroenterologist\u27s report and pathologist\u27s report. Only then would they be added to the pool of final selection that we could compute data from and draw conclusions. Results: Among HIV patients, those with less than 200 CD4 cells/µL counts had lower rates of diverticulosis and hemorrhoids, as compared with those with greater than 200 cells/µL counts. Other gross and histological findings (from either upper or lower endoscopy) were not statistically different between these two groups. In HIV-positive patients, gastritis, Conclusions: Physicians should consider the possibility that the GI symptoms in HIV-infected patients on HAART may be due to an opportunistic infection, even when the CD4 count is more than 200 cells/µL and the viral load is low

HIV-1 protease-substrate coevolution in nelfinavir resistance

Resistance to various human immunodeficiency virus type 1 (HIV-1) protease inhibitors (PIs) challenges the effectiveness of therapies in treating HIV-1-infected individuals and AIDS patients. The virus accumulates mutations within the protease (PR) that render the PIs less potent. Occasionally, Gag sequences also coevolve with mutations at PR cleavage sites contributing to drug resistance. In this study, we investigated the structural basis of coevolution of the p1-p6 cleavage site with the nelfinavir (NFV) resistance D30N/N88D protease mutations by determining crystal structures of wild-type and NFV-resistant HIV-1 protease in complex with p1-p6 substrate peptide variants with L449F and/or S451N. Alterations of residue 30\u27s interaction with the substrate are compensated by the coevolving L449F and S451N cleavage site mutations. This interdependency in the PR-p1-p6 interactions enhances intermolecular contacts and reinforces the overall fit of the substrate within the substrate envelope, likely enabling coevolution to sustain substrate recognition and cleavage in the presence of PR resistance mutations. IMPORTANCE: Resistance to human immunodeficiency virus type 1 (HIV-1) protease inhibitors challenges the effectiveness of therapies in treating HIV-1-infected individuals and AIDS patients. Mutations in HIV-1 protease selected under the pressure of protease inhibitors render the inhibitors less potent. Occasionally, Gag sequences also mutate and coevolve with protease, contributing to maintenance of viral fitness and to drug resistance. In this study, we investigated the structural basis of coevolution at the Gag p1-p6 cleavage site with the nelfinavir (NFV) resistance D30N/N88D protease mutations. Our structural analysis reveals the interdependency of protease-substrate interactions and how coevolution may restore substrate recognition and cleavage in the presence of protease drug resistance mutations

Molecular Basis for Drug Resistance in HIV-1 Protease

HIV-1 protease is one of the major antiviral targets in the treatment of patients infected with HIV-1. The nine FDA approved HIV-1 protease inhibitors were developed with extensive use of structure-based drug design, thus the atomic details of how the inhibitors bind are well characterized. From this structural understanding the molecular basis for drug resistance in HIV-1 protease can be elucidated. Selected mutations in response to therapy and diversity between clades in HIV-1 protease have altered the shape of the active site, potentially altered the dynamics and even altered the sequence of the cleavage sites in the Gag polyprotein. All of these interdependent changes act in synergy to confer drug resistance while simultaneously maintaining the fitness of the virus. New strategies, such as incorporation of the substrate envelope constraint to design robust inhibitors that incorporate details of HIV-1 protease’s function and decrease the probability of drug resistance, are necessary to continue to effectively target this key protein in HIV-1 life cycle

Co-evolution of nelfinavir-resistant HIV-1 protease and the p1-p6 substrate

The selective pressure of the competitive protease inhibitors causes both HIV-1 protease and occasionally its substrates to evolve drug resistance. We hypothesize that this occurs particularly in substrates that protrude beyond the substrate envelope and contact residues that mutate in response to a particular protease inhibitor. To validate this hypothesis, we analyzed substrate and protease sequences for covariation. Using the chi2 test, we show a positive correlation between the nelfinavir-resistant D30N/N88D protease mutations and mutations at the p1-p6 cleavage site as compared to the other cleavage sites. Both nelfinavir and the substrate p1-p6 protrude beyond the substrate envelope and contact residue 30, thus possibly making the p1-p6 cleavage site more vulnerable to co-evolution

Human Immunodeficiency Virus Type 1 Protease-Correlated Cleavage Site Mutations Enhance Inhibitor Resistance▿ §

Drug resistance is an important cause of antiretroviral therapy failure in human immunodeficiency virus (HIV)-infected patients. Mutations in the protease render the virus resistant to protease inhibitors (PIs). Gag cleavage sites also mutate, sometimes correlating with resistance mutations in the protease, but their contribution to resistance has not been systematically analyzed. The present study examines mutations in Gag cleavage sites that associate with protease mutations and the impact of these associations on drug susceptibilities. Significant associations were observed between mutations in the nucleocapsid-p1 (NC-p1) and p1-p6 cleavage sites and various PI resistance-associated mutations in the protease. Several patterns were frequently observed, including mutations in the NC-p1 cleavage site in combination with I50L, V82A, and I84V within the protease and mutations within the p1-p6 cleavage site in combination with D30N, I50V, and I84V within the protease. For most patterns, viruses with mutations both in the protease and in either cleavage site were significantly less susceptible to specific PIs than viruses with mutations in the protease alone. Altered PI resistance in HIV-1 was found to be associated with the presence of Gag cleavage site mutations. These studies suggest that associated cleavage site mutations may contribute to PI susceptibility in highly specific ways depending on the particular combinations of mutations and inhibitors. Thus, cleavage site mutations should be considered when assessing the level of PI resistance

Three Residues in HIV-1 Matrix Contribute to Protease Inhibitor Susceptibility and Replication Capacity▿

Other than cleavage site mutations, there is little data on specific positions within Gag that impact on HIV protease inhibitor susceptibility. We have recently shown that non-cleavage site mutations in gag, particularly within matrix protein can restore replication capacity and further reduce protease inhibitor drug susceptibility when coexpressed with a drug-resistant (mutant) protease. The matrix protein of this patient-derived virus was studied in order to identify specific changes responsible for this phenotype. Three amino acid changes in matrix (R76K, Y79F, and T81A) had an impact on replication capacity as well as drug susceptibility. Introduction of these three changes into wild-type (WT) matrix resulted in an increase in the replication capacity of the protease mutant virus to a level similar to that achieved by all the changes within the mutant matrix and part of the capsid protein. Pairs of changes to wild-type matrix led to an increased replication capacity of the protease mutant (although less than with all three changes). Having only these three changes to matrix in a wild-type virus (with wild-type protease) resulted in a 5- to 7-fold change in protease inhibitor 50% effective concentration (EC50). Individual changes did not have as great an effect on replication capacity or drug susceptibility, demonstrating an interaction between these positions, also confirmed by sequence covariation analysis. Molecular modeling predicts that each of the three mutations would result in a loss of hydrogen bonds within α-helix-4 of matrix, leading to the hypothesis that more flexibility within this region or altered matrix structure would account for our findings

Context surrounding processing sites is crucial in determining cleavage rate of a subset of processing sites in HIV-1 Gag and Gag-Pro-Pol polyprotein precursors by viral protease

Processing of the human immunodeficiency virus type 1 (HIV-1) Gag and Gag-Pro-Pol polyproteins by the HIV-1 protease (PR) is essential for the production of infectious particles. However, the determinants governing the rates of processing of these substrates are not clearly understood. We studied the effect of substrate context on processing by utilizing a novel protease assay in which a substrate containing HIV-1 matrix (MA) and the N-terminal domain of capsid (CA) is labeled with a FlAsH (fluorescein arsenical hairpin) reagent. When the seven cleavage sites within the Gag and Gag-Pro-Pol polyproteins were placed at the MA/CA site, the rates of cleavage changed dramatically compared with that of the cognate sites in the natural context reported previously. The rate of processing was affected the most for three sites: CA/spacer peptide 1 (SP1) ( approximately 10-fold increase), SP1/nucleocapsid (NC) ( approximately 10-30-fold decrease), and SP2/p6 ( approximately 30-fold decrease). One of two multidrug-resistant (MDR) PR variants altered the pattern of processing rates significantly. Cleavage sites within the Pro-Pol region were cleaved in a context-independent manner, suggesting for these sites that the sequence itself was the determinant of rate. In addition, a chimera consisting of SP1/NC P4-P1 and MA/CA P1\u27-P4\u27 residues (ATIM downward arrowPIVQ) abolished processing by wild type and MDR proteases, and the reciprocal chimera consisting of MA/CA P4-P1 and SP1/NC P1\u27-4\u27 (SQNY downward arrowIQKG) was cleaved only by one of the MDR proteases. These results suggest that complex substrate interactions both beyond the active site of the enzyme and across the scissile bond contribute to defining the rate of processing by the HIV-1 PR