Human Immunodeficiency Virus Type 1 Reverse Transcriptase and Protease Gene mutations: An Observational and Prediction study on Antiretroviral Interactions and Co-receptor Usage

Human immunodeficiency virus (HIV) belongs to the family Retroviridae and the subfamily lentivirinae. The other medically important members in this family include human Tlymphotropic viruses I and II. The HIV is of two types and are designated HIV-1 and HIV-2. HIV-1 and 2 are subdivided based on genetic differences and are divided into groups and clades/subtypes. Although the modes of transmission in humans are the same for HIV-1 and HIV-2, the disease progression differs for HIV-1 and HIV-2 infected individuals. HIV-1 infection is associated with a faster disease progression than HIV-2 infection. Globally there are 33 million HIV infected individuals according to the figures released by the Joint United Nations Program on HIV/AIDS (UNAIDS). India has about 3 million HIV infected individuals as per the latest National AIDS Control Organization (NACO) surveillance reports with an estimated prevalence of 0.36% in the general population. Studies have shown that ART has helped to increase the survival rate of individuals infected with HIV. Porter et al have shown that the proportion of person-time on ART has shown an increase from 22% in 1997 to 57% in 2001. Effective use of ART has increased the mean survival time of HIV infected individual significantly altering the lower survival time in the natural history of HIV infection. Reports from Africa and Asia have shown similar effects of ART on survival rate as compared to the pre-ART era. A HIV infected individual is put on combination of drugs which target varies proteins of HIV whereby the production of mature viral particles in the infected individual are inhibited. This combination of drugs is called antiretroviral therapy (ART) has been found to be effective in controlling HIV infections than administration of only one drug. The health agencies like NACO and other Non Governmental Organizations (NGOs) are taking a number of steps to control the spread of HIV infections in India. In the study reported in this thesis certain related aspects of HIV-1 drug resistance were investigated. It included sequencing the RT and Pr genes to detect mutations, assessment of the mutation effects on drug susceptibility of strains characterized from both treatment naive (n=101) and treatment failed (n=19) individuals. The effect of nucleotide mutations on amino acid changes and its bearing on drug susceptibility i.e., genotypic assay was achieved by using relevant web based databases designed to give information on HIV drug resistance. The amino acid sequences from selected strains (n=23) were taken for modelling to identify the specific effects of the mutations on the functioning of the RT (n=10) and Pr (n=13) molecules. The different genotypic algorithms were also applied on the same set of sequences to investigate any differences in interpretation of the effects of mutation on the susceptibility of the drug. HIV-1 strains have been shown to evolve showing difference in corecepetor usage i.e., CCR5 receptor utilizing R5 strains (primarily macrophage tropic, non syncitium inducing; NSI) to CXCR4 utilizing X4 strains (primarily dual tropic infecting CD4 cells and/or macrophages, syncitium inducing; SI) in a given individual during the course of the infection. Natural history studies prior to use of ART in western countries have shown a predominance of X4 strains in AIDS patient with low CD4 counts. This phenomenon is most marked for subtype B. Furthermore, coreceptor CCR5 blocking therapeutic agent Maraviroc, is now licensed for treating drug experienced individuals. The data on coreceptor usage is preliminary on subtype C which is predominant in India. Hence, an investigation was also carried out to characterize coreceptor usage based on examining the V3 sequences of 47 strains (subtype C) obtained from HIV-1 infected individuals to give information on X4 strains among the presently circulating strains. A study carried out a decade ago at National Institute of Virology, Pune had shown all type subtype C strains from individual to be NSI strains. The findings are important with the introduction of a CCR5 antagonist for the treatment of HIV-1 infections. Hence, in such scenarios it would be imperative to determine the co-receptor utilized by the strains before administration of these classes of drugs. Strains resistant to maraviroc have been detected in patients elsewhere in whom they have been administrated. These resistant strains were CXCR4 strains which had emerged from a preexisting population in the patient. It would be important to monitor the scenario when this group of drugs is introduced in India

HIV reverse transcriptase: Structural interpretation of drug resistant genetic variants from India

The reverse transcriptase (RT) enzyme is the prime target of nucleoside/ nucleotide (NRTI) and non-nucleoside (NNRTI) reverse transcriptase inhibitors. Here we investigate the structural basis of effects of drug-resistance mutations in clade C RT using three-dimensional structural modeling. Apropos the expectation was for unique mechanisms in clade C based on interactions with amino acids of p66 subunit in RT molecule. 3-D structures of RT with mutations found in sequences from 2 treatment naïve, 8 failed and one reference clade C have been modeled and analyzed. Models were generated by computational mutation of available crystal structures of drug bound homologous RT. Energy minimization of the models and the structural analyses were carried out using standard methods. Mutations at positions 75,101,118,190,230,238 and 318 known to confer drug resistance were investigated. Different mutations produced different effects such as alteration of geometry of the drugbinding pocket, structural changes at the site of entry of the drug (into the active site), repositioning the template bases or by discriminating the inhibitors from their natural substrates. For the mutations analyzed, NRTI resistance was mediated mainly by the ability to discriminate between inhibitors and natural substrate, whereas, NNRTI resistance affected either the drug entry or the geometry of the active site. Our analysis suggests that different mutations result in different structural effects affecting the ability of a given drug to bind to the RT. Our studies will help in the development of newer drugs taking into account the presence of these mutations and the structural basis of drug resistance

Structural Basis of Drug Resistance by Genetic Variants of HIV Type 1 Clade C Protease from India

Using computer modeling of three-dimensional structures and structural information available on the crystal structures of HIV-1 protease, we investigated the structural effects of mutations, in treatment-naive and treatment-exposed individuals from India and postulated mechanisms of resistance in clade C variants. A large number of models (14) have been generated by computational mutation of the available crystal structures of drug bound proteases. Localized energy minimization was carried out in and around the sites of mutation in order to optimize the geometry of interactions present. Most of the mutations result in structural differences at the flap that favors the semiopen state of the enzyme. Some of the mutations were also found to confer resistance by affecting the geometry of the active site. The E35D mutation affects the flap structure in clade B strains and E35N and E35K mutation, seen in our modeled strains, have a more profound effect. Common polymorphisms at positions 36 and 63 in clade C also affected flap structure. Apart from a few other residues Gln-58, Asn-83, Asn-88, and Gln-92 and their interactions are important for the transition from the closed to the open state. Development of protease inhibitors by structure-based design requires investigation of mechanisms operative for clade C to improve the efficacy of therapy