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This thesis project was designed to investigate the phenotypic and genotypic variability of human immunodeficiency virus type 1 (HIV-1) drug-naive clade C reverse transcriptase (RT) and its potential impact in the development of resistance against inhibitors of RT. Five treatment-naive HIV-1 Ethiopian isolates were classified as subtype C on the basis of env gene heteroduplex mobility assays (HMA) profile and phylogenetic analysis of RT sequences. In subtype C RT, a specific KVEQ motif of silent mutations (amino acid 65, 106, 138, 161) at resistance sites was present. Two Ethiopian strains were naturally resistant to non-nucleoside RT inhibitors (NNRTI), as well as to zidovudine (ZDV), based on the natural polymorphisms of G190A and K70R, respectively. The final drug concentration that selected for NNRTI primary resistance mutations in tissue culture assays was significantly higher for clade B than clade C for each of nevirapine (NW) (10 muM versus 2 or 4 muM), efavirenz (EFV) (1muM versus 0.01muM) and delaviridine (DLV) (10muM versus 1 or 4muM), respectively. In the middle of the selection period with all the NNRTIs, subtype B viruses were harboring a mixture of both wild type and mutated forms, whereas in clade C viruses, primary resistance mutations were fully generated. Thus, we have found that clade C isolates developed more rapidly resistance (8 or 9 weeks with NVP or DLV and 13 weeks with EFV) as compared with clade B controls (at least 15 weeks with NW or DLV and 30 weeks with EFV). Odd mutations were detected during selection with NNRTIs, such as S98I, and two mutations (A62V and V75E), at sites associated to multi-drug resistance against nucleoside inhibitors (NRTIs). The substitution A62V was initially observed as a drug-naive silent mutation A62A. NW and DLV mutants were broadly cross-resistants. Following in vitro selection for drug-resistance with NNRTIs (NVP, DLV and EFV) and NRTIs [lamivudine (3TC) and ZDV], RT immunodominant regions of 14 HIV-1 s

Diss. (sammanfattning) Stockholm : Karol. inst. / Härtill 6 uppsatser.

Reverse transcription of human immunodeficiency virus type-1 (HIV-1) is primed by cellular tRNALys3, which is selectively packaged into viral particles where it is bound at its 3' terminus to a complementary sequence of viral RNA termed the primer binding site (PBS). In addition to the PBS, other regions within the viral genome also interact with tRNALys3. Initiation of HIV-1 reverse transcription requires specific recognition of the viral genome, tRNA primer, and reverse transcriptase (RT). In this work, we study the important role played by the initiation complex in the initiation of HIV-1 reverse transcription. An "A-rich loop" located upstream of the PBS has been shown to interact with the anticodon loop of tRNALys3 and deletion of this A-rich loop caused diminished viral replication fitness. We have now studied the mechanisms involved in the altered replication capacities of the deletion-containing viruses in the context of both wild type HIV-1 and viruses also containing the M184V substitution in RT. We found that the M184V mutation in RT compromises the ability of deletion-containing viruses to restore wild-type replication. Further biochemical study indicates that both the M184V mutation in RT and deletion of sequences upstream of PBS caused diminished viral replication fitness by compromising the efficiency of reverse transcription initiation. / Since the initiation of DNA synthesis was shown to be a highly specific process, it represents a potential target for the development of novel antiviral agents. We developed strategies for inhibition of the HIV-1 replication via interference with the tRNALys3/viral RNA complex. To target primer tRNALys3, we employed oligodeoxyribonucleotides (ODNs) that are complementary to different parts of the tRNA primer. To target viral RNA, we devised a tRNALys3-like molecule, termed tRNA Lys*, that contained sequence alterations that direct initiation from a region distant from the natural PBS, designated PBS*. PBS* is involved in the formation of the natural tRNA/PBS complex and binding of tRNALys* was shown to interfere specifically with the initiation of reverse transcription. Inhibition of the synthesis of (-) strand strong-stop DNA was achieved successfully with both strategies by interfering with the formation of the initiation complex.

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Diss. (sammanfattning) Stockholm : Karol. inst., 2002. / Härtill 4 uppsatser.

Diss. (sammanfattning) Stockholm : Karol. inst., 2003. / Härtill 5 uppsatser.

Thesis (Ph. D.)--University of Washington, 2005. / Vita. Includes bibliographical references (leaves 112-127).

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