Connection domain mutations N348I and A360V in HIV-1 reverse transcriptase enhance resistance to 3'-azido-3'-deoxythymidine through both RNase H-dependent and -independent mechanisms. 2008

Maryam Ehteshami, and Greg L Beilhartz, and Brian J Scarth, and Egor P Tchesnokov, and Suzanne McCormick, and Brian Wynhoven, and P Richard Harrigan, and Matthias Götte
Department of Microbiology and Immunology, McGill University, Montreal, Quebec H3A 2B4, Canada.

Thymidine analogue-associated mutations (TAMs) in reverse transcriptase (RT) of the human immunodeficiency virus type 1 (HIV-1) cause resistance to 3'-azido-3'-deoxythymidine (AZT) through excision of the incorporated monophosphate. Mutations in the connection domain of HIV-1 RT can augment AZT resistance. It has been suggested that these mutations compromise RNase H cleavage, providing more time for AZT excision to occur. However, the underlying mechanism remains elusive. Here, we focused on connection mutations N348I and A360V that are frequently observed in clinical samples of treatment-experienced patients. We show that both N348I and A360V, in combination with TAMs, decrease the efficiency of RNase H cleavage and increase excision of AZT in the presence of the pyrophosphate donor ATP. The TAMs/N348I/A360V mutant accumulates transiently formed, shorter hybrids that can rebind to RT before the template is irreversibly degraded. These hybrids dissociate selectively from the RNase H-competent complex, whereas binding in the polymerase-competent mode is either not affected with N348I or modestly improved with A360V. Both connection domain mutations can compensate for TAM-mediated deficits in processive DNA synthesis, and experiments with RNase H negative mutant enzymes confirm an RNase H-independent contribution to increased levels of resistance to AZT. Moreover, the combination of diminished RNase H cleavage and increased processivity renders the use of both PP(i) and ATP advantageous, whereas classic TAMs solely enhance the ATP-dependent reaction. Taken together, our findings demonstrate that distinct, complementary mechanisms can contribute to higher levels of excision of AZT, which in turn can amplify resistance to this drug.

UI MeSH Term Description Entries
D008969 Molecular Sequence Data Descriptions of specific amino acid, carbohydrate, or nucleotide sequences which have appeared in the published literature and/or are deposited in and maintained by databanks such as GENBANK, European Molecular Biology Laboratory (EMBL), National Biomedical Research Foundation (NBRF), or other sequence repositories. Sequence Data, Molecular,Molecular Sequencing Data,Data, Molecular Sequence,Data, Molecular Sequencing,Sequencing Data, Molecular
D009154 Mutation Any detectable and heritable change in the genetic material that causes a change in the GENOTYPE and which is transmitted to daughter cells and to succeeding generations. Mutations
D011485 Protein Binding The process in which substances, either endogenous or exogenous, bind to proteins, peptides, enzymes, protein precursors, or allied compounds. Specific protein-binding measures are often used as assays in diagnostic assessments. Plasma Protein Binding Capacity,Binding, Protein
D004279 DNA, Viral Deoxyribonucleic acid that makes up the genetic material of viruses. Viral DNA
D001483 Base Sequence The sequence of PURINES and PYRIMIDINES in nucleic acids and polynucleotides. It is also called nucleotide sequence. DNA Sequence,Nucleotide Sequence,RNA Sequence,DNA Sequences,Base Sequences,Nucleotide Sequences,RNA Sequences,Sequence, Base,Sequence, DNA,Sequence, Nucleotide,Sequence, RNA,Sequences, Base,Sequences, DNA,Sequences, Nucleotide,Sequences, RNA
D012367 RNA, Viral Ribonucleic acid that makes up the genetic material of viruses. Viral RNA
D015215 Zidovudine A dideoxynucleoside compound in which the 3'-hydroxy group on the sugar moiety has been replaced by an azido group. This modification prevents the formation of phosphodiester linkages which are needed for the completion of nucleic acid chains. The compound is a potent inhibitor of HIV replication, acting as a chain-terminator of viral DNA during reverse transcription. It improves immunologic function, partially reverses the HIV-induced neurological dysfunction, and improves certain other clinical abnormalities associated with AIDS. Its principal toxic effect is dose-dependent suppression of bone marrow, resulting in anemia and leukopenia. AZT (Antiviral),Azidothymidine,3'-Azido-2',3'-Dideoxythymidine,3'-Azido-3'-deoxythymidine,AZT Antiviral,AZT, Antiviral,BW A509U,BWA-509U,Retrovir,3' Azido 2',3' Dideoxythymidine,3' Azido 3' deoxythymidine,Antiviral AZT,BWA 509U,BWA509U
D015497 HIV-1 The type species of LENTIVIRUS and the etiologic agent of AIDS. It is characterized by its cytopathic effect and affinity for the T4-lymphocyte. Human immunodeficiency virus 1,HIV-I,Human Immunodeficiency Virus Type 1,Immunodeficiency Virus Type 1, Human
D016914 Ribonuclease H A ribonuclease that specifically cleaves the RNA moiety of RNA:DNA hybrids. It has been isolated from a wide variety of prokaryotic and eukaryotic organisms as well as RETROVIRUSES. Endoribonuclease H,RNase H,Ribonuclease H, Calf Thymus,RNAase H
D017434 Protein Structure, Tertiary The level of protein structure in which combinations of secondary protein structures (ALPHA HELICES; BETA SHEETS; loop regions, and AMINO ACID MOTIFS) pack together to form folded shapes. Disulfide bridges between cysteines in two different parts of the polypeptide chain along with other interactions between the chains play a role in the formation and stabilization of tertiary structure. Tertiary Protein Structure,Protein Structures, Tertiary,Tertiary Protein Structures

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