Sharma PL, Chatis PA, Dogon AL, Crumpacker CS; National Conference on Human Retroviruses and Related Infections.
Program Abstr Second Natl Conf Hum Retrovir Relat Infect Natl Conf Hum Retrovir Relat Infect 2nd 1995 Wash DC. 1995 Jan 29-Feb 2; 139.
Harvard Medical School, Boston, MA.
Objectives: To define the mutations and related drug susceptibility, observed in clinical viral isolates exhibiting resistance to AZT and ddI. Methods: A 4.3 kb Sph1-Sal1 fragment of plasmid pNL4-3 carrying the entire RT gene was cloned into the polycloning site of the phagemid pALTERTM-1 (Promega). The site specific mutagenesis was carried out to obtain the 70 Lys-Arg and 65 Lys-Glu genotype in RT gene. Then the mutated fragment was exchanged with WT fragment of pNL4-3. The Wild type control and mutated full length plasmids were transfected in PBMC cultures and viral replication was monitored by measuring HIV-1 p24 antigen and reverse transcriptase activity. The virus obtained was tested for sensitivity to AZT and ddI. Results: After 3 weeks of culture virus was efficiently produced in cell lines transfected with both control and mutated plasmids (Lys70Arg). The drug susceptibility assays showed that mutation at codon 70 confers 6-fold decrease in susceptibility to AZT and a 2-fold decrease in susceptibility to ddI. Interestingly, the mutant virus containing the Lys65Glu mutation replicated poorly in PBMC cultures and drug susceptibility assay could not be performed due to insignificant amount of virus. Conclusions: 1) Lys70Arg mutation confers resistance to AZT and decreases susceptibility to ddI. 2) Lys65Glu mutation significantly impairs the ability of the HIV-1 virus to replicate in PBMC culture.
Publication Types:
Keywords:
- Codon
- Didanosine
- Genotype
- Glucose
- Glutamic Acid
- HIV-1
- Mutation
- RNA-Directed DNA Polymerase
- Zidovudine
- genetics
- therapy
Other ID:
UI: 102213435
From Meeting Abstracts