3rd Conference on Retroviruses and Opportunistic Infections Washington, DC - January 28-February 1, 1996

Conf Retroviruses Opportunistic Infect 1996 Jan 28-Feb 1; 3rd:53 (abstract no. 3)

Sharma PL, Crumpacker CS
Beth Israel Hospital.

OBJECTIVES: To evaluate the effect of the drug related mutations on the rate of viral replication in human PBMC.

METHODS: A 4.3 kb Sphl-Sal1 fragment of plasmid pNL4-3 carrying the entire RT gene was cloned into the polycloning site of the phagemid pALTERTM-1. Site specific mutagenesis was carried out to obtain RT-variants Lys70Arg, Lys70Thr and Leu74Val. The Wild type pNL4-3 and mutated full length clones were transfected into PBMC cultures and viral replication was monitored by measuring HIV-1 p24 antigen and RT-activity. Stimulated PBMC (PHA, 72h), 6X10⁶ cells were infected with equivalent amounts of RT units and viral replication was monitored by analysing the virion associated RT activity for 7 days. The RT assay was performed using template-primer polyA-d(T) and substrate³H dATP.

RESULTS: The comparison of replication kinetics of HIV-1 variants is shown in Figure.

CONCLUSIONS: 1. The replication rate for WT pNL4-3 as well as Lys70Arg viruses were similar in drug free medium. 2. The virus carrying ddl related mutation Leu74Val replicated less efficiently in comparison to WT virus in drug free medium. 3. When RT codon 70 was substituted by amino acid Thr instead of Arg, the viral replication was impaired considerably.

960128
3

Copyright © 1996 - Foundation for Retrovirology and Human Health . Reproduction of this abstract (other than one copy for personal reference) must be cleared through the Foundation for Retrovirology and Human Health.