AEGiS-15IAC: A new strategy based on recombinant viruses as a tool for the study of replicative capacity and determinants of viral fitness.

15th International AIDS Conference


Bangkok, Thailand - July 11-16, 2004

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A new strategy based on recombinant viruses as a tool for the study of replicative capacity and determinants of viral fitness.

Int Conf AIDS 2004 Jul 11-16; 15:(abstract no. WeOrA1273)

Alcami J, Garcia-Perez J, Sanchez-Palomino S, Gonzalez N, Fernandez B
ISCIII, Madrid, Spain

OBJECTIVE: To develop a system based on recombinant viruses harbouring reporter genes to analyse the replicative capacity of resistant viruses and the determinants of viral fitness. Material and

METHODS: The following modifications were introduced in the NL4.3 clone: cloning of renilla luciferase in the place of nef and the LacZ gene in different positions of the pol gene encompassing reverse transcriptase or protease sequences (LacZpol, LacZRT or LacZPr). Viral RNA was extracted from plasma of 10 heavily treated HIV patients in therapeutic failure and 2 naïve patients. cDNA was generated by reverse transcription. Full length pol gene, RT or Pr sequences were amplified by PCR, sequenced and used for generation of recombinant viruses (RV). For each patient were generated: RVpol, RVPr, and RVRT. Viral progeny was obtained through transfection in 293-t cells and normalysed by luciferase activity. MT-2 cells were infected with the different stocks and luciferase activity monitorised in cell lysates. Luciferase results from infection with mutated viruses were compared with wild-type viruses generated with the same strategy.

RESULTS: Renilla luciferase vectors produced multiple cycles of HIV replication in culture. A strong decrease (>90%) in the replicative capacity of RVpol population was observed in 6/10 heavily treated patients in virological failure as compared to wild type RV. The analysis of RV from these patients showed that the replicative capacity of RV containing RT sequences was particularly decreased in 4 of 6 patients whereas fitness was less reduced in RV harbouring mutated PR sequences. In the two remaining patients both cloned RT and PR sequences reduced the replicative capacity of RV at similar levels. Conclusion A new model to study the determinants of the replicative capacity of resistant viruses is described. In heavily-treated patients mutations in which viral fitness is severily decreased RT mutations are major determinants of this phenotype.

Keywords: AEGIS, HIV-1 Reverse Transcriptase, Luciferases, Renilla, Reverse Transcription, HIV Protease, Anti-HIV Agents, Virus Replication, HIV-1, HIV Protease Inhibitors, Acquired Immunodeficiency Syndrome, HIV Infections, Genes, pol, HIV Core Protein p24, HIV, RNA-Directed DNA Polymerase, Polymerase Chain Reaction, Humans, virology, genetics, immunology

040711
WeOrA1273

Copyright © 2004 - International AIDS Society (IAS). Reproduction of this abstract (other than one copy for personal reference) must be cleared through the IAS.