RESUMO
<p><b>OBJECTIVE</b>To compaire results of recombinant virus assay and live virus assay on evaluateing anti-HIV-1 drugs.</p><p><b>METHODS</b>The pseudoviruse was generated by cotransfection of the plasmid B01 containing gp160 genes and pSG3 delta env plasmid. After co-incubation of pseudovirus with serially diluted drug, the EC50 and ED50 were calculated according to RLU(relative light unit) for each drug. After co-incubation of live virus with serially diluted drug, the EC50 was calculated according to cytopathic effect.</p><p><b>RESULTS</b>EC50 of IDV measured by the recombinant virus assay and live virus assay was 88.9 nmol/L, 89.5 nmol/L, respectively, while EC50 of NVP measured by the recombinant virus assay and live virus assay was 0.36 micromol/L, 0.23 micromol/L, respectively. The recombinant virus assay showed good reproducibility with coefficient variation of 0, however coefficient variation of live virus assay reached to 60%. ED50 of IDV and NVP measured by the recombinant virus assay were 70.6 nmol/L and 0.62 micromol/L, respectively. Coefficient variations for IDV and NVP were 14.3% and 9.7%, respectively.</p><p><b>CONCLUSION</b>The pseudoviruses could be used in evaluating anti-HIV-1 drugs. The recombinant virus assay showed good reproducibility and could calculate not only the EC50 but also the ED50 of drugs.</p>
Assuntos
Fármacos Anti-HIV , Farmacologia , Avaliação de Medicamentos , HIV-1 , Recombinação GenéticaRESUMO
To find out whether the mutations of HIV-1 Env have influence on the assembly of pseudovirus and their abilities to infect cells, site-directed mutation (A457D)was performed using cycling mutagenesis and selection of mutants with DpnI. Transformation and plasmid purification technologies were used to obtain mutated env clone. Then both the prototype and the mutant were co-transfected with pSG3(delta(env)) to 293FT cells, respectively. Single-cycle infection assay was employed to analyze the effect of the prototype and the mutant on the ability of functional pseudovirus assembly. The transient expression of both the prototype S12-42-1 and mutant S12-42M were confirmed by Western blot essay. The S/CO value was less than 1 for S12-42-1 and 6.65 for S12-42M, demonstrating the functional pseudovirus was generated only for S12-42M. So mutation on HIV-1 Env has influence on the assembly of pseudovirus and their abilities to infect cells.