The AP-1 Binding Sites Located in the pol Gene Intragenic Regulatory Region of HIV-1 Are Important for Viral Replication
Figure 8
Viral particles from mutant and wild-type virus stocks are similar at the protein and at the RNA levels.
(A) Equivalent amounts of viral particles (as assessed by p24 ELISA assays) from the wild-type and each AP-1 mutant virus stocks were pelleted by centrifugation, lysed in Laemmli buffer and analyzed by Western blotting with an anti-HIV-1 immunoglobulin. The bands corresponding to the HIV-1 glycoprotein gp160, reverse transcriptase p66/p51, integrase p32 and capsid p24 proteins are indicated. MW, molecular weight (indicated in kDa). (B) Viral RNAs from equal amounts of viral particles were digested with DNase I and subsequently reverse-transcribed with random primers. First-strand viral cDNAs were then quantified by qPCR with primers hybridyzing in the TAR region (as described in the Materials and Methods section). An arbitrary value of 1 was assigned to the result obtained with the wild type virus stock HIV-1. Means and standard errors of the means from two independent experiments each performed in triplicate are indicated. (C) Mutations in the intragenic AP-1 binding sites affect HIV-1 expression. TZM-bl cells (6×103 cells) were infected or not with equal amounts of wild-type HIV-1 or mutant virus stocks. At 72 h post-infection, TZM-bl cells were lysed and luciferase activity was measured in cell lysates. Results are presented as histograms indicating the LuciferaseFirefly activity of the TZM-bl cells following infection with wild-type versus mutant viruses. An arbitrary value of 100% was attributed to the result obtained with the wild-type HIV-1. Means and standard errors of the means from one representative from three independent experiments each performed in triplicate are indicated. * indicates p<0.05 compared to the wild-type virus HIV-1.