The AP-1 Binding Sites Located in the pol Gene Intragenic Regulatory Region of HIV-1 Are Important for Viral Replication
Figure 9
The AP-1 binding sites located in the 5103 fragment are important for viral replication.
Jurkat (A and C) or U937 (B and D) cells were infected with equivalent amounts of p24 concentration of wild-type (HIV-1), totally mutated (HIV-1-AP-1totmut), partially mutated (HIV-1-AP-1#1+2mut) or individually mutated (HIV-1-AP-1#1mut, HIV-1-AP-1#2mut and HIV-1-AP-1#3mut) viral infectious stocks as described in the Materials and Methods section. (A and B) Viral production in cell supernatants was quantified by measuring p24 antigen concentration in the culture supernatants at different times following infection. The experiment shown is representative of at least 3 independent infection experiments (each performed in triplicate). The means are presented and the variation for a given mutant between different experiments was <15% in each case. (C and D) Total RNA was extracted from infected cells at days 5, 10 and 15 post-infection, digested with DNase I and reverse-transcribed using random primers. First-strand cDNAs were analyzed by qPCR with the comparative Ct (ΔΔCt) quantification method using the two following sets of primers: initiated transcripts (TAR primers) and elongated transcripts (Tat primers) were quantified using β-actin to normalize the results. Means and standard errors of the means from two independent experiments each performed in triplicate are indicated. * and ** indicate p<0.05 and p<0.1 compared to the wild-type virus HIV-1 at day 15 post-infection.