Figure 5: Downmodulation of CIB proteins does not impair late steps in the HIV-1 replication cycle.
From: CIB1 and CIB2 are HIV-1 helper factors involved in viral entry
HeLa cells were transduced with lentiviral vectors inducing the expression of the indicated shRNAs and transduced cells were selected by culture in medium with increasing concentrations of puromycin. Total RNA was extracted and cDNA was synthesized for quantification by quantitative real-time PCR of mRNA levels for CIB1 (A) or CIB2 (B), as described in Fig. 1 legend. Results are expressed relative to those obtained for non-transduced (NT) HeLa cells. Values represent mean ± SEM of 2 independent transductions. (C) The transduced HeLa cells were transfected with 1 μg of pHIV-1NL4-3, and 24 h later p24 levels were quantified by ELISA in both cell supernatants and cell lysates. Relative viral release was calculated by dividing the amount of p24 in the supernatant by the total amount of p24 (supernatant + cell lysate). Values represent mean ± SEM of 3 independent transfections and are expressed relative results obtained for non-transduced (NT) cells. (D) Culture supernatants recovered from transfected HeLa populations containing the indicated amounts of p24 were used to infect HeLaP4C5 cells, which carry the β-galactosidase gene under the control of the HIV-1 LTR. Tat-inducible β-galactosidase activity was quantified by colorimetric measurement of metabolized CPRG substrate. Values represent mean ± SEM of 3 independent infections, each done in triplicate and are expressed relative to results obtained for non-transduced (NT) cells.
