249 Caveolin-1 Blocks HIV-1 Expression Post-transcriptionally Manuel Llano*, Mary Peretz, Maria Vanegas, Eric Poeschla Mayo Clin Rochester, MN
Background: We previously reported that co-expression of the raft-organizing protein Caveolin-1 (Cav) specifically blocks virion production from cells co-transfected with HIV-1 proviral DNA. The effect mapped to the central membrane-associated domain of Cav, but the affected part of the HIV-1 productive phase was not established.
Methods: pCMV.Cav-1, or control DNA, was co-transfected into 293T cells with HIV-1 proviral DNA and supernatant RT was determined. Separately, pCMV.Cav-1 was co-transfected with luciferase reporters containing the HIV-1 LTR or other promoters. Total, nuclear, and cytoplasmic RNA fractions were analyzed by northern blotting. HIV-1 and reporter transcription rates were determined in nuclear run-on assays. Finally, HIV-1 and reporter mRNA half-lives were established by northern blotting after actinomycin-D treatment.
Results: Northern blotting showed that co-expression of Cav and HIV-1 reduced steady state spliced and unspliced HIV-1 mRNA levels with dose-dependence that correlated with inhibition of HIV-1 protein expression. Cav also inhibited luciferase expression directed by the HIV-1 LTR or the SV40 promoter. In contrast, the hCMV IE promoter, pol I and pol III promoters and the expression of endogenous mRNAs were not inhibited. Inhibition of HIV-1 LTR (basal or +Tat) and SV40 promoter-dependent expression involved reduced steady-state luciferase mRNA levels. Control protein or membrane domain-deleted Cav co-expression produced none of these effects. Nuclear run-on experiments with HIV-1 LTR reporters and HIV proviruses then showed that reduced transcription was not involved, indicating a post-transcriptional mechanism. Subcellular fractionation revealed that Cav co-transfection reduced cytoplasmic, but not nuclear viral mRNA levels. Consistent with this, Cav-expressing cells exhibited increased cytoplasmic HIV-1 mRNA decay rates, which affected spliced and unspliced viral mRNAs equivalently. In contrast, half-lives of LTR- or SV40-promoted luciferase mRNAs were unaltered.
Conclusions: Cav inhibits HIV-1 expression post-transcriptionally by inducing accelerated cytoplasmic decay of HIV-1 mRNAs. A separable inhibition of isolated LTR-directed expression does not involve accelerated mRNA decay, but is also post-transcriptional. Synergy of the 2 post-transcriptional effects results in the profound inhibition observed.
