Rev-dependent association of the intron-containing HIV-1 gag mRNA with the nuclear actin bundles and the inhibition of its nucleocytoplasmic transport by latrunculin-B.

BACKGROUND

A hallmark of HIV-1 gene expression is that unspliced genomic RNA, which also acts as mRNA for the expression of Gag/Pol, is exported to the cytoplasm. Rev directs this transport through the nuclear export signal (NES).

RESULTS

Fluorescence in situ hybridization and immunocytochemistry demonstrated that gag mRNA, Rev, and its NES receptor, CRM1, and RanGTPase formed nuclear tracks which were congruent with underlying beta-actin bundles. Actin bundle formation was confirmed electron-microscopically. These bundles were observed upon Rev-containing gag RNP formation. The loss of bundles was associated with the nuclear retention of gag mRNA. Reverse transcription-polymerase chain reaction analysis of both cytoplasmic and nuclear gag mRNAs demonstrated that disruption of nuclear actin filament formation by latrunculin-B (LAT-B), an F-actin depolymerizing compound, resulted in the dose-dependent inhibition of gag mRNA export. The differential subtyping of the mRNA-positive cells confirmed morphologically the effect of LAT-B treatment. The export inhibition was specific to gag mRNA and export of fully spliced HIV-1 tat/rev mRNAs as well as cellular GAPDH mRNA was not affected by the compound.

CONCLUSIONS

Nuclear beta-actin bundles are suggested to be functionally involved in the Rev-dependent nucleocytoplasmic transport of intron-containing HIV-1 gag mRNA.