Background:  HIV-1 intracellular reverse transcription complexes (RTC) are actively imported into the cell nucleus, but little is known on the cellular pathways involved in this process.

Methods:  To identify cellular factors required for their nuclear import, RTC were purified from acutely infected cells by sedimentation velocity centrifugation followed by density fractionation in sucrose gradients, Purified RTC were labelled fluorescently and used as substrate in the nuclear import assay. In this assay, cells are permeabilized with digitonin, which dissolves the plasma membrane but leaves the nuclear envelope intact. Nuclear import is then reconstituted by the addition of the labelled substrate, energy, the Ran system and import factors.

Results:  High-speed cytosolic extracts were fractionated by stepwise precipitation in ammonium sulfate. Nuclear import activity was recovered in the1.4M pellet, which contains importin 7 and Ran, and in the 2.4M supernatant. This supernatant was subjected to hydrophobic interaction and ion exchange chromatography resulting in a near-homogeneous fraction. Surprisingly, this fraction maintained nuclear import activity after treatment with proteinase K but lost it if treated with micrococcal nuclease. Further analyses revealed that the active fraction was sensitive to RNAse (DNAse free) digestion and could be end-labelled with T4 RNA ligase. The labelled fraction was examined in urea-denaturing PAGE. Eight distinct small RNA bands were detectable ranging in size from 140 to 20 bases. Size fractionation indicated that only the some 90 nucleotides band was active in RTC nuclear import and required in addition energy. The active RNA species have been cloned and appear to be made of several different tRNA-like molecules, the most abundant being tRNA-lys. Only a proportion of the cloned RNA molecules stimulated RTC nuclear import and they accumulated efficiently into the nuclei even in the absence of added RTC. Deletion and point mutation analyses revealed that the anticodon and D-loops are required for RTC recognition and nuclear import.

Conclusions:  These results indicate that tRNA-like molecules may be involved in HIV-1 RTC nuclear import and may represent a new class of cellular ribonucleoproteins.

Keywords: HIV-1; reverse transcription complex; nuclear transport