Novel nuclear import of Vpr promoted by importin alpha is crucial for human immunodeficiency virus type 1 replication in macrophages.

Monocytes/macrophages are major targets of human immunodeficiency virus type 1 (HIV-1) infection. The viral preintegration complex (PIC) of HIV-1 enters the nuclei of monocyte-derived macrophages, but very little PIC migrates into the nuclei of immature monocytes. Vpr, one of the accessory gene products of HIV-1, is essential for the nuclear import of PIC in these cells, although the role of Vpr in the entry mechanism of PIC remains to be clarified. We have shown previously that Vpr is targeted to the nuclear envelope and then transported into the nucleus by importin alpha alone, in an importin beta-independent manner. Here we demonstrate that the nuclear import of Vpr is strongly promoted by the addition of cytoplasmic extract from macrophages but not of that from monocytes and that the nuclear import activity is lost with immunodepletion of importin alpha from the cytoplasmic extract. Immunoblot analysis and real-time PCR demonstrate that immature monocytes express importin alpha at low levels, whereas the expression of three major importin alpha isoforms markedly increases upon their differentiation into macrophages, indicating that the expression of importin alpha is required for nuclear import of Vpr. Furthermore, interaction between importin alpha and the N-terminal alpha-helical domain of Vpr is indispensable, not only for the nuclear import of Vpr but also for HIV-1 replication in macrophages. This study suggests the possibility that the binding of Vpr to importin alpha, preceding a novel nuclear import process, is a potential target for therapeutic intervention.

Mutation effects of a conserved alanine (Ala510) in type I polyhydroxyalkanoate synthase from Ralstonia eutropha on polyester biosynthesis.

Type I polyhydroxyalkanoate (PHA) synthases, as represented by Ralstonia eutropha enzyme (PhaC(Re)), have narrow substrate specificity toward (R)-3-hydroxyacyl-coenzyme A with acyl chain length of C3-C5 to yield PHA polyesters. In this study, saturation point mutagenesis of a highly conserved alanine at position 510 (A510) in PhaC(Re) was carried out to investigate the effects on the polymerization activity and the substrate specificity for in vivo PHA biosynthesis in bacterial cells. A series of saturation mutants were first applied for poly[(R)-3-hydroxybutyrate] homopolymer synthesis in Escherichia coli and R. eutropha PHB(-)4 (PHA negative mutant) cells to assess the polymerization activity. All mutants showed quantitatively similar polymerization activities when R. eutropha PHB(-)4 was used for assay, whereas several mutants such as A510P showed low activities in E. coli. Further analysis has revealed that majority of mutants synthesize polyesters with higher molecular weights than the wild-type. In particular, substitution by acidic amino acids, A510D(E), led to remarkable increases in molecular weights. Subsequently, PHA copolymer synthesis from dodecanoate (C12 fatty acid) was examined. The copolymer compositions were varied depending on the mutants used. Significant increased fractions of long monomer units (C6 and C8) in PHA copolymers were observed for three mutants [A510M(Q,C)]. From these results, the mutations at this potion are beneficial to change the molecular weight of polyesters and the substrate specificity of PhaC(Re). Molecular weight distributions of PHA polymers synthesized by the wild-type enzyme (PhaC(Re)) and its mutants.