Inhibition of HIV-1 gene expression by Sam68ΔC: multiple targets but a common mechanism?

Initial experiments [7] identified Sam68ΔC as a dominant inhibitor of HIV-1 replication. While subsequent work determined that inhibition was dependent upon the cytoplasmic localization of Sam68ΔC and associated with formation of cytoplasmic granules around the outside of the nuclear envelope [8], the underlying mechanism remained unclear. However, the recent work of Marsh et al. [6] provided some detail as to the mechanism. Using various expression vectors, they showed that Sam68ΔC selectively inhibited mRNA expressing Gag exported via the exportin-1 pathway, with little to no effect on the same Gag coding sequence delivered to the cytoplasm by Nxf1. Co-expression of Sam68ΔC and an unspliced Env expressor resulted in translation inhibition of the latter and disruption of the cytoplasmic bundles failed to restore expression of the encoded protein. Rather, despite a normal polyA tail, inhibition by Sam68ΔC was attributed to a block in translation of the affected RNA due to reduced binding of PABP-1 (Fig. 1A). The ability of Sam68ΔC to selectively affect only those RNAs exported in a Rev- and exportin-1-dependent fashion suggested that it recognizes some features unique to the mRNPs exported by this pathway. In parallel work by Henao-Mejia et al. [5] and consistent with Marsh et al., it was shown that constructs functionally similar to Sam68ΔC had the capacity to repress Rev-dependent protein expression. Surprisingly, inhibition of Rev-independent Nef synthesis was also observed with little or no alteration in Tat or Rev levels. Given that these three proteins are expressed from multiply spliced HIV-1 RNAs that all use the Nxf1 export pathway (Fig. 1A), selective repression of Nef expression may require a different mechanism than that outlined by Marsh et al. Inhibition of Nef expression was reported to be associated with the accumulation of nef mRNA in cytoplasmic granules that co-stained with markers of stress granules (SGs); these observations led Henao-Mejia et al. to suggest that reduced Nef synthesis was due to sequestration in these bodies. At present, it is unclear whether the granules characterized by Henao-Mejia et al. are similar or distinct from those formed by Sam68ΔC and incompletely spliced HIV-1 RNAs and whether Sam68ΔC inhibition of Nef synthesis is dependent upon their integrity. The two studies suggest that, while the route different RNAs take to repressive sites can differ (the Exportin1 pathway for Rev-dependent RNAs versus the Nxf1 pathway for nef mRNA), a similar mechanism may underlie repression of HIV-1 structural protein and nef mRNAs by Sam68ΔC. However, whether the mechanism is simple sequestration in SGs or something more complex remains to be determined. This is based on the observation of Marsh et al. that RRE-containing RNAs are still repressed upon dispersal of Sam68ΔC granules, although dispersion into functional "nano" granules cannot be dismissed and should be investigated. In addition, ongoing studies (Marsh and Cochrane, unpublished) showing that Sam68ΔC-induced granules contain mRNAs whose expression is not repressed suggest that sequestration to such granules alone is insufficient to explain translational repression. Consequently, additional experiments are needed to assess whether common or distinct mechanisms underlie repression of HIV-1 structural protein and nef mRNAs by Sam68ΔC.