The HIV-1 Nef Protein Interacts with two components of the 40S small ribosomal subunit, the RPS10 protein and the 18S rRNA

To determine the effect of Nef on translation we used an in vitro translation assay. The detection of Nef/RPS10 complexes and the presence of 18S rRNA and tRNAs in the complexes were performed by coimmunoprecipitation and RT-PCR assay.We observed that the HIV-1 Nef protein specifically impaired translation in vitro. We observed the interaction of Nef with RPS10 by coimmunoprecipitation assay. In addition 18S rRNA and tRNAs were present in the Nef/RPS10 complexes.Our results are consistent with a model in which the Nef protein by binding to two components of the 40S small ribosomal subunit, RPS10 and 18S rRNA, and to a lesser extent to tRNAs, could lead to decreased protein synthesis.Ribosome biogenesis involves multiple coordinated steps: synthesis and processing of ribosomal RNA (rRNA), synthesis of ribosomal proteins and their import into the nucleus, the assembly of ribosome subunits, and the transport of the mature 40S and 60S subunits into the cytoplasm [1]. Ribosome biogenesis requires the synthesis, processing and assembly of several protein and RNA components. Structural RNA components of the ribosome include 5S, 5.8S, 18S and 28S rRNA [2]. The 18S, 5.8S and 28S rRNAs are encoded by ribosomal DNA (rDNA) that is organized as expanded chromosome loops in the nucleolus [3]. RNA polymerase I transcribes a single 47S rRNA precursor (pre-rRNA) which is subsequently processed through endonucleolytic and exonucleolytic cleavage into 18S, 5.8S and 28S rRNA [4,5]. Ribosomal proteins are synthesized in the cytoplasm by RNA polymerase II and then imported into the nucleus, where they are assembled into small and large ribosomal subunits [1,2]. The small ribosomal subunit contains one 18S rRNA and approximately 32 ribosomal proteins (known as RPS proteins). The large 60S subunit is composed of one of each 5S, 5.8S and 28S rRNA and approximately 47 ribosomal proteins (known as RPL proteins). The 40S and 60S subunits are then exported into the cytoplasm by exportin-1 and e