The screening of a Jurkat T cell cDNA library, using the yeast two-hybrid selection and HIV-1Rev as a bait protein, led to the isolation of two new Rev-interacting cDNA-encoded proteins (11.5.1 and 16.4.1). The computer analysis of available sequence databases revealed a 100% identity of the 11.5.1 cDNA with the C-terminal part of a previously reported human nuclear single strand DNA binding protein (Sakura et al., 1988). A partial sequence identity of 16.4.1 cDNA was observed with two previously identified human genes with unknown function: a cDNA clone from the EST database (ref) and the 3’end of a large brain-derived cDNA clone (Nagase et al., 1998).
The subsequent study of the 16.4.1 clone led to the characterization of one fragment of a larger protein, consisting of 171 amino acids that interacted specifically with Rev. Because of its ability to shuttle between the nucleus and the cytoplasm it was termed Risp (Rev-interacting shuttle protein).
The yeast two-hybrid system has revealed a number of useful applications (Allen et al., 1995), including the capacity to isolate novel genes encoding proteins that associate with a known protein of interest. Previous studies have already successfully used Rev or its activation domain in the yeast two-hybrid selection to identify Rev- interacting proteins (table 2.3) (Bogerd et al., 1995; Farjot et al., 1999; Fritz and Green, 1996; Fritz et al., 1995; Luo et al., 1994; Neville et al., 1997; Stutz et al., 1995). The two-hybrid system is able to detect in vivo binding that is often undetectable in vitro by methods such as immunoprecipitation. In fact, the two-hybrid system gives very sensitive results. First of all, the hybrid-proteins are generally over-expressed by strong promoters present in high-copy-number plasmids, which favor a complex formation. In addition, the stability of the hybrid-protein complex is probably enhanced by the interaction of the activation domain with proteins from the transcriptional initiation complex, which also associate with the promoter DNA. Therefore, the overall stability of the complex on DNA further increases the sensitivity of detection of the bait-prey protein interaction.
However, the two main obstacles preventing the success of the yeast two-hybrid system are the activation of transcription of the reporter gene by the bait protein itself and the failure to express the bait properly. In our case, the Rev protein could be proven to be a suitable bait, since it was not able to activate the transcription of the reporter genes LEU2 and lacZ without the presence of the B42 activation domain.
Moreover, the expression of the Rev protein fused to the binding domain LexA was demonstrated by western blot analysis using a Rev-specific antibody.
Another major problem of the yeast two-hybrid system is the risk to detect “false positive” interactors. In fact, some proteins contain regions that have low affinities for many different proteins and therefore may form stable complexes with several bait proteins, which result in an unspecific detection.
To limit the number of “false positive” interactions, a dual screen selection based on the use of two distinct reporter genes, lacZ and LEU2 was performed. Moreover, for additional controls the screening was performed in presence of the testing bait (Rev sense) and two unspecific baits (Rev antisense and Lexcd2). While the lacZ expression provided a detectable blue color phenotype on plates containing Xgal, the LEU2 expression allowed a nutritional selection on plates lacking leucine. It is known that the requirement for the expression of two reporter genes driven by different promoters significantly reduces the number of false-positive signals, since separate transcriptional events must be maintained at distinct chromosomal loci.
The screening of a Jurkat T cell cDNA library led us to sequence 6 yeast clones: two were assumed to be clear Rev-interacting (11.5.1 and 16.4.1) and 4 “putative” Rev- interacting clones (6.2.1, 11.2.1, 15.2.2 and 16.3.15) (see tables 3.5 and 3.6).
The sequence analysis and the subsequent BLAST search with available sequence databases suggested that 3 out of the 4 “putative” clones were “false positive”. In fact they were very similar (85%) or 100% identical to proteins generally known as common false positive in the yeast two-hybrid system (i.e. ribosomal proteins, zinc finger containing proteins or heat shock proteins) (Hengen, 1997). The “putative” clone 16.3.15 was 100% identical with a HLA-DR associated protein (924bp) and the phosphatase 2A inhibitor I2PP2a (833 bp).
I2PP2a is a truncated form of SET, a largely nuclear protein that is fused to nucleoporin Nup214 in acute non-lymphocytic myeloid leukemia (Li et al., 1996). However, even though this clone could be of certain interest - Nup214 is one of the nucleoporins known to interact with Rev (Zolotukhin and Felber, 1999)- in this work the other two Rev-interacting clones were further characterized for several reasons. First, the yeast clone 16.3.10 (representing the same cDNA-encoded protein of 16.3.15) showed clearly unspecific binding to Rev. Second, the two clones 11.5.1 and 16.4.1 showed a certain specific binding to Rev. Third, the absence of homology of the clone 16.4.1 to any proteins with known function.
It is worth noting that in our screen none of the known Rev-interacting proteins were detected, maybe because of the insufficient number of analyzed clones.