Development and application of a DNA microarray-based yeast two-hybrid system.

The yeast two-hybrid (Y2H) system is the most widely applied methodology for systematic protein-protein interaction (PPI) screening and the generation of comprehensive interaction networks. We developed a novel Y2H interaction screening procedure using DNA microarrays for high-throughput quantitative PPI detection. Applying a global pooling and selection scheme to a large collection of human open reading frames, proof-of-principle Y2H interaction screens were performed for the human neurodegenerative disease proteins huntingtin and ataxin-1. Using systematic controls for unspecific Y2H results and quantitative benchmarking, we identified and scored a large number of known and novel partner proteins for both huntingtin and ataxin-1. Moreover, we show that this parallelized screening procedure and the global inspection of Y2H interaction data are uniquely suited to define specific PPI patterns and their alteration by disease-causing mutations in huntingtin and ataxin-1. This approach takes advantage of the specificity and flexibility of DNA microarrays and of the existence of solid-related statistical methods for the analysis of DNA microarray data, and allows a quantitative approach toward interaction screens in human and in model organisms.

Molecular determinants of multi-nucleoside analogue resistance in HIV-1 reverse transcriptases containing a dipeptide insertion in the fingers subdomain: effect of mutations D67N and T215Y on removal of thymidine nucleotide analogues from blocked DNA primers.

Human immunodeficiency virus type 1 isolates having dipeptide insertions in the fingers subdomain of the reverse transcriptase (RT) show high level resistance to 3 '-azido-3 '-deoxythymidine (AZT) and other nucleoside analogues. Insertions are usually associated with thymidine analogue resistance mutations, such as T215Y. The resistance phenotype correlates with increased ATP-dependent phosphorolytic activity, which facilitates removal of thymidine analogues from inhibitor-terminated primers. In this report, we show that substituting Thr, Ser, or Asn for Tyr-215 in a multidrug-resistant RT, bearing a Ser-Ser insertion between codons 69 and 70, leads to AZT and stavudine resensitization through the loss of the ATP-mediated removal activity. The mutation D67N, which is rarely found in insertion-containing strains, had no effect on excision and a minor influence on resistance. Substituting Tyr-215 had a larger effect than deleting the dipeptide insertion. The presence of both the insertion and mutation T215Y in the wild-type BH10 RT conferred significant ATP-mediated removal activity and moderate resistance to AZT. However, resistance levels and unblocking activities were lower than those observed with the multidrug-resistant enzyme. Removal reactions can be inhibited by the next complementary dNTP. Both Tyr-215 and the dipeptide insertion affect RT-DNA.DNA-dNTP ternary complex formation, an effect that was not detected in the presence of foscarnet. Based on crystal structures of binary and ternary complexes of HIV-1 RT, we propose that Tyr-215 exerts its action by facilitating a proper orientation of the pyrophosphate donor molecule, whereas the effects on dNTP binding are indirect and could be related to significant conformational changes occurring during polymerization.

Multidrug-resistant HIV-1 reverse transcriptase: involvement of ribonucleotide-dependent phosphorolysis in cross-resistance to nucleoside analogue inhibitors.

Human immunodeficiency virus type 1 (HIV-1) strains having a dipeptide insertion between codons 69 and 70 of the viral reverse transcriptase (RT) have been observed in isolates from patients treated with 3'-azido-3'-deoxythymidine (AZT) and other nucleoside analogues. These viruses contain additional mutations related to drug resistance and display reduced susceptibility to most nucleoside analogue inhibitors, including AZT. The mechanism of AZT resistance implies an increased ability of the multidrug-resistant (SS) RT to remove AZT-monophosphate (AZTMP) from blocked primers through a nucleotide-dependent reaction. We show that its higher ATP-dependent phosphorolytic activity is also detectable with primers terminated with 2',3'-didehydro-3'-deoxythymidine-5'-monophosphate (d4TMP) or 2',3'-dideoxythymidine-5'-monophosphate (ddTMP), but is significantly reduced when the dipeptide insertion is deleted. Removal of AZTMP, d4TMP and ddTMP can be inhibited by the next complementary deoxynucleoside triphosphate (dNTP). AZTMP removal reactions catalysed by SS RT were highly resistant to dNTP inhibition (IC(50)>0.25mM), while unblocking of d4TMP- and ddTMP-terminated primers was around tenfold more sensitive to inhibition by the next complementary dNTP. Both SS and mutant 2S0S RTs were able to unblock and extend primers terminated with 2',3'-dideoxycytidine-5'-monophosphate (ddCMP) in the presence of ATP, albeit very poorly. Under these conditions, none of the RTs was able to remove 2',3'-dideoxy-3'-thiacytidine-5'-monophosphate (3TCMP) from a terminated DNA primer. Resistance mediated by ATP-dependent phosphorolysis depends on the intracellular levels of dNTP. High levels as found in transformed cell lines (i.e. H-9, CEM lymphoblasts, SupT1 cells, etc.) may prevent repair of primers terminated with d4TMP. However, ATP-dependent phosphorolysis could be relevant for d4T resistance in cells having low levels of dNTPs. This proposal could explain why insertion-containing HIV-1 variants have been detected in the absence of AZT, during d4T treatment.