Nef Alleles from All Major HIV-1 Clades Activate Src-Family Kinases and Enhance HIV-1 Replication in an Inhibitor-Sensitive Manner
Figure 3
Purified primary Nef proteins bind to the Hck SH3 domain and activate downregulated Hck in vitro.
A) SDS-PAGE of recombinant purified proteins. Each of the indicated Nef subtypes were expressed in bacteria and purified with N-terminal His-tags (left). GST and GST fusion proteins with the Hck SH3 domain (wild-type and W93A mutant) were also expressed in bacteria and immobilized on glutathione-agarose beads (right). Protein aliquots were resolved by SDS-PAGE and stained with Coomassie blue. B) Nef-SH3 binding assay. Recombinant His-tagged Nef proteins (1 µg) were incubated with equimolar amounts of immobilized GST and GST-SH3 fusion proteins. The agarose beads were then washed, and associated Nef proteins were detected by immunoblotting using antibodies to the His-tags. C) Nef-induced Hck activation. Kinase activity of recombinant downregulated Hck (Hck-YEEI; [44], [52], [53] was determined in the absence or presence of the indicated Nef proteins using the Z-Lyte assay as described under Materials and Methods. Results are expressed as the mean percent of maximum substrate phosphorylation ± S.D.; this experiment was repeated twice with comparable results.