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Background:  Genetic libraries, in particular random peptide libraries are a powerful tool for the study and interference of signaling pathways and for drug discovery. Random peptides possess shapes that bind to biological surfaces and interfere with normal signaling cascades. Using a screen-based HIV-1 infectivity, one can envision the existence of shapes that will decrease or block viral infection.

Methods:  We have generated random peptide libraries, of linear and constrained structures, for cytosolic expression in mammalian cells. These libraries were transferred into Supt-1 cells using retroviral vectors for stable expression of the peptides. The library-expressing cells were then subjected to viral infection to screen for peptides that block HIV-1 infection. Instead of the wild type HIV we used a surrogate particle produced in trans by a lentiviral self-inactivating transfer vector carrying a CMV promoter-green fluorescence protein cassette as tracking device for infection. The cells were sorted by FACS and the non-fluorescent cells were collected and amplified. This process was repeated eight times. Individual clones were amplified and their peptide sequence rescued by PCR.

Results:  We rescued by PCR the peptide sequence integrated into the genome of clonal cell populations. Some of the sequences were reintroduced into naïve cells to corroborate infection blockade. Peptide candidates were synthesized as GST-fusion proteins for pull-down experiments. One of the peptides has been shown to interact with the signalosome complex through the JAB-1/subunit 3. Interestingly this complex has previously been shown in our laboratory to be implicated in HIV-1 infection.

Conclusions:  We rescued several peptides that interfere with viral infection. One of the peptides binds the JAB-1/subunit 3 of the signalosome. The use of random peptide libraries can be efficiently applied in the search for novel molecules with pharmacologic properties against HIV-1 and other infectious pathogens.

Keywords: peptide libraries; antiretroviral therapy; pharmacophore