Traptamer screening: a new functional genomics approach to study virus entry and other cellular processes

Abstract

Historically, the genetic analysis of mammalian cells entailed the isolation of randomly arising mutant cell lines with altered properties, followed by laborious genetic mapping experiments to identify the mutant gene responsible for the phenotype. In recent years, somatic cell genetics has been revolutionized by functional genomics screens, in which expression of every protein-coding gene is systematically perturbed, and the phenotype of the perturbed cells is determined. We outline here a novel functional genomics screening strategy that differs fundamentally from commonly used approaches. In this strategy, we express libraries of artificial transmembrane proteins named traptamers and select rare cells with the desired phenotype because, by chance, a traptamer specifically perturbs the expression or activity of a target protein. Active traptamers are then recovered from the selected cells and can be used as tools to dissect the biological process under study. We also briefly describe how we have used this new strategy to provide insights into the complex process by which human papillomaviruses enter cells.

Retromer stabilizes transient membrane insertion of L2 capsid protein during retrograde entry of human papillomavirus

“…Because the first four traptamers we isolated inhibit entry at different steps, it is likely that we can isolate additional inhibitory traptamers with other sites of action. It should be possible to design other selection strategies to isolate traptamers that perturb a wide variety of cellular processes, including entry of other viruses ( 37 ). Traptamer screening may allow the interrogation of proteins required for cell viability, which are difficult to identify by knockdown/knockout screening, because a traptamer might modulate an activity of a cellular protein required for the studied process without disrupting other essential activities of the protein.…”

“…To further characterize HPV entry, we developed a functional genetics screen to isolate artificial TM proteins that modulate the activity of natural TM proteins ( 36 , 37 ). In this approach, we construct libraries that express millions of proteins containing a 15- to 26-residue stretch of randomized hydrophobic amino acids and screen these libraries in cells to select rare proteins that by chance bind to the TM domain of a cellular or viral protein and modulate its activity.…”

Recent Advances in Our Understanding of the Infectious Entry Pathway of Human Papillomavirus Type 16

“…As stated before, retromer complex binding after L2 pierces through the membrane is required to stabilize the membrane insertion of L2 [96]. Thus, infection can be blocked at this step using cell-penetrating peptides derived from the CPP and a retromer binding moiety from L2 or by the aforementioned traptamers that interfere with retromer association with L2 [67,104]. Complicating this part of the trafficking picture a bit more is the recent finding that L2 interacts with both the retromer and retriever complexes.…”

“…These proteins are comprised of primarily hydrophobic amino acids and are small, less than 25 residues in length. They are called "traptamers", short for "transmembrane protein aptamers" [67]. The use of traptamers in HPV entry experiments is fairly new, with current data supporting traptamers affecting HPV entry later than the internalization step.…”

Introducing Emerging Methods and Technologies

“…Jian Xe and Daniel DiMaio report a screening strategy that relies on artificial transmembrane proteins, which localize in cells and perturb cellular pathways, yielding a desired phenotype. These so‐called traptamer libraries can be used to identify new proteins or protein–protein interactions in cellular processes and the authors show how selective traptamers can inhibit the human papillomavirus (HPV) pathway and identify the Rab GAP TBC1D5 as a factor needed for endosome exit during HPV entry [1].…”