Abstract
Retromer, a cellular protein trafficking complex, sorts human papillomaviruses (HPVs) into the retrograde pathway for transport of HPV to the nucleus during virus entry. Here, we conducted a protein modulation screen to isolate four artificial transmembrane proteins called traptamers that inhibit different steps of HPV entry. By analyzing cells expressing pairs of traptamers, we ordered the trafficking steps during entry into a coherent pathway. One traptamer stimulates ubiquitination of the L2 capsid protein or associated proteins and diverts incoming virus to the lysosome, whereas the others act downstream by preventing sequential passage of the virus through retrograde compartments. Complex genetic interactions between traptamers revealed that a cell-penetrating peptide (CPP) on L2 mediates transient insertion of L2 into the endosome membrane, which is stabilized by retromer-L2 binding. These results define the retrograde entry route taken by HPV and show that retromer can play a role in CPP-mediated membrane insertion.