Highlights d Traptamer screening identifies TBC1D5 as a human papillomavirus entry factor d TBC1D5 is a Rab7 GTPase-activating protein that activates retromer during HPV entry d Rab7 cycling between GTP-and GDP-bound forms is required for HPV entry d HPV trafficking displays different Rab7 requirements than cellular cargo

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.

Human papillomaviruses are important pathogens responsible for approximately 5% of cancer as well as other important human diseases, but many aspects of the papillomavirus life cycle are poorly understood. To undergo genome replication, HPV DNA must traffic from the cell surface to the nucleus. Recent findings have revolutionized our understanding of HPV entry, showing that it requires numerous cellular proteins and proceeds via a series of intracellular membrane-bound vesicles that comprise the retrograde transport pathway. This paper reviews the evidence supporting this unique entry mechanism with a focus on the crucial step by which the incoming virus particle is transferred from the endosome into the retrograde pathway. This new understanding provides novel insights into basic cellular biology and suggests novel rational approaches to inhibit HPV infection.Pathogens 2020, 9, 267 2 of 11 with wild-type or mutant capsid proteins, and the convenience of assaying successful infection by measuring reporter gene expression. Nevertheless, authentic papillomaviruses should be used to validate crucial findings obtained with pseudoviruses because there may be important differences between authentic viruses produced in stratified epithelial cells and pseudoviruses produced in a monolayer cell culture [13]. Role of Retrograde Trafficking and Retromer in HPV EntryKnockdown and inhibitor studies have identified numerous cell proteins required for HPV entry and trafficking, including but not limited to furin, kallikrein-8, ADAM17, cyclophilin B, SNX17, SNX27, γ-secretase, VPS4, obscurin-like1, TSG101,TRAPPC8, growth factor receptors, DCT, annexin A2/S100A10, and tetraspanins CD131 and CD63 [8,10,[14][15][16][17][18][19][20][21][22][23][24][25][26][27][28][29]. The proteases furin and kallikrein-8 cleave L2 and L1, respectively. Furin-catalyzed cleavage near the N terminus of L2 occurs at the cell surface, but in the absence of cleavage, intracellular trafficking events are disrupted [30]. ADAM17 appears to assemble an entry platform containing CDC151 and an epidermal growth factor receptor that mediates internalization of HPV [27]. In most cases, however, little is known about the mechanism by which these proteins support HPV infection.To comprehensively identify cellular proteins involved in HPV entry, we conducted a genome-wide siRNA screen. In this screen,~20,000 different siRNA pools were transfected into HeLa cells in a microtiter plate format to repress individual cellular genes, and the ability of an HPV16 pseudovirus to express GFP from the encapsidated reporter plasmid was measured in each well of cells [31]. The results showed that efficient HPV infection requires numerous cellular proteins localized to vesicular compartments such as the trans-Golgi network (TGN) and the endoplasmic reticulum (ER). Subsequent validation studies and more targeted analysis of candidate genes confirmed that these proteins were required for HPV entry [30,31]. Among the required factors identified in these experiments were Rab proteins...

Human papillomaviruses (HPVs) are the most common sexually transmitted viruses and one of the most important infectious causes of cancers worldwide. While prophylactic vaccines are effective against certain strains of HPV, established infections still cause deadly cancers in both men and women. HPV traffics to the nucleus via the retrograde transport pathway, but the mechanism of intracellular transport of non-enveloped viruses such as HPV is incompletely understood. Using an overexpression screen, we identify several genes that control HPV16 entry. We focused on the mechanism by which one of the screen hits, stannin, blocks HPV16 infection. Stannin has not been previously implicated in virus entry. Overexpression of stannin specifically inhibits infection by several HPV types, but not other viruses tested. Stannin is constitutively expressed in human keratinocytes, and its basal levels limit entry by HPV16. Stannin is localized to the endolysosomal compartment and does not affect HPV16 binding to cells, virus uptake, or virus uncoating, but inhibits the entry of HPV into the trans-Golgi network (TGN) and stimulates HPV degradation. We further show that stannin interacts with L1 major capsid protein and impairs the interaction of the L2 minor capsid protein with retromer, which is required for virus trafficking to the TGN. Our findings shed light on a novel cellular protein that interferes with HPV entry and highlight the role of retrograde transport in HPV entry.

Intracellular trafficking of human papillomavirus (HPV) during virus entry requires γ-secretase, a cellular protease consisting of a complex of four cellular transmembrane (TM) proteins. γ-secretase typically cleaves substrate proteins but it plays a non-canonical role during HPV entry. γ-secretase binds to the HPV minor capsid protein L2 and facilitates its insertion into the endosomal membrane. After insertion, L2 protrudes into the cytoplasm, which allows HPV to bind other cellular factors required for proper virus trafficking into the retrograde transport pathway. Here, we further characterize the interaction between γ-secretase and HPV L2. We show that γ-secretase is required for cytoplasmic protrusion of L2 and that L2 associates strongly with the PS1 catalytic subunit of γ-secretase and stabilizes the γ-secretase complex. Mutational studies revealed that a putative TM domain in HPV16 L2 cannot be replaced by a foreign TM domain, that infectivity of HPV TM mutants is tightly correlated with γ-secretase binding and stabilization, and that the L2 TM domain is required for protrusion of the L2 protein into the cytoplasm. These results provide new insight into the interaction between γ-secretase and L2 and highlight the importance of the native HPV L2 TM domain for proper virus trafficking during entry.