γ-secretase facilitates retromer-mediated retrograde transport.

The retromer complex mediates retrograde transport of protein cargos from endosomes to the trans-Golgi network (TGN). γ-secretase is a multisubunit protease that cleaves the transmembrane domain of its target proteins. Mutations in genes encoding subunits of retromer or γ-secretase can cause familial Alzheimer disease (AD) and other degenerative neurological diseases. It has been reported that retromer interacts with γ-secretase, but the consequences of this interaction are not known. Here, we report that retromer-mediated retrograde protein trafficking in cultured human epithelial cells is impaired by inhibition of γ-secretase activity or by genetic elimination of γ-secretase. γ-secretase inhibitor XXI and knockout of PS1, the catalytic subunit of γ-secretase, inhibit endosome to TGN trafficking of retromer-dependent retrograde cargos, divalent metal transporter 1 isoform II (DMT1-II), cation-independent mannose-6-phosphate receptor (CIMPR), and shiga toxin. Trafficking of retromer-independent cargos, such as cholera toxin and a CIMPR mutant that does not bind to retromer was not affected by γ-secretase inhibition. XXI treatment and PS1 KO inhibit interaction of γ-secretase with retromer but do not inhibit the association of cargo with retromer or with γ-secretase in intact cells. Similarly, these treatments do not affect the level of Rab7-GTP, which regulates retromer-cargo interaction. These results suggest that the γ-secretase-retromer interaction facilitates retromer-mediated retrograde trafficking.

Human Papillomavirus L2 Capsid Protein Stabilizes γ-Secretase during Viral Infection.

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.

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

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.

TBC1D5-Catalyzed Cycling of Rab7 Is Required for Retromer-Mediated Human Papillomavirus Trafficking during Virus Entry.

During virus entry, human papillomaviruses are sorted by the cellular trafficking complex, called retromer, into the retrograde transport pathway to traffic from the endosome to downstream cellular compartments, but regulation of retromer activity during HPV entry is poorly understood. Here we selected artificial proteins that modulate cellular proteins required for HPV infection and discovered that entry requires TBC1D5, a retromer-associated, Rab7-specific GTPase-activating protein. Binding of retromer to the HPV L2 capsid protein recruits TBC1D5 to retromer at the endosome membrane, which then stimulates hydrolysis of Rab7-GTP to drive retromer disassembly from HPV and delivery of HPV to the retrograde pathway. Although the cellular retromer cargos CIMPR and DMT1-II require only GTP-bound Rab7 for trafficking, HPV trafficking requires cycling between GTP- and GDP-bound Rab7. Thus, ongoing cargo-induced membrane recruitment, assembly, and disassembly of retromer complexes drive HPV trafficking.

Papillomaviruses Go Retro.

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.

The cellular endosomal protein stannin inhibits intracellular trafficking of human papillomavirus during virus entry.

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.