The Cytoskeletal Adaptor Obscurin-Like 1 Interacts with the Human Papillomavirus 16 (HPV16) Capsid Protein L2 and Is Required for HPV16 Endocytosis

Abstract

The human papillomavirus (HPV) capsid protein L2 is essential for viral entry. To gain a deeper understanding of the role of L2, we searched for novel cellular L2-interacting proteins. A yeast two-hybrid analysis uncovered the actin-depolymerizing factor gelsolin, the membrane glycoprotein dysadherin, the centrosomal protein 68 (Cep68), and the cytoskeletal adaptor protein obscurin-like 1 protein (OBSL1) as putative L2 binding molecules. Pseudovirus (PsV) infection assays identified OBSL1 as a host factor required for gene transduction by three oncogenic human papillomavirus types, HPV16, HPV18, and HPV31. In addition, we detected OBSL1 expression in cervical tissue sections and noted the involvement of OBSL1 during gene transduction of primary keratinocytes by HPV16 PsV. Complex formation of HPV16 L2 with OBSL1 was demonstrated in coimmunofluorescence and coimmunoprecipitation studies after overexpression of L2 or after PsV exposure. We observed a strong colocalization of OBSL1 with HPV16 PsV and tetraspanin CD151 at the plasma membrane, suggesting a role for OBSL1 in viral endocytosis. Indeed, viral entry assays exhibited a reduction of viral endocytosis in OBSL1-depleted cells. Our results suggest OBSL1 as a novel L2-interacting protein and endocytosis factor in HPV infection. IMPORTANCEHuman papillomaviruses infect mucosal and cutaneous epithelia, and the high-risk HPV types account for 5% of cancer cases worldwide. As recently discovered, HPV entry occurs by a clathrin-, caveolin-, and dynamin-independent endocytosis via tetraspanin-enriched microdomains. At present, the cellular proteins involved in the underlying mechanism of this type of endocytosis are under investigation. In this study, the cytoskeletal adaptor OBSL1 was discovered as a previously unrecognized interaction partner of the minor capsid protein L2 and was identified as a proviral host factor required for HPV16 endocytosis into target cells. The findings of this study advance the understanding of a so far less well-characterized endocytic pathway that is used by oncogenic HPV subtypes. Human papillomaviruses (HPVs) are small, nonenveloped DNA viruses that infect dividing basal keratinocytes of skin and mucosa via microlesions of the tissue. HPV is capable of inducing benign epithelial warts on the skin and mucosa, and infection with a high-risk HPV type may cause cervical and other anogenital and oropharyngeal cancers (1, 2). Cervical cancer is the third most common cancer in women worldwide and is associated with HPV infection, more precisely with high-risk HPV types such as HPV16, HPV18, and HPV31 (3). HPV is composed of a viral capsid with the major capsid protein L1, the minor capsid protein L2, and the viral genome. One icosahedral capsid contains 360 copies of L1, which can self-assemble to 72 pentamers, and up to 72 copies of the minor capsid protein L2, located inside the L1 shell (4-6). The capsid proteins L1 and L2 are key players in early events of infection, such as virus binding at the plasma membrane, cell entry, and t...

Human Papillomavirus Major Capsid Protein L1 Remains Associated with the Incoming Viral Genome throughout the Entry Process

“…(ii) The L2 protein may even span the TGN membrane twice, whereas both the N and C termini and the viral genome reside in the lumen during trafficking. Theoretically, this would allow for the L2 protein to interact with its cytosolic binding partners needed for intracellular trafficking preceding true translocation of the L2/DNA complex (29)(30)(31)(33)(34)(35)(36)(37), while at the same time allowing the carboxyl-terminal putative DNA binding domain on the luminal side to interact with viral genome. However, at the onset of mitosis, the carboxyl terminus has been shown by the Campos group to be accessible in the cytosol, suggesting that at this time the L2 protein assumes the possible type I transmembrane configuration previously suggested by us.…”

“…During this time, data suggest that the L2 protein undergoes conformational changes resulting in translocation of the majority of the protein across the endosomal membrane through possible solo or concerted efforts of a membrane-destabilizing (residues 445 to 467) and a transmembrane-like (residues 45 to 65) domain (26)(27)(28)(29). This translocation event allows for the cytoplasmic region of the L2 protein (likely residues 65 to 473) to directly interact with cytoplasmic factors, most notably, the retromer complex, which facilitates the trafficking of the L2/DNA complex to the trans-Golgi network (TGN) (29)(30)(31)(32)(33)(34)(35)(36)(37). Residues 13 to 45 are located on the luminal side of transport vesicles, whereas residues 45 through 65 span the membrane.…”

Cruising the cellular highways: How human papillomavirus travels from the surface to the nucleus

“…The virion is internalized by a previously unknown uptake pathway, which has the most commonalities with micropinocytosis resulting in the formation of HPV-harboring small, smooth endocytic vesicles (Schelhaas et al, 2012; Spoden et al, 2008; Spoden et al, 2013). Uptake does not depend on clathrin, caveolin, dynamin, or flotillin but requires reorganization of the actin cytoskeleton and may involve the cytoskeletal adaptor obscurin-like 1 (OBSL1) (Schelhaas et al, 2008; Schelhaas et al, 2012; Smith et al, 2008b; Wustenhagen et al, 2016). Activation of PI3 kinase has been implicated, likely participating in reorganization of the actin cytoskeleton during uptake (Fothergill and McMillan, 2006; Schelhaas et al, 2012; Surviladze et al, 2013).…”

“…C-terminal 40 amino acids of the HPV16 and HPV33 L2 protein have been shown to also interact with the dynein light chains DYNLT1 and DYNLT3, an interaction that seems to be important for nuclear delivery (Florin et al, 2006; Schneider et al, 2011). Recently, it was identified that obscurin-like 1 protein, a cytosolic cytoskeletal adaptor protein, forms complexes with the C-terminus of L2 (residues 280-473), which is important for efficient endocytosis (Wustenhagen et al, 2016). Currently, it is unknown precisely when the L2 protein becomes transmembranous.…”

Tetraspanin CD9 affects HPV16 infection by modulating ADAM17 activity and the ERK signalling pathway

“…Initial experiments showed that CD9 is involved after virus primary attachment to the cell surface and before capsid disassembly occurs. Inhibited CD63 colocalization as well as blocked capsid disassembly were both observed after CD9 knockdown and are strong indications of disturbed virus entry as also observed after depletion of other crucial entry mediators such as the annexin A2/S100A10 heterotetramer (A2t) [51], the obscurin-like protein OBSL1 [52], or the tetraspanin CD151 [13,14].…”