Inhibition by Cellular Vacuolar ATPase Impairs Human Papillomavirus Uncoating and Infection

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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...

Section: Hpv16 L2 Forms a Complex With Obsl1supporting

confidence: 65%

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confidence: 99%

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

Wüstenhagen

Hampe

Boukhallouk

et al. 2016

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“…After internalization, the virus traffics through the endosomal system, requiring interactions with sorting nexin 17 in the endosome as well as the retromer complex and ␥-secretase in the trans-Golgi network (21)(22)(23)(24)(25)(26)(27). During this process, acidification and further conformational changes induced by CyPB result in L1 and L2 dissociation and viral uncoating (28,29). L2 then interacts with and permeabilizes the host cell membrane, actions that are dependent upon prior furin cleavage at the cell surface (30,31).…”

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confidence: 99%

Alpha-Defensin HD5 Inhibits Furin Cleavage of Human Papillomavirus 16 L2 To Block Infection

Wiens

Smith

2015

Human papillomavirus (HPV) is a significant oncogenic virus, but the innate immune response to HPV is poorly understood. Human ␣-defensin 5 (HD5) is an innate immune effector peptide secreted by epithelial cells in the genitourinary tract. HD5 is broadly antimicrobial, exhibiting potent antiviral activity against HPV at physiologic concentrations; however, the specific mechanism of HD5-mediated inhibition against HPV is unknown. During infection, the HPV capsid undergoes several critical cell-mediated viral protein processing steps, including unfolding and cleavage of the minor capsid protein L2 by host cyclophilin B and furin. Using HPV16 pseudovirus, we show that HD5 interacts directly with the virus and inhibits the furin-mediated cleavage of L2 at the cell surface during infection at a step downstream of the cyclophilin B-mediated unfolding of L2. Importantly, HD5 does not affect the enzymatic activity of furin directly. Thus, our data support a model in which HD5 prevents furin from accessing L2 by occluding the furin cleavage site via direct binding to the viral capsid. IMPORTANCEOur study elucidates a new antiviral action for ␣-defensins against nonenveloped viruses in which HD5 directly interferes with a critical host-mediated viral processing step, furin cleavage of L2, at the cell surface. Blocking this key event has deleterious effects on the intracellular steps of virus infection. Thus, in addition to informing the antiviral mechanisms of ␣-defensins, our studies highlight the critical role of furin cleavage in HPV entry. Innate immune control, mediated in part by ␣-defensins expressed in the genital mucosa, may influence susceptibility to HPV infections that lead to cervical cancer. Moreover, understanding the mechanism of these natural antivirals may inform the design of therapeutics to limit HPV infection. Defensins are effector peptides of the human innate immune system. They are divided into two classes, ␣-and ␤-defensins, based on the pattern of disulfide bonds that stabilize their tertiary structure (1, 2). HD5 is one of six human ␣-defensins and is constitutively expressed and secreted in the female and male genitourinary tracts (3-5). Concentrations of HD5 in vaginal lavage fluid of healthy women have been reported to be 16.5 Ϯ 10.5 M (3). Although originally discovered due to their antibacterial activity, defensin antiviral activity against both enveloped and nonenveloped viruses has also been described. Neutralization of enveloped viruses, such as human immunodeficiency virus 1 (HIV-1), herpes simplex virus (HSV), and respiratory syncytial virus (RSV), is largely dependent on direct interactions of defensins with both viral attachment proteins and cellular receptors, as well as envelope damage, fusion inhibition, and modulation of host responses (6). Inhibition of these viruses may be due to multiple defensin actions rather than a single overriding inhibitory mechanism. While less is known about the mechanisms of defensin antiviral activity against nonenveloped viruses, human adeno...

“…Internalized virions enter the endosomal pathway, where acidification due to the V-ATPase proton pump triggers L1 uncoating (18) and the L2/vDNA complex separates from the dissociated L1 capsid and retrograde traffics to the trans-Golgi network (TGN) in a furin-, cyclophilin-, ␥-secretase-, and retromer-dependent manner (19)(20)(21)(22)(23)(24). Transport of L2/vDNA to the TGN is essential, and prolonged residence in the endosomal compartment may be detrimental, as the acid-dependent cathepsins B and L restrict infection (25).…”

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confidence: 99%

Furin Cleavage of L2 during Papillomavirus Infection: Minimal Dependence on Cyclophilins

Bronnimann

Calton

Chiquette

et al. 2016

Despite an abundance of evidence supporting an important role for the cleavage of minor capsid protein L2 by cellular furin, direct cleavage of capsid-associated L2 during human papillomavirus 16 (HPV16) infection remains poorly characterized. The conserved cleavage site, close to the L2 N terminus, confounds observation and quantification of the small cleavage product by SDS-PAGE. To overcome this difficulty, we increased the size shift by fusing a compact protein domain, the Propionibacterium shermanii transcarboxylase domain (PSTCD), to the N terminus of L2. The infectious PSTCD-L2 virus displayed an appreciable L2 size shift during infection of HaCaT keratinocytes. Cleavage under standard cell culture conditions rarely exceeded 35% of total L2. Cleavage levels were enhanced by the addition of exogenous furin, and the absolute levels of infection correlated to the level of L2 cleavage. Cleavage occurred on both the HaCaT cell surface and extracellular matrix (ECM). Contrary to current models, experiments on the involvement of cyclophilins revealed little, if any, role for these cellular enzymes in the modulation of furin cleavage. HPV16 L2 contains two consensus cleavage sites, Arg5 ( 2 RHKR 5 ) and Arg12 ( 9 RTKR 12 ). Mutant PSTCD-L2 viruses demonstrated that although furin can cleave either site, cleavage must occur at Arg12, as cleavage at Arg5 alone is insufficient for successful infection. Mutation of the conserved cysteine residues revealed that the Cys22-Cys28 disulfide bridge is not required for cleavage. The PSTCD-L2 virus or similar N-terminal fusions will be valuable tools to study additional cellular and viral determinants of furin cleavage. IMPORTANCEFurin cleavage of minor capsid protein L2 during papillomavirus infection has been difficult to directly visualize and quantify, confounding efforts to study this important step of HPV infection. Fusion of a small protein domain to the N terminus greatly facilitates direct visualization of the cleavage product, revealing important characteristics of this critical process. Contrary to the current model, we found that cleavage is largely independent of cyclophilins, suggesting that cyclophilins act either in parallel to or downstream of furin to trigger exposure of a conserved N-terminal L2 epitope (RG-1) during infection. Based on this finding, we strongly caution against using L2 RG-1 epitope exposure as a convenient but indirect proxy of furin cleavage. H uman papillomaviruses (HPVs) are currently the most common sexually transmitted infection in the United States (1). These viruses infect and replicate in differentiating mucosal and cutaneous epithelia, and a subset of the mucosa-tropic viruses, the high-risk HPVs, cause Ͼ99% of cervical cancers in women and are associated with other anogenital and nasopharyngeal cancers in both women and men (2). In all, the high-risk HPVs account for an astounding 5% of total cancers worldwide (3).HPVs are nonenveloped viruses with a 55-nm icosahedral capsid composed of 72 pentamers of the major capsid p...