Conformational and linear epitopes on virus-like particles of human papillomavirus type 33 identified by monoclonal antibodies to the minor capsid protein L2

BPHE-1 cells, which harbor 50 to 200 viral episomes, encapsidate viral genome and generate infectious bovine papillomavirus type 1 (BPV1) upon coexpression of capsid proteins L1 and L2 of BPV1, but not coexpression of BPV1 L1 and human papillomavirus type 16 (HPV16) L2. BPV1 L2 bound in vitro via its C-terminal 85 residues to purified L1 capsomers, but not with intact L1 virus-like particles in vitro. However, when the efficiency of BPV1 L1 coimmunoprecipitation with a series of BPV1 L2 deletion mutants was examined in vivo, the results suggested that residues 129 to 246 and 384 to 460 contain independent L1 interaction domains. An L2 mutant lacking the C-terminal L1 interaction domain was impaired for encapsidation of the viral genome. Coexpression of BPV1 L1 and a chimeric L2 protein composed of HPV16 L2 residues 1 to 98 fused to BPV1 L2 residues 99 to 469 generated infectious virions. However, inefficient encapsidation was seen when L1 was coexpressed with either BPV1 L2 with residues 91 to 246 deleted or with BPV1 L2 with residues 1 to 225 replaced with HPV16 L2. Impaired genome encapsidation did not correlate closely with impairment of the L2 proteins either to localize to promyelocytic leukemia oncogenic domains (PODs) or to induce localization of L1 or E2 to PODs. We conclude that the L1-binding domain located near the C terminus of L2 may bind L1 prior to completion of capsid assembly, and that both L1-binding domains of L2 are required for efficient encapsidation of the viral genome.Papillomaviruses are nonenveloped double-stranded DNA tumor viruses. Their capsid comprises 360 molecules of the major capsid protein L1, arranged as 72 pentamers, or capsomers, in a Tϭ7d icosahedral surface lattice (2). Expression of L1 protein results in the self-assembly of virus-like particles (VLPs), which have the size, shape, and conformational epitopes of virion capsids (14). Bovine papillomavirus type 1 (BPV1) virions in the presence of low ionic strength and dithiothreitol (DTT) (18) and human papillomavirus type 11 (HPV11) and HPV33 VLPs in the presence of reducing agents (21, 25) are disassembled into capsomers. Recombinant HPV11 L1 protein with a Cys-to-Gly mutation in the C terminus of L1 forms pentamers but cannot assemble into capsidlike structures (18). Taken together, the data imply that both ionic and disulfide bonds mediate interpentamer binding in the papillomavirus capsid.Virions also contain L2, the minor capsid protein (5). The number of L2 molecules per capsid has been estimated at between 12 (30) and 36 (5) molecules per virion. If L2 is coexpressed with L1, the L2 protein is coassembled into VLPs with a stoichiometry similar to that seen in authentic virions (15). Three-dimensional reconstruction of cryo-electron micrographs of quench-frozen BPV virions has revealed the capsid architecture to 9-Å resolution. This analysis detected a protein density within the central cavity of the pentavalent capsomers, suggesting that L2 may be associated with these 12 vertex capsomers (30).Rodent fibroblasts main...

Section: Resultssupporting

confidence: 50%

L1 Interaction Domains of Papillomavirus L2 Necessary for Viral Genome Encapsidation

Okun

Day

Greenstone

et al. 2001

“…The 33L1-7 MAb has been previously characterized and recognizes a linear epitope at amino acids 303 to 313 that is buried inside the capsid structure (11,25,58,71). The L2 protein was detected with mouse MAb 33L2-1, which recognizes an epitope at amino acids 163 to 170 (25,27,72). The TGN was detected using rabbit polyclonal antibody (pAb) anti-TGN46, which recognizes a luminal epitope within amino acids residues 200 to 350 (PA5-23068; Thermo Fisher Scientific).…”

Section: Methodsmentioning

confidence: 99%

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

DiGiuseppe

Bienkowska-Haba

Guion

et al. 2017

During infectious entry, acidification within the endosome triggers uncoating of the human papillomavirus (HPV) capsid, whereupon host cyclophilins facilitate the release of most of the major capsid protein, L1, from the minor capsid protein L2 and the viral genome. The L2/DNA complex traffics to the trans-Golgi network (TGN). After the onset of mitosis, HPV-harboring transport vesicles bud from the TGN, followed by association with mitotic chromosomes. During this time, the HPV genome remains in a vesicular compartment until the nucleus has completely reformed. Recent data suggest that while most of L1 protein dissociates and is degraded in the endosome, some L1 protein remains associated with the viral genome. The L1 protein has DNA binding activity, and the L2 protein has multiple domains capable of interacting with L1 capsomeres. In this study, we report that some L1 protein traffics with L2 and viral genome to the nucleus. The accompanying L1 protein is mostly full length and retains conformation-dependent epitopes, which are recognized by neutralizing antibodies. Since more than one L1 molecule contributes to these epitopes and requires assembly into capsomeres, we propose that L1 protein is present in the form of pentamers. Furthermore, we provide evidence that the L1 protein interacts directly with viral DNA within the capsid. Based on our findings, we propose that the L1 protein, likely arranged as capsomeres, stabilizes the viral genome within the subviral complex during intracellular trafficking.IMPORTANCE After internalization, the nonenveloped human papillomavirus virion uncoats in the endosome, whereupon conformational changes result in a dissociation of a subset of the major capsid protein L1 from the minor capsid protein L2, which remains in complex with the viral DNA. Recent data suggest that some L1 protein may accompany the viral genome beyond the endosomal compartment. We demonstrate that conformationally intact L1 protein, likely still arranged as capsomeres, remains associated with the incoming viral genome throughout mitosis and transiently resides in the nucleus until after the viral DNA is released from the transport vesicle.KEYWORDS HPV entry, HPV16, L1 protein, L2 protein, mitosis, nuclear transport, virus trafficking P apillomaviruses are a family of nonenveloped DNA viruses that infect a wide range of hosts with a preference for epithelial cells. The papillomaviruses that infect humans (HPVs) are a particular health burden. While most infections with HPVs are cleared by the immune system, a persistent infection with the high-risk types is associated with increased risk of carcinomas. The high-risk type 16 accounts for

“…For detection of V5-tagged OBSL1 after Western blotting or immunofluorescence staining, either monoclonal antibody (MAb) SV5-Pk1 (Bio-Rad, Hercules, CA) or polyclonal antibody ab9116 (Abcam, Cambridge, UK) was used. HPV16 L1-specific mouse monoclonal antibody 16L1-312F and rabbit polyclonal antibody K75 and 33L1-7 as well as HPV16 L2-specific mouse antibody 33L2-1 have been previously described (L2-1 [60], K75 [61], 312F [62], and L1-7 [63]). Obscurin-like 1-specific polyclonal rabbit antibody HPA036404 and FLAG (M2)-and ␤-actin (A5441)-specific mouse antibodies were purchased from SigmaAldrich, St. Louis, MO.…”

Section: Methodsmentioning

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

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...