scholarly journals Human Papillomavirus Type 16 Minor Capsid Protein L2 N-Terminal Region Containing a Common Neutralization Epitope Binds to the Cell Surface and Enters the Cytoplasm

2001 ◽  
Vol 75 (5) ◽  
pp. 2331-2336 ◽  
Author(s):  
Yukiko Kawana ◽  
Kei Kawana ◽  
Hiroyuki Yoshikawa ◽  
Yuji Taketani ◽  
Kunito Yoshiike ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Human Papillomavirus ◽  
Cell Surface ◽  
Capsid Protein ◽  
Hela Cells ◽  
Human Cancer ◽  
Hpv Infection ◽  
Terminal Region ◽  
Neutralization Epitope ◽  
Minor Capsid Protein

ABSTRACT The first step of papillomavirus infection is believed to be binding of major capsid protein L1 to the cell surface without involvement of minor capsid protein L2, but the viral infectivity can be neutralized either by anti-L1 or anti-L2 antibody. To understand the role of L2 in human papillomavirus (HPV) infection, we examined a segment of HPV type 16 (HPV16) L2, which contains a neutralization epitope common to HPV6, for its involvement in adsorption and penetration of the capsids. Preincubation of monkey COS-1 cells with a synthetic peptide having amino acids (aa) 108 to 120 of HPV16 L2 reduced the susceptibility of COS-1 cells to infection with HPV16 pseudovirions. Confocal microscopy showed that the green fluorescence protein (GFP) fused with the L2 peptide was found to bind to the surface of a HeLa cell, an HPV18-positive human cancer cell line, at 4°C and to enter the cytoplasm after subsequent incubation at 37°C. Flow cytometry showed that fused GFP did not bind to HeLa cells that had been treated with trypsin. Besides COS-1 and HeLa cells, some human and rodent cell lines were detected by flow cytometry to be susceptible to binding with fused GFP, showing a tendency of epithelial cells toward higher susceptibility. Substitutions at aa 108 to 111 inhibited fused GFP from binding to HeLa cells and reduced the infectivity in COS-1 cells of the in vitro-constructed pseudovirions. The results suggest that L2 plays an important role in enhancing HPV infection through interaction between the N-terminal region and a cellular surface protein, facilitating penetration of the virions and determining part of the tropism of HPVs.

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

2016 ◽  
Vol 90 (14) ◽  
pp. 6224-6234 ◽  
Author(s):  
Matthew P. Bronnimann ◽  
Christine M. Calton ◽  
Samantha F. Chiquette ◽  
Shuaizhi Li ◽  
Mingfeng Lu ◽  
...  
Keyword(s):  
Capsid Protein ◽  
Current Model ◽  
Hpv Infection ◽  
Cleavage Product ◽  
Protein Domain ◽  
Furin Cleavage ◽  
Minor Capsid Protein ◽  
Papillomavirus Infection ◽  
Successful Infection ◽  
N Terminus

ABSTRACTDespite 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, thePropionibacterium shermaniitranscarboxylase 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 (2RHKR5) and Arg12 (9RTKR12). 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.

scholarly journals Neutralization of Human Papillomavirus with Monoclonal Antibodies Reveals Different Mechanisms of Inhibition

2007 ◽  
Vol 81 (16) ◽  
pp. 8784-8792 ◽  
Author(s):  
Patricia M. Day ◽  
Cynthia D. Thompson ◽  
Christopher B. Buck ◽  
Yuk-Ying S. Pang ◽  
Douglas R. Lowy ◽  
...  
Keyword(s):  
Extracellular Matrix ◽  
Monoclonal Antibodies ◽  
Human Papillomavirus ◽  
Cell Surface ◽  
Binding Sites ◽  
Neutralizing Antibodies ◽  
Hpv Infection ◽  
Heparan Sulfate Proteoglycans ◽  
Hacat Cells ◽  
Block Association

ABSTRACT The mechanisms of human papillomavirus (HPV) neutralization by antibodies are incompletely understood. We have used HPV16 pseudovirus infection of HaCaT cells to analyze how several neutralizing monoclonal antibodies (MAbs) generated against HPV16 L1 interfere with the process of keratinocyte infection. HPV16 capsids normally bind to both the cell surface and extracellular matrix (ECM) of HaCaT cells. Surprisingly, two strongly neutralizing MAbs, V5 and E70, did not prevent attachment of capsids to the cell surface. However, they did block association with the ECM and prevented internalization of cell surface-bound capsids. In contrast, MAb U4 prevented binding to the cell surface but not to the ECM. The epitope recognized by U4 was inaccessible when virions were bound to the cell surface but became accessible after endocytosis, presumably coinciding with receptor detachment. Treatment of capsids with heparin, which is known to interfere with binding to cell surface heparan sulfate proteoglycans (HSPGs), also resulted in HPV16 localization to the ECM. These results suggest that the U4 epitope on the intercapsomeric C-terminal arm is likely to encompass the critical HSPG interaction residues for HPV16, while the V5 and E70 epitopes at the apex of the capsomer overlap the ECM-binding sites. We conclude that neutralizing antibodies can inhibit HPV infection by multiple distinct mechanisms, and understanding these mechanisms can add insight to the HPV entry processes.

scholarly journals Identification of a Dynein Interacting Domain in the Papillomavirus Minor Capsid Protein L2

2006 ◽  
Vol 80 (13) ◽  
pp. 6691-6696 ◽  
Author(s):  
Luise Florin ◽  
Katrin A. Becker ◽  
Carsten Lambert ◽  
Thorsten Nowak ◽  
Cornelia Sapp ◽  
...  
Keyword(s):  
Amino Acids ◽  
Human Papillomavirus ◽  
Capsid Protein ◽  
Viral Genome ◽  
Promyelocytic Leukemia ◽  
Protein Bodies ◽  
Viral Dna ◽  
Microtubule Network ◽  
Promyelocytic Leukemia Protein ◽  
Minor Capsid Protein

ABSTRACT Papillomaviruses enter cells via endocytosis (H. C. Selinka et al., Virology 299:279-287, 2002). After egress from endosomes, the minor capsid protein L2 accompanies the viral DNA to the nucleus and subsequently to the subnuclear promyelocytic leukemia protein bodies (P. M. Day et al., Proc. Natl. Acad. Sci. USA 101:14252-14257, 2004), suggesting that this protein may be involved in the intracytoplasmic transport of the viral genome. We now demonstrate that the L2 protein is able to interact with the microtubule network via the motor protein dynein. L2 protein was found attached to microtubules after uncoating of incoming human papillomavirus pseudovirions. Based on immunofluorescence and coimmunoprecipitation analyses, the L2 region interacting with dynein is mapped to the C-terminal 40 amino acids. Mutations within this region abrogating the L2/dynein interaction strongly reduce the infectivity of pseudoviruses, indicating that this interaction mediates the minus-end-directed transport of the viral genome along microtubules towards the nucleus.

scholarly journals Zombies in TCGA

2015 ◽  
Vol 89 (8) ◽  
pp. 4044-4046 ◽  
Author(s):  
Daniel DiMaio
Keyword(s):  
Human Papillomavirus ◽  
Next Generation Sequencing ◽  
Hela Cells ◽  
Somatic Mutations ◽  
Hpv Infection ◽  
Next Generation ◽  
Human Cancers ◽  
Human Papillomavirus 18 ◽  
Tumor Types ◽  
Generation Sequencing

Next-generation sequencing results obtained to detect somatic mutations in human cancers can also be searched for viruses that contribute to cancer. Recently, human papillomavirus 18 RNA was detected in tumor types not typically associated with HPV infection. Analyses reported in this issue ofJournal of Virologydemonstrate that the apparent presence of HPV18 RNA in these atypical tumors is due in at least some cases to contamination of samples with HeLa cells, which harbor HPV18.

scholarly journals Observations on the expression of human papillomavirus major capsid protein in HeLa cells

2015 ◽  
Vol 15 (1) ◽  
Author(s):  
Chang-Yi Xiao ◽  
Bing-Bing Fu ◽  
Zhi-Ying Li ◽  
Gohar Mushtaq ◽  
Mohammad Amjad Kamal ◽  
...  
Keyword(s):  
Human Papillomavirus ◽  
Capsid Protein ◽  
Hela Cells ◽  
Major Capsid Protein

scholarly journals Topography of the Human Papillomavirus Minor Capsid Protein L2 during Vesicular Trafficking of Infectious Entry

2015 ◽  
Vol 89 (20) ◽  
pp. 10442-10452 ◽  
Author(s):  
Stephen DiGiuseppe ◽  
Timothy R. Keiffer ◽  
Malgorzata Bienkowska-Haba ◽  
Wioleta Luszczek ◽  
Lucile G. M. Guion ◽  
...  
Keyword(s):  
Human Papillomavirus ◽  
Host Cell ◽  
Capsid Protein ◽  
Conformational Changes ◽  
Transmembrane Protein ◽  
Vesicular Trafficking ◽  
Minor Capsid Protein ◽  
Intracellular Membranes ◽  
Cytosolic Side ◽  
Infectious Entry

ABSTRACTThe human papillomavirus (HPV) capsid is composed of the major capsid protein L1 and the minor capsid protein L2. During entry, the HPV capsid undergoes numerous conformational changes that result in endosomal uptake and subsequent trafficking of the L2 protein in complex with the viral DNA to thetrans-Golgi network. To facilitate this transport, the L2 protein harbors a number of putative motifs that, if capable of direct interaction, would interact with cytosolic host cell factors. These data imply that a portion of L2 becomes cytosolic during infection. Using a low concentration of digitonin to selectively permeabilize the plasma membrane of infected cells, we mapped the topography of the L2 protein during infection. We observed that epitopes within amino acid residues 64 to 81 and 163 to 170 and a C-terminal tag of HPV16 L2 are exposed on the cytosolic side of intracellular membranes, whereas an epitope within residues 20 to 38, which are upstream of a putative transmembrane region, is luminal. Corroborating these findings, we also found that L2 protein is sensitive to trypsin digestion during infection. These data demonstrate that the majority of the L2 protein becomes accessible on the cytosolic side of intracellular membranes in order to interact with cytosolic factors to facilitate vesicular trafficking.IMPORTANCEIn order to complete infectious entry, nonenveloped viruses have to pass cellular membranes. This is often achieved through the viral capsid protein associating with or integrating into intracellular membrane. Here, we determine the topography of HPV L2 protein in the endocytic vesicular compartment, suggesting that L2 becomes a transmembrane protein with a short luminal portion and with the majority facing the cytosolic side for interaction with host cell transport factors.

scholarly journals Research Progress of HPV L1 Capsid Protein in Prediction of Cervical Lesions

2020 ◽  
Vol 4 (6) ◽  
Author(s):  
Jing Cheng ◽  
Lin Xu ◽  
Beibei Liu ◽  
Biao Wang ◽  
Xicui Long ◽  
...  
Keyword(s):  
Cervical Cancer ◽  
Human Papillomavirus ◽  
Capsid Protein ◽  
Hpv Infection ◽  
Research Progress ◽  
Patient Treatment ◽  
Structural Protein ◽  
Cervical Lesions ◽  
Cervical Cells ◽  
Cellular Immune

Cervical cancer is one of the most common malignant gynecological tumors and has the second highest incidence of all malignancies in females. Chronic and persistent infection with High Risk Human Papillomavirus (HR-HPV) is the main cause of cervical cancer. There is a distinct lack of methodology by which to determine whether cervical epithelial dysplasia is cancerous following HPV infection. HPV L1 capsid protein is a major structural protein of human papillomavirus (HPV)?and it is the main target of the local cellular immune response aiming to combat human papillomavirus after HPV infection within cervical cells. Greater understanding of HPV L1 capsid protein and its association with cervical cytology, histopathology, patient age and human papillomavirus viral load has the potential to contribute toward improved the improved diagnosis and management of cervical cancer, providing useful information for gynecological clinicians in the hope of improving patient treatment and quality of life. This article reviews the predictive utility of HPV L1 capsid protein for cervical lesions.

scholarly journals Interaction of tSNARE Syntaxin 18 with the Papillomavirus Minor Capsid Protein Mediates Infection

2005 ◽  
Vol 79 (11) ◽  
pp. 6723-6731 ◽  
Author(s):  
Ioannis Bossis ◽  
Richard B. S. Roden ◽  
Ratish Gambhira ◽  
Rongcun Yang ◽  
Mitsuo Tagaya ◽  
...  
Keyword(s):  
Cell Surface ◽  
Capsid Protein ◽  
Ectopic Expression ◽  
Bovine Papillomavirus ◽  
Perinuclear Region ◽  
Minor Capsid Protein ◽  
Protein Receptor ◽  
Virion Morphogenesis ◽  
Target Membrane

ABSTRACT The papillomavirus capsid mediates binding to the cell surface and passage of the virion to the perinuclear region during infection. To better understand how the virus traffics across the cell, we sought to identify cellular proteins that bind to the minor capsid protein L2. We have identified syntaxin 18 as a protein that interacts with bovine papillomavirus type 1 (BPV1) L2. Syntaxin 18 is a target membrane-associated soluble N-ethylmaleimide-sensitive factor-attachment protein receptor (tSNARE) that resides in the endoplasmic reticulum (ER). The ectopic expression of FLAG-tagged syntaxin 18, which disrupts ER trafficking, blocked BPV1 pseudovirion infection. Furthermore, the expression of FLAG-syntaxin 18 prevented the passage of BPV1 pseudovirions to the perinuclear region that is consistent with the ER. Genetic studies identified a highly conserved L2 domain, DKILK, comprising residues 40 to 44 that mediated BPV1 trafficking through the ER during infection via an interaction with the tSNARE syntaxin 18. Mutations within the DKILK motif of L2 that did not significantly impact virion morphogenesis or binding at the cell surface prevented the L2 interaction with syntaxin 18 and disrupted BPV1 infection.

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