Furin Cleavage of L2 during Papillomavirus Infection: Minimal Dependence on Cyclophilins
Autor: | Matthew P. Bronnimann, Mingfeng Lu, Christine M. Calton, Janice A. Chapman, Samuel K. Campos, Kristin N. Bratton, Shuaizhi Li, Samantha F. Chiquette, Angela M. Schlegel |
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Jazyk: | angličtina |
Rok vydání: | 2016 |
Předmět: |
0301 basic medicine
Keratinocytes viruses Immunology Protein domain Mutant Cleavage (embryo) Microbiology Epitope 03 medical and health sciences Cyclophilins Epitopes Virology Humans Amino Acid Sequence RNA Small Interfering Peptide sequence Furin Human papillomavirus 16 biology Sequence Homology Amino Acid Papillomavirus Infections RNA Oncogene Proteins Viral Virus Internalization Cell biology Virus-Cell Interactions 030104 developmental biology Capsid Insect Science Mutation biology.protein Mutagenesis Site-Directed Capsid Proteins |
Popis: | 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. IMPORTANCE Furin 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. |
Databáze: | OpenAIRE |
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