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...

No abstract

No abstract

This study aims to explore the structure characteristic of random polypeptides constructed by origin early amino acid alphabet, as well as the effects of cofactors on conformation transition of random peptides. DNA library R8-4 encoding VNM random peptides were constructed by small cassette strategy. Subsequently, a random polypeptide library was constructed using in vitro translation. Expression and purification of VNM random peptides were also performed by a conventional method of recombinant. CD spectrum analysis indicated that VNM random polypeptides have a secondary structure characteristic of protein, such as the content of α-helix is greater than 60%, random coil is about 20% β sheet, and β turn is less than 10%. CD spectrum changed with the addition of 10-40 µM ATP and NADP, but slightly changed by NAD; no influence was observed with MgSO4. Bis-ANS binding assay indicated that fluorescent intensity of bis-ANS was strengthened slightly by 10 VNM random peptides. Fluorescent intensity was strengthened fourfold by adding 10-40 µM ATP, NAD, and NADH, whereas the inducing effect of NADPH and MgSO4 were negligible. VNM random peptides have a classic secondary structure and hydrophobic domain in water solution. Moreover, conformation transition and hydrophobic domain could be induced by cofactor, indicating the preliminary evidence for the hypothesis that "the origin of primitive protein was induced by small molecule."