ABSTRACT
The binding affinity of the human papillomavirus type 6 E2 protein is strongly mediated by the sequence of the DNA linker region, with high affinity for the AATT linker and low affinity for the CCGG linker. When two terminal leucine residues are removed from the protein, the level of binding to both strands increases, but unequally, resulting in a significant decrease in selectivity for the AATT linker strand. To rationalize this behavior, we performed molecular dynamics simulations of the wild-type and mutant protein in the apo state and bound to DNA with high-affinity AATT and low-affinity CCGG linker strands. While no stable contacts were made between the ß2-ß3 loop and DNA in the wild type, this loop was repositioned in the mutant complexes and formed electrostatic contacts with the DNA backbone. More contacts were formed when the mutant was bound to the CCGG linker strand than to the AATT linker strand, resulting in a more favorable change in interaction energy for the CCGG strand. In addition, significant differences in correlated motions were found, which further explained the differences in binding. The simulations suggest that ß2-ß3 loop motions are responsible for the increased affinity and decreased selectivity of the mutant protein.
Subject(s)
Amino Acid Sequence , DNA, Viral/chemistry , DNA-Binding Proteins/chemistry , Human papillomavirus 6/chemistry , Oncogene Proteins, Viral/chemistry , Sequence Deletion , DNA, Viral/genetics , DNA, Viral/metabolism , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Human papillomavirus 6/genetics , Human papillomavirus 6/metabolism , Humans , Oncogene Proteins, Viral/genetics , Oncogene Proteins, Viral/metabolism , Protein Binding/genetics , Protein Structure, SecondaryABSTRACT
Human papillomavirus 6b L1 virus-like particles (VLPs) were successfully expressed using Bombyx mori nucleopolyhedrovirus (BmNPV) bacmid expression system and rapidly purified using size exclusion chromatography after ultracentrifugation procedure and characterized by capillary zone electrophoresis (CZE). The average capillary electrophoresis migration time was 11 min with the relative standard deviation (RSD) of 0.3% of human papillomavirus 6b L1 VLPs. After this threefold fractionation, the CZE samples were still further investigated by dynamic light scattering and immuno blotting. The versatile technique, CZE not only proved to be a valuable tool for VLP characterization, but was also found to be reliable and precise. Thus CZE will also be an important option for the quality control of VLPs in pharmaceutical research level.
Subject(s)
Bombyx/chemistry , Capsid Proteins/chemistry , Human papillomavirus 6/chemistry , Papillomavirus Vaccines/biosynthesis , Vaccines, Virus-Like Particle/biosynthesis , Animals , Bombyx/cytology , Bombyx/virology , Capsid Proteins/genetics , Capsid Proteins/isolation & purification , Capsid Proteins/metabolism , Electrophoresis, Capillary , Gene Expression , Humans , Nucleopolyhedroviruses/geneticsABSTRACT
UNLABELLED: Human papillomavirus (HPV) can successfully evade the host immune response to establish a persistent infection. We show here that expression of the E7 oncoprotein in primary human keratinocytes results in increased production of interleukin-18 (IL-18) binding protein (IL-18BP). This anti-inflammatory cytokine binding protein is a natural antagonist of IL-18 and is necessary for skin homeostasis. We map increased IL-18BP production to the CR3 region of E7 and demonstrate that this ability is shared among E7 proteins from different HPV types. Furthermore, mutagenesis shows that increased IL-18BP production is mediated by a gamma-activated sequence (GAS) in the IL-18BP promoter. Importantly, the increased IL-18BP levels seen in E7-expressing keratinocytes are capable of diminishing IL-18-mediated CD4 lymphocyte activation. This study provides the first evidence for a virus protein that targets IL-18BP and further validates E7 as a key component of the HPV immune evasion armor. IMPORTANCE: Infection with human papillomavirus is a leading cause of morbidity and mortality worldwide. This study demonstrates that the E7 protein increases production of the anti-inflammatory IL-18BP, a major regulator of epithelial homeostasis. A number of E7 proteins can increase IL-18BP production, and a region within the CR3 of E7 is necessary for mediating the increase. A consequence of increased IL-18BP production is a reduction in CD4-positive lymphocyte activation in response to IL-18 costimulation. These findings may shed light on the immune evasion abilities of HPV.
