Characterization and interaction of the HPV16 major capsid protein L1 and minor capsid protein L2

Abstract

The Human Papillomavirus (HPV) Type 16 capsid consists of 360 monomer copies of the major capsid protein L1 and is arranged in 72 pentamers to a T = 7 icosahedral symmetry. L1 pentamers itself can assemble to virus-like-particles, which are successfully used as a prophylactic vaccine against HPV infections. The minor capsid protein L2 is located in the inner part of the HPV 16 capsid and the amount differs from 12-72 L2 monomer copies. This arrangement of L1 and L2 suggests an interaction of these two capsid proteins. This interaction has already been shown in cell-based experiments but also partially qualitatively at the structural level. However, until now it is not known with which binding affinity this interaction takes place and to what extent different influences on the cellular level play a role. Characterization of the L1 and L2 interaction is important for a better understanding of the viral entry of HPV16 into the cell. To characterize the interaction of L1 and L2 and to analyze the two capsid proteins at the biochemical level, we optimized recombinant protein expression in E. coli and purification of the viral structural proteins. For interaction studies, a L1-non-assembly variant (L1nav) was used since this variant forms only L1-pentamers and no capsids. Subsequently, characterization of the L1nav and L2 protein was performed in terms of stability, secondary protein structure, oligomerization state and homogeneity under different buffer conditions. Subsequently, the binding affinity (Kd) of the purified capsid proteins can be determined qualitatively and quantitatively by different methods (EL(2)ISA, SPR or anisotropy measurement) and under different conditions. The results demonstrate an interaction of L1 and L2 under pH 7.4 with a binding affinity of 620 nM ± 296 nM in contrast, pH 5.5 shows a binding affinity of 36 nM ± 69 nM. Previously published results indicated that pH in infected cervical tissue increases with HPV infection and this is thought to reduce the stability of L1. This decrease in stability may lead to promote a conformational change and expose the N-terminus of L2. This conformational change plays an important role in viral HPV entry into the cell. In addition, our results show a similar Kd for an L2 mutant. This L2 mutant (L2ΔL1bs) lacks the L1 binding domain and for this reason L2ΔL1bs should no longer bind to L1. However, L2ΔL1bs binds at 39 nM ± 229 nM compared to the L2 full-length protein at pH 7.4. These results suggest that a second L1 binding domain may be present on the L2 protein. These results are important for deeper and biochemical understanding of viral entry into the cell. We were also able to locate additional interaction partners (L1nav monomer and CypA) of L2 by the EL(2)ISA "screening tool". Up to this point, it is assumed that not all interaction partners of L2 are known during viral entry into the cell. After initial "screening", further characterization of the interaction, in terms of binding affinity or complex properties, can be performed. Fully understanding the intracellular transport of HPV16 into the nucleus is important, as most likely not only one type of virus hijacks this transport pathway. For this reason, viral protein studies have long contributed to the discovery and characterization of cellular proteins and signaling pathways.