The T = 1 particle structure shows that h2, h3, and h4 near the C-terminal end of L1 are the solely responsible for making inter-pentameric contacts required for the particle assembly . There are the obvious differences between the T1 structure and the proposed assembly model of T7 , and the roles of h2, h3, and h4 in T7 assembly are not defined. We have selectively deleted each of the three helices from papillomavirus L1. The biochemical and structural characterization of the deletion mutant proteins demonstrated the important roles of these C-terminal helices in the folding and assembly of L1 capsid.
The deletion mutants of HPV16 L1 lacking h4, h3, or h2 were expressed as GST-fusions in E. coli. The mutant proteins with partial or entire h4 deletion, D1-L1 and D2-L2, were similar to the full-length wild-type L1 in protein expression and behavior by size-exclusion chromatography. Like wild type, D1-L1 and D2-L2 were expressed at quite high levels, and a good fraction of each expressed proteins was soluble and could be purified as pentamers (Fig. 2C). This result demonstrated that h4 is not required for L1 folding and pentamerization.
In contrast, when h3 and h2 were deleted and fusion proteins (GST-D3-L1 and GST-D4-L1) were expressed, little intact GST-L1 fusion protein was detected (Fig. 2C). Instead, multiple smaller protein bands were present in the SDS-PAGE gels. Because only a GST-fusion can bind the glutathione resin of the column used for purification, the smaller protein bands must contain intact GST plus L1 fragments of various lengths due to C-terminal degradation. Thrombin treatment of these GST-fusions did not produce discreet L1 fragments, indicating that both D3-L1 and D4-L1 were unfolded and sensitive to protease degradation. Consistent with this severe degradation of these deletion mutants of L1 is that there are six potential thrombin cleavage sites within the HPV16 L1 sequence and many Arg and Lys residues can serve as cleavage sites for other proteases. Thus, we conclude that h2 and h3 are important for the folding and integrity of HPV L1 structure.
Interestingly, a small helix at the C-terminus, h5 (Fig. 1B), was also shown to be critical for L1 folding [1,2]. A common feature of h2, h3, and h5 in the x-ray structure  is that all helices have a highly hydrophobic side that packs with the core structure of L1. Disrupting these hydrophobic packing interactions will obviously affect the folding of L1.
The deletion of h4 in another papillomavirus, HPV18, resulted in a mutant that behaved like the HPV16 D2-L1 mutant. This result confirmed that the L1 structure is conserved among different HPV types, which is not surprising considering the amino acid sequences of L1 proteins are highly conserved. We predict that the same D2 deletion of any other papillomavirus L1 should have the same properties.
When the D1-L1 and D2-L2 proteins of HPV16 and 18 were incubated under assembly conditions, pentamer structures, but no T = 7 particles, were visualized by EM examination (Fig. 3C–E). If the deletion mutants assemble into particles, these particles might be unstable to the acidic conditions used during EM sample preparation, which requires uranyl acetate treatment. To rule out this possibility, size-exclusion column chromatography was used to analyze the deletion constructs after assembly treatment (Fig. 4). The size-exclusion analysis showed that, in contrast to the full length L1, the D1-L1 and D2-L2 mutants lacking h4 failed to form assemblies larger than the pentamers, confirming the results from EM study.
In conclusion, we have demonstrated that the structural elements h2, h3, and h4 near the C-terminal end of L1 are important for the assembly of papillomaviruses into particles. The h2 and h3 regions are essential for L1 folding and pentamer formation, whereas the h4 region is indispensable for the assembly of not only T1, but also of the T7 virus-like particle.