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The outer shell of the papillomavirus particle is comprised of 72 pentamers of …
Biology Articles » Virology » Structure-based engineering of papillomavirus major capsid L1: controlling particle assembly » Results
The crystal structure of the T1 particle of HPV16 L1 capsid protein shows 60 L1 molecules assembled through pentamerization interactions and inter-pentameric bonding . The L1 protein with β-barrel core oligomerizes into pentamers (Fig. 1A). The pentamers further associate with each other to assemble into icosahedral particles of different T numbers. In the T = 1 crystal structure, the pentamer-pentamer interactions are formed through interactions of three helices near the C-terminus, h2, h3, and h4 (Fig. 1B, C). However, it is not clear if these helices are important for the T = 7 virion particle assembly. To determine the role of h2, h3, and h4 in the HPV16 papillomavirus particle assembly, four internal deletions, D1, D2, D3, and D4, were constructed (Fig. 2A, 2B). Briefly, in the D1 and D2 constructs h4 was deleted, in D3 both h4 and h3 were deleted, and in D4 all three helices were deleted. Because the full-length L1 can be expressed and purified successfully as GST-L1 fusion , the deletion mutants were constructed to express GST-L1 proteins.
Because the L1 expression level and solubility could not be easily detected by directly analyzing the cell lysates by SDS-PAGE, we performed partial purification of the GST-fusion proteins from the cell lysates using affinity chromatography. The GST-L1 protein from the supernatant fraction of the lysates was passed through a glutathione resin column. After washing the column with 10× bed volumes of buffer L (25 mM Tris-Cl, pH 8.0, 0.25 M NaCl, 1 mM EDTA, 2 mM DTT), the resin was incubated with thrombin to cleave L1 from the GST fusion.
The proteins bound to the resins before and after thrombin treatment, as well as those in the eluate after thrombin treatment, were analyzed by SDS-PAGE (Fig. 2C). D1 and D2 constructs produced large quantities of soluble GST-L1 fusion proteins (lanes 2 and 5, respectively). D3 produced some GST-D3-L1 fusion (lane 8 in Fig. 2C), but many degradation bands were observed in the fraction before thrombin treatment. These are presumably L1 degradation products that are still fused with GST at the N-terminus of L1. There was almost no detectable full-length GST-D4-L1 fusion (lane 11 in Fig. 2B), indicating that this protein was even more severely degraded than the GST-D3-L1 protein (compare lanes 8 and 11).
After thrombin digestion of the GST-L1 fusions on the glutathione resin, soluble D1- and D2-L1 were eluted from the column (lanes 3 and 6, respectively). For D3-L1, however, no intact protein was present in the elution after thrombin cleavage; D3-L1 may be degraded by thrombin due to an unfolded or misfolded conformation.
The L1 proteins eluted from the glutathione column were analyzed using size-exclusion chromatography on Superdex-200 (Fig. 2D). A single major peak with the elution position expected for a pentamer was present for both D1- and D2-L1. These results demonstrated that h4 deletion mutants D1-L1 and D2-L1 were soluble and stable and that they fold and oligomerize into pentamers. However, when the deletion extended to h3 (in D3) or to h2 (in D4), the proteins were degraded into multiple fragments, before and during thrombin treatment.
Since both D1 and D2 deletion constructs of HPV16 produced soluble L1 proteins, we attempted the equivalent of a D2 deletion on HPV18 L1 (Fig. 2A, 2B). As expected, the D2 deletion mutant of HPV18 was expressed and purified using the same protocol for HPV16 L1, and the protein behaved like a pentamer in size-exclusion chromatography in a buffer containing 25 mM Tris-Cl, pH 8.0, 0.2 M NaCl (data not shown). All the purified deletion mutants of the L1 proteins can be concentrated to approximately 20 mg/mL (Fig. 3A). The yield of the purified L1 proteins for D1 and D2 deletion constructs of HPV16 and HPV18 were roughly comparable, varying in the range between 0.7 mg to 1.3 mg per litters of cell culture.
We used two methods, electron microscopy (EM) and size-exclusion chromatography, to examine whether the purified proteins from the deletion constructs of HPV16 and 18 assembled into particles. The L1 proteins (at a concentration of ~0.1 mg/mL) were first incubated in a buffer known to promote icosahedral particle assembly  for 30 minutes at room temperature. The proteins were then examined using EM and size-exclusion column chromatography on Superdex-200. As shown in Fig. 3C–E, after incubation in assembly buffer, only pentamers, and no viral particles, were detected by EM examination of the D1-L1 and D2-L1 deletions of HPV16 and D2-L1 of HPV18. Under the same conditions, T = 7 particles, as well as some free pentamers, were observed for the full-length HPV16 L1 (Fig. 3B). Because of the potentially disruptive acidic conditions in the negative staining for EM examination, the particle assemble reaction was also examined by size-exclusion chromatography. The chromatography result showed that each of the three L1 mutants eluted as a single pentamer peak after assembly treatment (Fig. 4). In contrast, the full length HPV16 L1 showed an additional peak at void volume after assembly treatment, consistent with T7 particle assembly observed using EM.
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