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. 2008 Jun;82(11):5190-7.
doi: 10.1128/JVI.02726-07. Epub 2008 Mar 26.

Arrangement of L2 within the papillomavirus capsid

Christopher B Buck  1 , Naiqian Cheng, Cynthia D Thompson, Douglas R Lowy, Alasdair C Steven, John T Schiller, Benes L Trus
Affiliations

Arrangement of L2 within the papillomavirus capsid

Christopher B Buck et al. J Virol. 2008 Jun.

Abstract

Papillomaviruses are a family of nonenveloped DNA tumor viruses. Some sexually transmitted human papillomavirus (HPV) types, including HPV type 16 (HPV16), cause cancer of the uterine cervix. Papillomaviruses encode two capsid proteins, L1 and L2. The major capsid protein, L1, can assemble spontaneously into a 72-pentamer icosahedral structure that closely resembles native virions. Although the minor capsid protein, L2, is not required for capsid formation, it is thought to participate in encapsidation of the viral genome and plays a number of essential roles in the viral infectious entry pathway. The abundance of L2 and its arrangement within the virion remain unclear. To address these questions, we developed methods for serial propagation of infectious HPV16 capsids (pseudoviruses) in cultured human cell lines. Biochemical analysis of capsid preparations produced using various methods showed that up to 72 molecules of L2 can be incorporated per capsid. Cryoelectron microscopy and image reconstruction analysis of purified capsids revealed an icosahedrally ordered L2-specific density beneath the axial lumen of each L1 capsomer. The relatively close proximity of these L2 density buttons to one another raised the possibility of homotypic L2 interactions within assembled virions. The concept that the N and C termini of neighboring L2 molecules can be closely apposed within the capsid was supported using bimolecular fluorescence complementation or "split GFP" technology. This structural information should facilitate investigation of L2 function during the assembly and entry phases of the papillomavirus life cycle.

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Figures

FIG. 1.
FIG. 1.
L2 is integrally associated with L1. Cells were transfected with the plasmid shown at the top of the figure. After 48 h, the cells were lysed and capsids were allowed to mature in crude lysate (except in lanes marked “immature”). Capsids were then purified using iodixanol gradients. For immature capsids, this procedure results in a loss of encapsidated DNA and associated histone proteins. In some instances, purified capsid preparations were treated briefly with trypsin. The capsids were separated by SDS-PAGE and visualized with Sypro Ruby fluorescent protein staining.
FIG. 2.
FIG. 2.
Capsids with an average of 72 molecules of L2 per capsid. A small pilot batch of capsids with greater L2 occupancy was generated as described in the text. The capsids were separated by SDS-PAGE and stained with Sypro Ruby. Visual inspection and digital analysis of L1 and L2 band intensities suggested an L1:L2 ratio of 5:1.
FIG. 3.
FIG. 3.
L2 content of capsids used for cryo-EM analysis. A scaled-up capsid preparation was diluted and separated by SDS-PAGE. Replicate gels were stained with Sypro-Ruby (top panel) or colloidal Coomassie (bottom panel). Visual inspection and digital analysis of L1 and L2 band intensities suggested an L1:L2 ratio of 6:1.
FIG. 4.
FIG. 4.
3DR of more capsids with an average of 60 molecules of L2 per capsid. Computerized reconstructions of L1-only capsids (a, c, and e) and L1+L2 capsids (b, d, and f) are shown. The top images (a and b) are of the central section through vertex (pentavalent) capsomers. An area of additional density present in L1+L2 capsids (b) is marked with a red asterisk. Panels c and d show exterior views of each type of capsid. Panels e and f show interior/cutaway views of an L1-only or L1+L2 capsid, respectively. DNA and histone density have been computationally removed from the interior views. The bottom images show L2-specific density in red either alone (h) or superimposed on an interior view of the L1-only 3DR in blue (g). Bar = 200 Å.
FIG. 5.
FIG. 5.
Bimolecular fluorescence complementation. The graph depicts the fluorescence of purified capsids (gray bars) produced in 293TT cells by cotransfecting an L1 expression plasmid together with the L2 constructs shown to the left of the figure. Light gray bars show the fluorescence of live HeLa cells transfected with the L2 constructs shown (without any L1 plasmid). The terms “NYFP” and “CYFP” refer to N-terminal and C-terminal fragments of YFP, respectively. Fluorescence intensity was standardized to the fluorescence of L2 fused to full-length YFP (L2-YFP). The relative infectious titer of each capsid stock is given to the right of the graph. w.t., wild type.
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