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. 2005 Mar;79(5):2839-46.
doi: 10.1128/JVI.79.5.2839-2846.2005.

Maturation of papillomavirus capsids

Christopher B Buck  1 , Cynthia D Thompson, Yuk-Ying S Pang, Douglas R Lowy, John T Schiller
Affiliations

Maturation of papillomavirus capsids

Christopher B Buck et al. J Virol. 2005 Mar.

Abstract

The papillomavirus capsid is a nonenveloped icosahedral shell formed by the viral major structural protein, L1. It is known that disulfide bonds between neighboring L1 molecules help to stabilize the capsid. However, the kinetics of inter-L1 disulfide bond formation during particle morphogenesis have not previously been examined. We have recently described a system for producing high-titer papillomavirus-based gene transfer vectors (also known as pseudoviruses) in mammalian cells. Here we show that papillomavirus capsids produced using this system undergo a maturation process in which the formation of inter-L1 disulfide bonds drives condensation and stabilization of the capsid. Fully mature capsids exhibit improved regularity and resistance to proteolytic digestion. Although capsid maturation for other virus types has been reported to occur in seconds or minutes, papillomavirus capsid maturation requires overnight incubation. Maturation of the capsids of human papillomavirus types 16 and 18 proceeds through an ordered accumulation of dimeric and trimeric L1 species, whereas the capsid of bovine papillomavirus type 1 matures into more extensively cross-linked forms. The presence of encapsidated DNA or the minor capsid protein, L2, did not have major effects on the kinetics or extent of capsid maturation. Immature capsids and capsids formed from L1 mutants with impaired disulfide bond formation are infectious but physically fragile. Consequently, capsid maturation is essential for efficient purification of papillomavirus-based gene transfer vectors. Despite their obvious morphological differences, mature and immature capsids are similarly neutralizable by various L1- and L2-specific antibodies.

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Figures

FIG. 1.
FIG. 1.
Electron micrographs of HPV16 capsids. (A and B) Lysates of cells transfected with HPV16 L1 and L2 expression vectors were incubated for 1 or 24 h, respectively, and then subjected to purification through Optiprep gradients. The samples were then examined by transmission EM. (C) The purified material used in panel A was incubated at 37°C for 24 h and then subjected to EM using the same grid preparation methods and magnification.
FIG. 2.
FIG. 2.
DNA content of Optiprep-purified HPV16 capsids. Nuclease-resistant DNA was extracted from Optiprep-fractionated capsids subjected to prior incubation at 37°C for the time shown. During the extraction, the samples were spiked with 0.5 ng of a 1-kb DNA marker fragment to monitor recovery. The purified DNA samples were digested with BglII, separated on an agarose gel, and stained with SYBR Green I. It was necessary to dilute the sample recovered from the material incubated for 24 h in order to compare it to the 1- and 4-h samples. In the chart at the bottom of the figure, the total titer of the starting crude cell lysate is compared to the titer recovered in the fractionated material. Markers (annotated in kilobase pairs) are shown in the far left lane.
FIG. 3.
FIG. 3.
Western blots of transfected cell lysates. Cells transfected only with a plasmid expressing HPV16 L1 (left panels) or cotransfected with plasmids expressing both HPV16 L1 and L2 (right panels) were lysed in PBS supplemented with Brij 58. The lysate was then incubated for the time shown at the bottom of the figure. (A) The lysate was separated on a nonreducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis gel and then subjected to anti-L1 Western blotting. (B) The lysate was briefly digested with trypsin and then subjected to conventional reducing Western blotting to detect L1. (C) Untreated lysate was subjected to reducing Western blotting to detect L1.
FIG. 4.
FIG. 4.
Western blots of transfected cell lysates. Panels A to C are arranged in the same way as in Fig. 3, except that the far left lane of panel A shows a sample of BPV1 virions purified from infected bovine tissue.
FIG. 5.
FIG. 5.
Nuclease-resistant DNA in transfected cell lysates. Cells transfected with L1 alone or cotransfected with L1 and L2 were lysed in the presence of a nuclease cocktail. DNA was then extracted from the lysate, separated on an agarose gel, and stained with SYBR Green I. (A) Untreated nuclease-resistant DNA samples; (B) the same samples digested with BglII. Purified target plasmid control is shown in the far right lanes.
FIG. 6.
FIG. 6.
Western blots of transfected cell lysates supplemented with GSSG. Cells were cotransfected with HPV16 L1 and L2 and then lysed with Brij 58. Immediately after harvest of the time zero sample, the lysate was spiked with 5 mM GSSG and then incubated for the times shown at the bottom of the figure. (A) Nonreducing Western blot of the samples; (B and C) reducing Western blots of trypsin-digested or untreated lysate, respectively.
FIG. 7.
FIG. 7.
(A to C) Western blots of cells transfected with cysteine mutant L1 constructs. Cells were cotransfected with a construct expressing HPV16 L2 together with wild-type or cysteine mutant L1 constructs C175S or C428S. The cells were lysed and then incubated at 37°C for 10 min or 18 h. The chart at the bottom of the figure shows the titer of the lysates standardized to the titer found in the wild-type L1 lysate after 10 min of incubation at 37°C.
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