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. 2002 Oct;76(19):10009-14.
doi: 10.1128/jvi.76.19.10009-10014.2002.

Assembly and translocation of papillomavirus capsid proteins

Luise Florin  1 , Cornelia Sapp, Rolf E Streeck, Martin Sapp
Affiliations

Assembly and translocation of papillomavirus capsid proteins

Luise Florin et al. J Virol. 2002 Oct.

Abstract

The major and minor capsid proteins of polyomavirus are preassembled in the cytoplasm and translocated to the nucleus only as a VP1-VP2/VP3 complex. In this study, we describe independent nuclear translocation of the L1 major protein and the L2 minor capsid protein of human papillomavirus type 33 by several approaches. First, we observed that expression and nuclear translocation of L2 in natural lesions precede expression of L1. Second, using a cell culture system for coexpression, we found that accumulation of L2 in nuclear domain 10 (ND10) subnuclear structures precedes L1 by several hours. In contrast, complexes of L2 and mutants of L1 forced to assemble in the cytoplasm are translocated directly to ND10, like L2 expressed alone. Interestingly, accumulation of wild-type L1 is observed only after L2-induced release of the ND10-associated protein Sp100. Third, nuclear translocation of L2 but not of L1 was blocked by the proteasome inhibitor MG132. Our data suggest that L1 and L2 interaction occurs after L2-induced reorganization of ND10 subnuclear domains.

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Figures

FIG. 1.
FIG. 1.
Cellular localization of L1 and L2 proteins in CIN II lesions induced by HPV33. Thin sections were deparaffinized and immunostained for L1 and L2. (A) Cross-section of a lesion stained for L1 using dye precipitation and hematoxylin counterstaining as described previously (15) to indicate the orientation of the sections shown in panels B and C. The pictures in panels B and C were taken of keratinocyte layers at the onset of capsid protein expression. Two magnifications from different areas are shown. Arrows indicate nuclei exclusively displaying L2 staining.
FIG. 2.
FIG. 2.
Cellular localization of wt L1 and L2 proteins. (A) COS-7 cells were infected with vaccinia viruses recombinant for HPV33 L1 or HPV33 L2 and immunostained after 8 h. Colocalization of L2 with promyelocytic leukemia protein (PML) is shown. (B) COS-7 cells were coinfected with both viruses; at the indicated times after infection the cells were fixed, and the capsid proteins were visualized by immunofluorescence.
FIG. 3.
FIG. 3.
Cellular localization of mutant L1 and wt L2 proteins. (A) COS-7 cells were infected for 10 h with recombinant vaccinia virus encoding mutant L1-1/470 or L1-1/470-C427S or were transfected with pEGFPGFP-NLS coding for the HPV33 L1-NLS fused to the carboxy terminus of dimeric GFP (GFP-NLS). Capsid proteins were visualized after 10 h by immunofluorescence. GFP was visualized 40 h after transfection. (B) COS-7 cells were coinfected with recombinant vaccinia viruses encoding wt L2 and mutant L1-1/470, respectively. Capsid proteins were visualized by immunofluorescence at the indicated times after infection. (C) COS-7 cells were infected with recombinant viruses encoding wt L2 and mutant L1-1/470-C427S, respectively. Cells were fixed 8 h after infection, and the capsid proteins were visualized by immunostaining.
FIG. 4.
FIG. 4.
Cellular localization of L1 and Sp100. HuTK− cells were coinfected with recombinant vaccina viruses encoding wt L1 and L2, respectively. Cells were fixed at the indicated times after infection. L1 and Sp100 were visualized by immunostaining.
FIG. 5.
FIG. 5.
Localization of wt L1 and L2 in cells treated with MG132. HuTK− cells were coinfected with vaccinia viruses encoding wt L1 and L2, respectively. MG132 was added 3 h after infection, and the capsid proteins were visualized 6 h later using immunofluorescence. The cells were infected with one virus (A) or two viruses (B). (A) The nuclear dots lacking L1, as seen in the L1 +MG132 panel, represent nucleoli.
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