Possible role for cellular karyopherins in regulating polyomavirus and papillomavirus capsid assembly

Abstract

Polyomavirus and papillomavirus (papovavirus) capsids are composed of 72 capsomeres of their major capsid proteins, VP1 and L1, respectively. After translation in the cytoplasm, L1 and VP1 pentamerize into capsomeres and are then imported into the nucleus using the cellular alpha and beta karyopherins. Virion assembly only occurs in the nucleus, and cellular mechanisms exist to prevent premature capsid assembly in the cytosol. We have identified the karyopherin family of nuclear import factors as possible "chaperones" in preventing the cytoplasmic assembly of papovavirus capsomeres. Recombinant murine polyomavirus (mPy) VP1 and human papillomavirus type 11 (HPV11) L1 capsomeres bound the karyopherin heterodimer alpha2beta1 in vitro in a nuclear localization signal (NLS)-dependent manner. Because the amino acid sequence comprising the NLS of VP1 and L1 overlaps the previously identified DNA binding domain, we examined the relationship between karyopherin and DNA binding of both mPy VP1 and HPV11 L1. Capsomeres of L1, but not VP1, bound by karyopherin alpha2beta1 or beta1 alone were unable to bind DNA. VP1 and L1 capsomeres could bind both karyopherin alpha2 and DNA simultaneously. Both VP1 and L1 capsomeres bound by karyopherin alpha2beta1 were unable to assemble into capsids, as shown by in vitro assembly reactions. These results support a role for karyopherins as chaperones in the in vivo regulation of viral capsid assembly.

FIG. 1.

The karyopherin heterodimer α2β1 binds VP1 and L1 in an NLS-dependent manner. (A) Schematic representation of the mPy VP1 and HPV11 L1 proteins depicting the location of the NLS and the DNA binding domain (DBD). (B) GST-β1 or GST bound to glutathione-Sepharose incubated with VP1 or VP1NΔ6 in the presence of karyopherin α2. (C) L1 or L1CΔ29 in the presence of karyopherin α2. Bound protein complexes were identified by SDS-PAGE and Coomassie staining.

FIG. 2.

Karyopherins differentially affect the DNA binding of VP1 and L1 to DNA. Shown are results for recombinant mPy VP1 or VP1NΔ6 (A) or L1 or L1CΔ29 (B) capsomeres incubated with karyopherins α2, β1, and α2β1 and DNA. (C) Recombinant HPV11 L1 capsomeres incubated with karyopherins and DNA (top panel). Associated proteins were detected with antibodies against L1 (panel second from top), karyopherin α2 (panel second from bottom), or karyopherin β1 (bottom panel). DPC designates migration of the DNA-protein complex. (D) GST-L1 or GST bound to glutathione-Sepharose incubated with karyopherin α2, β1, or α2β1. Bound protein complexes were identified by SDS-PAGE followed by Western blotting.

FIG. 3.

mPy VP1 and HPV11 L1 capsomeres bind DNA and karyopherin α2 simultaneously. Shown are results for recombinant mPy VP1 (A) or HPV11 L1 (B) capsomeres incubated with DNA and karyopherin α2 at equal molar concentrations relative to capsid protein (1×) or karyopherin α2 in molar excess (2×, 4×, 8×, and 16×). DPC designates migration of the DNA-protein complex.

FIG. 4.

DNA competes karyopherin α2 from the NLS of mPy VP1. Shown are EMSA results for VP1-karyopherin α2 binding to DNA in the presence of completing duplexed DNA oligonucleotides ranging from 3 to 24 μM (A) and from 3 to 6 μM (B). DPC designates migration of the DNA-protein complex.

FIG. 5.

Karyopherin α2β1 inhibits in vitro assembly of VP1. Assembly reactions of VP1 with purified karyopherin α2, β1, or α2β1 were visualized by negative staining and TEM. (A) Electron micrograph of a VP1 assembly reaction (top row) or VP1 with karyopherin α2β1 (bottom row) Magnifications, ×4,800 and ×49,000. (B) Electron micrograph of a VP1-karyopherin α2 (top row) or VP1-karyopherin β1 (bottom row) assembly reaction. Magnifications, ×4,800 and ×49,000. (C) Quantitation of the average number of assembled particles per image (n = 6) from four independent assembly reactions. The scale bars are 1 μm at ×4,800 and 100 nm at ×49,000.

FIG. 6.

Karyopherins α2 and α2β1 inhibit in vitro assembly of L1. In vitro assembly reactions of L1 capsomeres in the presence of purified karyopherin α2, β1, or α2β1 were analyzed by electron microscopy. (A) L1 assembly reaction (top row) or L1 assembled in the presence of karyopherin α2β1 (bottom row) Magnifications, ×4,800 and ×49,000. (B) L1-karyopherin α2 (top row) or L1-karyopherin β1 (bottom row) assembly reaction. Magnifications, ×4,800 and ×49,000. (C) Quantitation of the average number of assembled particles per image (n = 5) from three independent assembly reactions. The scale bars are 1 μm at ×4,800 and 100 nm at ×49,000.