The l2 minor capsid protein of human papillomavirus type 16 interacts with a network of nuclear import receptors

Abstract

The L2 minor capsid proteins enter the nucleus twice during viral infection: in the initial phase after virion disassembly and in the productive phase when, together with the L1 major capsid proteins, they assemble the replicated viral DNA into virions. In this study we investigated the interactions between the L2 protein of high-risk human papillomavirus type 16 (HPV16) and nuclear import receptors. We discovered that HPV16 L2 interacts directly with both Kapbeta(2) and Kapbeta(3). Moreover, binding of Ran-GTP to either Kapbeta(2) or Kapbeta(3) inhibits its interaction with L2, suggesting that the Kapbeta/L2 complex is import competent. In addition, we found that L2 forms a complex with the Kapalpha(2)beta(1) heterodimer via interaction with the Kapalpha(2) adapter. In agreement with the binding data, nuclear import of L2 in digitonin-permeabilized cells could be mediated by either Kapalpha(2)beta(1) heterodimers, Kapbeta(2), or Kapbeta(3). Mapping studies revealed that HPV16 L2 contains two nuclear localization signals (NLSs), in the N terminus (nNLS) and C terminus (cNLS), that could mediate its nuclear import. Together the data suggest that HPV16 L2 interacts via its NLSs with a network of karyopherins and can enter the nucleus via several import pathways mediated by Kapalpha(2)beta(1) heterodimers, Kapbeta(2), and Kapbeta(3).

FIG. 1.

HPV16 L2 forms a complex with the Kapα₂β₁ heterodimer via interaction with the Kapα₂ adapter. Purified L2 was subjected to SDS-PAGE and transferred onto a nitrocellulose membrane. Two L2 blots were detected with anti-HPV16 L2 (lane 1) and anti-His (lane 2) antibodies. Other L2 blots were incubated with increasing concentrations of Kapα₂ in the absence (lanes 3 to 5) or the presence (lanes 6 to 11) of 5 μg of GST-Kapβ₁ per ml. Separate L2 blots were incubated with GST-Kapβ₁ alone (lane 12) or with GST as a negative control (lane 13). Bound Kapα₂ was detected with an anti-Kapα₂ antibody (lanes 3 to 8), and bound GST-Kapβ₁ and GST were detected with an anti-GST antibody (lanes 9 to 13).

FIG. 2.

HPV16 L2 interacts specifically with Kapβ₂, and Ran-GTP inhibits the interaction. (A) L2 (lanes 1 to 7), BSA (lane 8), and GST (lane 9) were subjected to SDS-PAGE and transferred onto nitrocellulose membrane. Two L2 blots were detected with anti-HPV16 L2 (lane 1) and anti-His (lane 2) antibodies. Other L2 blots were incubated with increasing concentrations of GST-Kapβ₂ (lanes 4 to 7) or with 2 μg of GST per ml (lane 3). The BSA blot was incubated with 5 μg of GST-Kapβ₂ per ml (lane 8). Bound GST-Kapβ₂ and GST were detected with an anti-GST antibody. (B) L2 blots were incubated with GST-Kapβ₂ in the absence (lane 1) or presence of Ran-GTP (lane 2), and bound GST-Kapβ₂ was detected with an anti-GST antibody. GST-M9 blots were incubated with Kapβ₂ in the absence (lane 4) or presence (lane 5) of Ran-GTP, and bound Kapβ₂ was detected with an anti-Kapβ₂ antibody. GST-M9 was detected with an anti-GST antibody (lane 3).

FIG. 3.

HPV16 L2 interacts specifically with Kapβ₃, and Ran-GTP inhibits the interaction. (A) L2 (lanes 1 to 7), BSA (lane 8), and GST (lane 9) were subjected to SDS-PAGE and transferred onto a nitrocellulose membrane. Two L2 blots were detected with anti-HPV16 L2 (lane 1) and anti-His (lane 2) antibodies. Other L2 blots were incubated with increasing concentrations of GST-Kapβ₃ (lanes 4 to 7) or with 2 μg of GST per ml (lane 3). The BSA blot was incubated with 5 μg of GST-Kapβ₃ per ml (lane 8). Bound GST-Kapβ₃ and GST were detected with an anti-GST antibody. (B) L2 blots were incubated with GST-Kapβ₃ in the absence (lane 1) or presence (lane 2) of Ran-GTP, and bound GST-Kapβ₃ was detected with an anti-GST antibody. GST-M9 blots were incubated with Kapβ₂ in the absence (lane 4) or presence (lane 5) of Ran-GTP, and bound Kapβ₂ was detected with an anti-Kapβ₂ antibody. GST-M9 was detected with an anti-GST antibody (lane 3). (C) Kapβ₃ competes with Kapβ₂ for binding to HPV16 L2 minor capsid protein. L2 blots were incubated with either 5 μg of GST-Kapβ₂ per ml in the absence or presence of increasing concentrations of Kapβ₃ (lanes 3 to 6) or 2 μg of GST-Kapβ₃ per ml in the absence or presence of increasing concentrations of Kapβ₂ (lanes 7 to 10). Bound GST-Kapβ₂ and GST-Kapβ₃ were detected with an anti-GST antibody.

FIG. 4.

HPV16 L2 can enter the nucleus via Kapα₂β₁-, Kapβ₂-, and Kapβ₃-mediated pathways. (A) Digitonin-permeabilized HeLa cells were incubated with either wild-type L2 (panels A to D) or GST-NLS_HPV16L1 (panels E to H) in the presence of either transport buffer (panels A and E), cytosol (panels B and F), Kapβ₁ plus Ran-GDP (panels C and G), or Kapα₂β₁ plus Ran-GDP (panels D and H). Note the nuclear import in panels B, D, F, and H. (B) Digitonin-permeabilized HeLa cells were incubated with wild-type L2 in the presence of either transport buffer (panel A), Ran-GDP (panel B), Kapβ₂ plus Ran-GDP (panel C), or Kapβ₃ plus Ran-GDP (panel D). Note the nuclear import in panels C and D.

FIG. 5.

Both the nNLS and cNLS of HPV16 L2 can mediate the nuclear import of a GST reporter protein. Digitonin-permeabilized HeLa cells were incubated with either GST-nNLS (panels A and E), GST-mNLS (panels B and F), GST-cNLS (panels C and G), or GST (panels D and H) in the presence of either only transport buffer (panels A to D) or HeLa cytosol (panels E to H). Note the nuclear import in panels E and G.

FIG. 6.

Deletion of both the N and C termini of HPV16 L2 abolishes interaction with the Kapα₂ adapter. Blots containing wild-type L2 protein (lanes 1 and 7), L2ΔN mutant protein (lanes 2 and 8), L2ΔM mutant protein (lanes 3 and 9), L2ΔC mutant protein (lanes 4 and 10), L2ΔNΔC double mutant protein (lanes 5 and 10), and L2ΔNΔMΔC triple mutant protein (lanes 6 and 12) were either probed with an anti-His antibody (lanes 1 to 6) or incubated with 2.5 μg of Kapα₂ per ml, and bound Kapα₂ was detected with an anti-Kapα₂ antibody (lanes 7 to 12).

FIG. 7.

Both the nNLS and cNLS of HPV16 L2 interact with the Kapα₂ adapter and form a complex with Kapα₂β₁ heterodimers. (A) Blots containing GST-nNLS_16L2 (lanes 2 to 4), GST-mNLS_16L2 (lanes 6 to 8), GST-cNLS_16L2 (lanes 10 to 12), GST-NLS_16L1 (positive control; lanes 14 to 16), or GST (negative control; lane 18) were incubated with increasing amounts of Kapα₂, and bound Kapα₂ was detected with an anti-Kapα₂ antibody. The different GST-NLS proteins and GST were detected with an anti-GST antibody (lanes 1, 5, 9, 13, and 17, respectively). (B) GST-nNLS_16L2 (lanes 1 to 3), GST-cNLS_16L2 (lanes 4 to 6), GST-NLS_HPV16L1 (lanes 7 to 9), and GST (lanes 10 to 12) immobilized on glutathione-Sepharose (2 μg/10 μl of beads) were incubated with either Kapα₂ (lanes 1, 4, 7, and 10), Kapβ₁ (lanes 2, 5, 8, and 11), or Kapβ₁ plus Kapα₂ (lanes 3, 6, 9, and 12). Bound proteins were eluted with sample buffer and analyzed by SDS-PAGE and Coomassie blue staining. The input of Kapα₂ and Kapβ₁ is shown in lanes 13 and 14.

FIG. 8.

The nNLS of HPV16 L2 efficiently mediates the nuclear import of a GST reporter via the Kapα₂β₁ pathway. Digitonin-permeabilized cells were incubated with either GST-nNLS (panels A to C), GST-cNLS (panels D to F), or GST-NLS_HPV16L1 (panels G to I) in the presence of either transport buffer (panels A, D, and G), Kapβ₁ plus Ran-GDP (panels B, E, and H), or Kapα₂ plus Kapβ₁ plus Ran-GDP (panels C, F, and I). Nuclear import of the GST-NLSs was detected with an anti-GST antibody.

FIG. 9.

The N terminus of HPV16 L2 is essential for the interaction with Kapβ₂ and Kapβ₃ nuclear import receptors. (A) Blots containing wild-type L2 protein (lanes 1 and 7), L2ΔN mutant protein (lanes 2 and 8), L2ΔM mutant protein (lanes 3 and 9), L2ΔC mutant protein (lanes 4 and 10), L2ΔNΔC double mutant protein (lanes 5 and 10), and L2ΔNΔMΔC triple mutant protein (lanes 6 and 12) were either probed with anti-His antibody (lanes 1 to 6) or incubated with 10 μg of GST-Kapβ₂ per ml, and bound GST-Kapβ₂ was detected with an anti-GST antibody (lanes 7 to 12). (B) Blots containing wild-type L2 protein (lanes 1 and 7), L2ΔN mutant protein (lanes 2 and 8), L2ΔM mutant protein (lanes 3 and 9), L2ΔC mutant protein (lanes 4 and 10), L2ΔNΔC double mutant protein (lanes 5 and 10), and L2ΔNΔMΔC triple mutant protein (lanes 6 and 12) were either probed with an anti-His antibody (lanes 1 to 6) or incubated with 10 μg of GST-Kapβ₃ per ml, and bound GST-Kapβ₃ was detected with an anti-GST antibody (lanes 7 to 12).

FIG. 10.

The nNLS is essential for Kapβ₂- or Kapβ₃-mediated nuclear import of HPV16 L2. Digitonin-permeabilized HeLa cells were incubated with either wild-type L2 protein (panels A to C) or L2ΔN mutant protein (panels D to F) in the presence of either transport buffer (panels A and D), Kapβ₂ plus Ran-GDP (panels B and E), or Kapβ₃ plus Ran-GDP (panels C and F). Note the nuclear import in panels B and C.