Degradation of Nup62 during HRV2 infection. A, schematic representation of Nup62 and regions recognized by anti-Nup62 antibodies. Vertical white bars indicate the location of FG repeats. Nup62(N) and Nup62(C) indicate the regions of Nup62 used to raise antibodies to the N and C terminus, respectively. B, HRV2 infection induces Nup62 degradation. Twenty-five μg of whole cell lysates prepared from mock-infected cells or cells that had been infected with HRV2 for the indicated length of time were analyzed by immunoblotting with Nup62(N) or Nup62(C). Immunoblots were stripped and reprobed to detect nucleolin and eIF4GI. Molecular mass markers are indicated in kilodaltons. % remaining, band intensities were quantitated by densitometry, and the amounts relative to mock-infected cells are indicated. ‡, eIF4GI cleavage products. longer exposure, a longer exposure of the Nup62(N) blot.

HRV2 2A^pro induces cleavage of Nup62. A, 2A^pro causes cleavage of Nup62 in cell lysates. Uninfected HeLa whole cell lysates were incubated in the presence or absence of the indicated amount of purified HRV2 2A^pro for 4 h at 30 °C. After incubation, extracts were analyzed by immunoblotting to detect Nup62 using mAb414. Immunoblots were sequentially stripped and reprobed to detect Nup155 and eIF4GI. B, cleavage kinetics of in vitro Nup62 degradation. Uninfected HeLa whole cell lysates were incubated with or without 0.1 μg of purified HRV2 2A^pro for the indicated times and analyzed for cleavage of Nup62 by immunoblotting with Nup62(N). Immunoblots were sequentially stripped and reprobed with anti-eIF4G antibody and anti-nucleolin antibody. Cleavage products are indicated with an asterisk. C, Nup62 is resistant to cleavage by 3C^pro. Uninfected HeLa whole cell lysates were incubated in the presence or absence of the indicated amount of purified HRV14 3C^pro at 30 °C for the indicated amount of time. After incubation, extracts were analyzed by immunoblotting to detect Nup62 using Nup62(N). The immunoblot was then stripped and reprobed to detect NF-κB. % remaining, band intensities were quantitated by densitometry, and the amounts relative to mock-infected cells are indicated.

2A^pro cleaves Nup62 directly. A, cleavage of in vitro translated Nup62 by 2A^pro. Radiolabeled Nup62 was generated by in vitro translation in the presence of [³⁵S]methionine. 1 μl of the translation reaction was either prepared for loading immediately (0 h incubation) or incubated for 4 h at 30 °C in the presence or absence of 0.3 μg of purified 2A^pro. After incubation, samples were electrophoresed, transferred to PVDF membrane, and analyzed by autoradiography. B, 2A^pro can cleave purified Nup62. Nup62 was expressed and purified from bacteria and then incubated with or without 0.3 μg of purified 2A^pro for the indicated times at 30 °C. In some cases, 100 μm N-(methoxysuccinyl)-Ala-Ala-Pro-Val-chloromethyl ketone (MPCMK) was included to inhibit 2A^pro activity. After incubation, samples were electrophoresed, transferred to PVDF membrane, and stained with Coomassie Brilliant Blue. C, characterization of in vitro Nup62 cleavage products. Nup62 was purified from bacteria and incubated in the presence or absence of 0.3 μg of purified HRV2 2A^pro for the indicated times at 30 °C. After incubation, immunoblotting was performed with Nup62(N), followed by striping and reprobing with Nup62(C) to detect N- and C-terminal cleavage products, respectively. Untreated, 16-h incubation of purified Nup62 in PBS. Reaction buffer, 16-h incubation of purified Nup62 in 2A^pro reaction buffer without protease. Cleavage products reactive with Nup62(N) are indicated by normal type, those reactive with Nup62(C) are indicated in boldface type, and those reactive with both antibodies are underlined. Molecular mass markers are indicated in kilodaltons.

Identification of 2A^pro cleavage sites in Nup62. A, analysis of 2A^pro cleavage sites identified by N-terminal sequencing or sequence homology. In vitro translated radiolabeled WT or mutant Nup62s were incubated with 0.3 μg of purified 2A^pro for 4 h at 30 °C. Constructs are designated by the P1′ amino acid and its location in the Nup62 primary sequence followed by the mutant amino acid inserted at that position. Cleavage products that change in abundance due to a mutation are indicated with an asterisk. Load, the amount of full-length protein used in each cleavage assay. Molecular mass markers are indicated in kilodaltons. B, schematic representation of Nup62 showing the location of cleavage sites and corresponding cleavage products. The amino acid position of identified cleavage sites are indicated along with corresponding cleavage products and their predicted molecular mass and cp designation (as shown in Fig. 3A). Cleavage products that arise due to processing at unidentified cleavage sites are indicated by a gray bar.

Nup62 is cleaved at Ala¹⁰³ in HRV2-infected cells. Plasmids encoding GFP or GFP fused to wild type or mutant Nup62 (GFP-Nup62Wt or GFP-NUP62A103D, respectively) were transfected into HeLa cells, and 24 h later were infected with HRV2. Whole cell lysates prepared 18 h later were electrophoresed, transferred to PVDF membrane, and analyzed by immunoblotting with antibodies recognizing GFP, Nup62(N), eIF4GI, or nucleolin. Molecular mass markers are indicated in kilodaltons. None, cells were treated with transfection reagent without plasmid. Cleavage product arising due to proteolysis at Ala¹⁰³ is indicated with an asterisk.

Nup62 association with the NPC in infected cells. A, HRV2-infected cells. Indirect immunofluorescence was performed with mAb414 (N terminus) and Nup62(C) (C terminus) on cells that had been infected with HRV2 for the indicated amount of time. Seven different fields were analyzed, and the percentage of cells showing the indicated pattern of N- and C-terminal staining is indicated. B, poliovirus-infected cells. Cells infected with poliovirus were analyzed as described above. mAb414 and Nup62(C) were detected with FITC and TRITC filters, respectively. Hoechst-stained DNA (Nuclei) was visualized with a UV filter. All images were acquired with identical exposure times and adjustments.