Fig 1.
B12 binds to the ER membrane proteins Erlin1 and Erlin2.
(A) Parental Flp-In 293 T-REx cells and cell lines stably expressing 3xFLAG-B12 were lysed and the resulting whole cell extracts subjected to SDS-PAGE followed by immunoblotting with an antibody against B12. (B) Strategy used to identify B12-interacting partners. (C) Cell lysates derived from the indicated cell lines were subjected to affinity purification as depicted in B, and the precipitated material analyzed with SDS-PAGE followed by silver staining. (D) Cell lysates derived from the indicated cell lines were subjected to FLAG immunoprecipitation, and the precipitated samples eluted with SDS sample buffer and analyzed by immunoblotting with the indicated antibodies.
Table 1.
A list of proteins identified with more than 20 peptides from the 3xFLAG-B12 affinity purified sample.
Fig 2.
Erlin1 and Erlin2 execute critical functions during SV40 infection.
(A) CV-1 cells were transfected with the indicated siRNAs, fractionated with digitonin, and the resulting membrane fractions subjected to SDS-PAGE, followed by immunoblotting with the indicated antibodies. (B) Reverse transcription-PCR (RT-PCR) analysis of the unspliced (u) and spliced (s) forms of the XBP1 mRNA derived from CV-1 cells transfected with the indicated siRNA or treated with DTT. (C) Large T antigen-positive nuclei were scored in SV40-infected CV-1 cells transfected with the indicated siRNAs. Data are normalized against the scrambled siRNA, and represent the means ± standard deviations of data from at least 3 independent experiments. Student two-tailed t test was used to determine statistical significance. (D) CV-1 cells initially transfected with scrambled or PAN-Erlin siRNA were subsequently transfected with GFP-FLAG, Erlin1*-FLAG, or Erlin2*-FLAG. Cells were then infected with SV40 for 24 h and subjected to immunofluorescence analyses using antibodies against FLAG and large T antigen. Only cells expressing the indicated FLAG protein were scored for large T antigen expression. Data are normalized against cells that were initially transfected with scrambled siRNA and subsequently transfected with GFP-FLAG, and represent the means ± standard deviations of data from at least 3 independent experiments. Student two-tailed t test was used to determine statistical significance.
Fig 3.
Erlin1 and Erlin2 support SV40 ER-to-cytosol transport and virus-induced foci formation.
(A) CV-1 cells transfected with the indicated siRNA were infected with SV40 for 12 h, harvested, treated with digitonin, and centrifuged. The resulting supernatant fraction containing cytosolic proteins (cytosol) and the pellet fraction containing membranous organelles (membrane) were analyzed by SDS-PAGE followed by immunoblotting with the indicated antibodies. (B) The VP1 band intensity in the cytosol fraction in A was quantified using ImageJ (NIH). Data were normalized against cells transfected with scrambled siRNA, and represent the means ± standard deviations of data from at least 3 independent experiments. Student two-tailed t test was used to determine statistical significance. (C) The membrane fraction in A was further solubilized with Triton X-100 and centrifuged. The resulting supernatant fractions (Triton X-100 soluble membrane) containing ER-localized SV40 were analyzed by immunoblotting with the indicated antibodies. (D) CV-1 cells transfected with the scrambled or PAN-Erlin siRNA were infected with SV40 for 12 h, and subjected to a fractionation approach using digitonin as in A. The membrane fraction was further solubilized with Triton X-100, and centrifuged as in C. The resulting supernatant fraction was analyzed by immunoblotting with the indicated antibodies. (E) The VP1 band intensity of ER-localized SV40 in cells transfected with scrambled or PAN-Erlin siRNA in C and D was quantified using ImageJ (NIH). Data were normalized against cells transfected with the scrambled siRNA, and represent the means ± standard deviations of data from at least 3 independent experiments. Student two-tailed t test was used to determine statistical significance. (F) CV-1 cells transfected with scrambled or PAN-Erlin siRNA were infected with SV40 for 8 h, fixed, and subjected to immunofluorescence staining with antibodies against B12 and BAP31, and DAPI (4, 6-diamidino-2-phenylindole) staining. Images were taken by epifluorescence microscopy. Arrows indicate SV40-induced foci. Bar represents 10 μm. (G) Numbers of cells with at least one B12 and BAP31 double-positive focus in SV40-infected cells transfected with the indicated siRNA were counted. Data are normalized against cells transfected with the scrambled siRNA, and represent the means ± standard deviations of data from at least 3 independent experiments. Student two-tailed t test was used to determine statistical significance.
Fig 4.
Interactions with Erlin1 and Erlin2 are critical for B12 to mobilize into the foci and support infection.
(A) Schematics of the N-terminal FLAG-tagged B12 constructs used in this figure. Wild type B12 (B12 [WT]) is composed of 375 amino acids with three domains: J-domain (J, 112–176 aa), glycine/phenylalanine-rich domain (G/F, 184–213 aa) and transmembrane domain (TM, 244–264 aa). Hsp70-binding defective B12 (B12 [H138Q]) harbors a mutation in which histidine at position 138 is changed to glutamine. Mutant B12 (B12 [1–355]) lacks the last 20 amino acids from the C-terminus. (B) 293T cells transfected with the indicated FLAG-tagged constructs were lysed and the resulting whole cell lysates were subjected to immunoprecipitation using FLAG antibody-conjugated agarose beads. Whole cell lysate (input) and immunoprecipitated material were analyzed by immunoblotting with the indicated antibodies. (C) CV-1 cells transfected with the indicated FLAG-tagged constructs were infected with SV40 for 16 h, fixed, and subjected to the immunofluorescence assay using antibodies against BAP31 and FLAG. Images were taken by epifluorescence microscopy. Inset shows a 3x enlarged image corresponding to the enclosed white square. Bar represents 10 μm. (D) Numbers of cells with at least one BAP31 and FLAG double-positive focus in SV40-infected cells were counted, as in Fig 3E. Data are normalized against cells transfected with FLAG-B12 [WT], and represent the means ± standard deviations of data from at least 3 independent experiments. Student two-tailed t test was used to determine statistical significance. (E) Parental and B12 CRISPR KO CV-1 cells were lysed and the resulting whole cell lysates were analyzed by immunoblotting with the indicated antibodies. (F) Parental and B12 CRISPR KO CV-1 cells transfected with the indicated FLAG-tagged constructs were infected with SV40 and subjected to immunofluorescence analysis using antibodies against FLAG and large T antigen. FLAG and large T antigen double-positive cells were counted and analyzed as in Fig 2D. Data are normalized against parental cells transfected with GFP-FLAG, and represent the means ± standard deviations of data from at least 3 independent experiments. Student two-tailed t test was used to determine statistical significance.
Fig 5.
Erlin1 and Erlin2 do not reorganize into the SV40-induced foci structures.
(A) CV-1 cells were infected with SV40 for 16 h, fixed, and subjected to immunofluorescence analysis using antibodies against BAP31 and Erlin1. Images were taken by epifluorescence microscopy. Inset shows a 3x enlarged image corresponding to the enclosed white square. Bar represents 10 μm. (B) CV-1 cells transfected with Erlin2*-FLAG were infected with SV40 for 16 h and analyzed as in A, except that antibodies against B12, BAP31, and FLAG were used.
Fig 6.
SV40 induces the release of B12 from Erlin1 and Erlin2.
(A) CV-1 cells transfected with GFP-FLAG or Erlin2-FLAG were mock-infected or infected with SV40 for 16 h in the absence or presence of BFA. Cells were then harvested and lysed. The resulting whole cell lysates were subjected to immunoprecipitation using FLAG antibody-conjugated agarose beads and the immunoprecipitated samples analyzed by immunoblotting with the indicated antibodies. (B) As in A, except that Erlin1-FLAG was used. (C) As in A, except that an antibody against TMUB1 was used for immunoblotting. (D) The immunoprecipitated B12 band intensity in A was quantified using ImageJ (NIH). Data were normalized against mock-infected cells expressing Erlin2-FLAG, and represent the means ± standard deviations of data from at least 3 independent experiments. Student two-tailed t test was used to determine statistical significance. (E) As in D, except the immunoprecipitated B12 band intensity in B was quantified. (F) As in D, except the immunoprecipitated TMUB1 band intensity in C was quantified. (G) SV40 (WT) and SV40 (ΔVP3) were analyzed with SDS-PAGE, followed by immunoblotting with antibodies against VP1 and VP3. (H) As in A, except that CV-1 cells transfected with Erlin2-FLAG were mock-infected, infected with SV40 (WT), or infected with SV40 (ΔVP3) for 16 h. The immunoprecipitated B12 band intensity in A was quantified using ImageJ (NIH) and shown below the B12 band.
Fig 7.
Trapping B12 to Erlin2 prevents B12 from mobilizing into SV40-induced foci and blocks infection.
(A) A schematic describing the FKBP-FRB dimerization system used in this study. B12’s last 20 amino acids from the C-terminus that interact with Erlin1/2 are indicated in red. Although B12-FRB-S binds to Erlin2-FKBP-FLAG, B12-FRB-S is expected to dissociate from Erlin2-FKBP-FLAG during SV40 infection in the absence of raplog-1. However, in the presence of raplog-1 during SV40 infection, B12-FRB-S is trapped with Erlin2-FKBP-FLAG due to FKBP-FRB dimerization. (B) B12 CRISPR KO cells transfected with B12 [WT]-FRB-S with or without Erlin2-FKBP-FLAG were infected with SV40 or mock-infected for 6 h, and further incubated in the presence or absence of rapalog-1 for 10 h. Cells were harvested, lysed, and the resulting cell lysates subjected to immunoprecipitation using FLAG antibody-conjugated agarose beads. The immunoprecipitated samples were analyzed by immunoblotting with the indicated antibodies. (C) Parental and B12 CRISPR KO cells transfected with a vector co-expressing Erlin2-FKBP-FLAG and either GFP-S, B12 [WT]-S, or B12 [WT]-FRB-S were infected with SV40 for 6 h, further incubated in the absence or presence of rapalog-1 for 18 h, and subjected to immunofluorescence analysis using antibodies against S-tag and large T antigen. S-tag and large T antigen double-positive cells were counted and analyzed as in Fig 2C. Data are normalized against parental cells co-expressing Erlin2-FKBP-FLAG and GFP-S in the absence of rapalog-1, and represent the means ± standard deviations of data from at least 3 independent experiments. Student two-tailed t test was used to determine statistical significance. (D) B12 CRISPR KO cells transfected with a vector co-expressing Erlin2-FKBP-FLAG and B12 [WT]-FRB-S were infected with SV40 for 6 h, further incubated in the absence or presence of rapalog-1 for 10 h, and subjected to immunofluorescence analysis using antibodies against S-tag and BAP31. Images were taken by epifluorescence microscopy. Inset shows a 3x enlarged image corresponding to the enclosed white box. Bar represents 10 μm. (E) Numbers of cells with at least one S-tag and BAP31 double-positive focus in SV40-infected cells co-expressing Erlin2-FKBP-FLAG and either B12 [WT]-S or B12 [WT]-FRB-S in the absence or presence of rapalog-1 were counted. Data are normalized against cells co-expressing Erlin2-FKBP-FLAG and B12 [WT]-S in the absence of rapalog-1, and represent the means ± standard deviations of data from at least 3 independent experiments. Student two-tailed t test was used to determine statistical significance.
Fig 8.
The Erlin1 and Erlin2 complex anchors B12 in the ER.
(A) CV-1 cells transfected with scrambled or PAN-Erlin siRNA were fixed and subjected to immunofluorescence analysis using antibodies against B12 and BAP31. Images were taken by epifluorescence microscopy. Bar represents 10 μm. (B) Cells that have at least five B12-positive aggregates in the nuclei were scored. Data represent the means ± standard deviations of data from at least 3 independent experiments. Student two-tailed t test was used to determine statistical significance. (C) CV-1 cells transfected with scrambled or PAN-Erlin siRNA were infected with SV40, fixed, and subjected to immunofluorescence analysis using antibodies against B12 and large T antigen. Large T antigen-positive nuclei were analyzed as Fig 2C, except that in cells transfected with PAN-siRNA, the number of large T antigen-positive nuclei in cells displaying at least five B12-positive aggregates were counted. At least 300 nuclei from scrambled and PAN Erlin siRNA-transfected cells (total) and at least 100 nuclei from PAN Erlin siRNA-transfected cells displaying B12-positive nuclear aggregates were counted. Data are normalized against cells transfected with scrambled siRNA, and represent the means ± standard deviations of data from at least 3 independent experiments. Student two-tailed t test was used to determine statistical significance. (D) Cells transfected with truncated CMV-driven FLAG-B12 [WT] or FLAG-B12 [1–355] were fixed and subjected to immunofluorescence analysis using antibodies against FLAG. Cells with at least five B12-positive aggregates in the nuclei were scored as in A. Data represent the means ± standard deviations of data from at least 3 independent experiments. Student two-tailed t test was used to determine statistical significance.
Fig 9.
Model depicting the role of the Erlin1/2-B12 interaction during SV40 ER membrane penetration.
The Erlin1 and Erlin2 (Erlin1/2) complex is identified as a binding partner of B12. When SV40 traffics from the cell surface to the ER, it triggers the release of B12 from the Erlin1/2 complex (step 1). The released B12 in turn mobilizes to the foci (step 2) where B12 promotes ER-to-cytosol membrane penetration of SV40 by recruiting a cytosolic extraction complex [6,21] that ejects the virus into the cytosol. In the absence of the Erlin1/2 complex, B12 can mislocalize to the nucleus, suggesting that Erlin1/2 acts to anchor B12 in the ER.