A PDI family network acts distinctly and coordinately with ERp29 to facilitate polyomavirus infection

Abstract

Endoplasmic reticulum (ER)-to-cytosol membrane transport is a decisive infection step for the murine polyomavirus (Py). We previously determined that ERp29, a protein disulfide isomerase (PDI) member, extrudes the Py VP1 C-terminal arm to initiate ER membrane penetration. This reaction requires disruption of Py's disulfide bonds. Here, we found that the PDI family members ERp57, PDI, and ERp72 facilitate virus infection. However, while all three proteins disrupt Py's disulfide bonds in vitro, only ERp57 and PDI operate in concert with ERp29 to unfold the VP1 C-terminal arm. An alkylated Py cannot stimulate infection, implying a pivotal role of viral free cysteines during infection. Consistent with this, we found that although PDI and ERp72 reduce Py, ERp57 principally isomerizes the virus in vitro, a reaction that requires viral free cysteines. Our mutagenesis study subsequently identified VP1 C11 and C15 as important for infection, suggesting a role for these residues during isomerization. C11 and C15 also act together to stabilize interpentamer interactions for a subset of the virus pentamers, likely because some of these residues form interpentamer disulfide bonds. This study reveals how a PDI family functions coordinately and distinctly to promote Py infection and pinpoints a role of viral cysteines in this process.

FIG. 1.

ERp57, ERp72, and PDI facilitate Py infection. (A) Knockdown of PDI family members. NIH 3T3 cells were transfected with the indicated siRNA, and lysates derived from the cells were subjected to SDS-PAGE and immunoblotted with the indicated antibodies. (B) Induction of XBP1 splicing. Shown is RT-PCR analysis of the unspliced (u) and spliced (s) forms of the XBP1 mRNA from cells treated with DTT or tunicamycin or transfected with the indicated siRNA. (C) Py infection in knockdown cells. Cells transfected with scrambled siRNA or one of two independent siRNAs against ERp57, ERp72, and PDI (siRNAs 1 and 2) were challenged with Py (100 PFU/cell), and the large T antigen expression was analyzed by standard immunofluorescence microscopy. The values were normalized to scrambled siRNA. The data represent the means and standard deviations (SD) of at least three independent experiments. (D) As for panel A, except the indicated siRNAs were used. (E) As for panel B, except the indicated siRNAs were used. (F) As for panel C, except the indicated siRNAs were used. (G) Average infection of single- and double-knockdown cells. A two-tailed t test was used.

FIG. 2.

ERp57, PDI, and ERp72 disrupt Py's disulfide bonds in vitro. (A) Expression and purification of ERp57, PDI, and ERp72. N-terminally His-tagged ERp57, PDI, and ERp72 constructs were expressed in bacteria, purified to homogeneity, and subjected to Coomassie staining. (B) ERp57 acts on Py directly. Where indicated, purified Py was incubated with ERp57, heat-treated ERp57, or NEM-treated ERp57. The samples were subjected to nonreducing SDS-PAGE and immunoblotted with an I-58 VP1 antibody. The asterisk indicates a nonspecific band recognized by the VP1 antibody. (C) As for panel B, except Py was pretreated at pH 5. (D) PDI acts on Py directly. PDI or NEM-treated PDI was incubated with or without purified Py and analyzed as for panel B. (E) As for panel D, except Py was pretreated at pH 5. (F) ERp72 acts on Py directly. ERp72 or NEM-treated ERp72 was incubated with or without purified Py and analyzed as for panel B. (G) As for panel F, except Py was pretreated at pH 5.

FIG. 3.

ERp57 and PDI function coordinately with ERp29 to unfold Py in vitro. (A) VP1 digestion pattern. Crude Py was incubated with DTT, EGTA, and either BSA or an ER lumenal extract, followed by trypsin addition where indicated. The samples were subjected to SDS-PAGE, followed by immunoblotting with a VP1 antibody. (B) Purified Py was pretreated with either calmodulin, ERp57, ERp72, or PDI in the presence of EGTA. The samples were then incubated with an ERp29-enriched ER lumenal extract, supplemented with trypsin, subjected to SDS-PAGE, and immunoblotted with a VP1 antibody. A 10% input for the amounts of ERp57, PDI, and the ERp29-enriched ER lumenal extract used is shown. (C) As for panel B, except NEM-treated ERp57 and PDI were used where indicated. (D) NIH 3T3 cells transfected with either a rat ERp29 or an N-terminally FLAG-tagged rat ERp29 construct were treated with the DSP cross-linker or left untreated. The resulting cell lysates were subjected to immunoprecipitation using an antibody directed against the FLAG epitope conjugated to agarose (IP:FLAG). The precipitates, as well as the cell lysates (input), were subjected to SDS-PAGE and immunoblotted with antibodies against ERp57 and ERp29. (E) As for panel D, except antibodies against ERp72 and PDI were used instead of antibodies against ERp57.

FIG. 4.

ERp57 principally isomerizes Py, while PDI and ERp72 reduce the virus in vitro. (A) Free cysteines in Py are required for infection. NIH 3T3 cells were incubated with either WT or NEM-treated Py (100 PFU/cell), and the infection efficiency was analyzed as for Fig. 1C. (B) NEM-treated virus is not grossly misfolded. WT and NEM-treated Py were incubated with the indicated concentrations of proteinase K, and the samples were subjected to SDS-PAGE and immunoblotted with a VP1 antibody. (C) ERp57 largely isomerizes Py. WT or NEM-treated Py was incubated with ERp57 or DTT (where indicated), and the samples were analyzed by nonreducing SDS-PAGE, followed by immunoblotting with a VP1 antibody. (D) PDI reduces Py directly. As for panel C, except PDI was used instead of ERp57. (E) ERp72 reduces Py directly. As for panel C, except ERp72 was used instead of ERp57. The asterisk indicates a nonspecific band recognized by the M1 VP1 antibody.

FIG. 5.

Characterization of the C11A, C15A, and C11A-C15A Py mutants. (A) VP1 C11 and C15 are important for Py infection. NIH 3T3 cells were incubated with similar levels of crude WT Py, Py (C11A), Py (C15A), and Py (C11A-C15A), and the infection efficiency was analyzed as for Fig. 1C. (B) Mutant viruses are not misfolded globally. The viruses in panel A were subjected to limited proteolysis as for Fig. 4B. (C) Native agarose gel analyses of mutant Py. The viruses in panel A were subjected to 0.4% native agarose gel electrophoresis, transferred to a nitrocellulose membrane, and immunoblotted with an antibody against VP1. (D) VP1 C11 and C15 mediate interpentamer interaction for a subset of the pentamers. The viruses in panel A were subjected to nonreducing and reducing SDS-PAGE, followed by immunoblotting with an antibody against VP1.