A nucleotide exchange factor promotes endoplasmic reticulum-to-cytosol membrane penetration of the nonenveloped virus simian virus 40

Abstract

The nonenveloped simian polyomavirus (PyV) simian virus 40 (SV40) hijacks the endoplasmic reticulum (ER) quality control machinery to penetrate the ER membrane and reach the cytosol, a critical infection step. During entry, SV40 traffics to the ER, where host-induced conformational changes render the virus hydrophobic. The hydrophobic virus binds and integrates into the ER lipid bilayer to initiate membrane penetration. However, prior to membrane transport, the hydrophobic SV40 recruits the ER-resident Hsp70 BiP, which holds the virus in a transport-competent state until it is ready to cross the ER membrane. Here we probed how BiP disengages from SV40 to enable the virus to penetrate the ER membrane. We found that nucleotide exchange factor (NEF) Grp170 induces nucleotide exchange of BiP and releases SV40 from BiP. Importantly, this reaction promotes SV40 ER-to-cytosol transport and infection. The human BK PyV also relies on Grp170 for successful infection. Interestingly, SV40 mobilizes a pool of Grp170 into discrete puncta in the ER called foci. These foci, postulated to represent the ER membrane penetration site, harbor ER components, including BiP, known to facilitate viral ER-to-cytosol transport. Our results thus identify a nucleotide exchange activity essential for catalyzing the most proximal event before ER membrane penetration of PyVs.

Importance: PyVs are known to cause debilitating human diseases. During entry, this virus family, including monkey SV40 and human BK PyV, hijacks ER protein quality control machinery to breach the ER membrane and access the cytosol, a decisive infection step. In this study, we pinpointed an ER-resident factor that executes a crucial role in promoting ER-to-cytosol membrane penetration of PyVs. Identifying a host factor that facilitates entry of the PyV family thus provides additional therapeutic targets to combat PyV-induced diseases.

FIG 1

Release of BiP from SV40 promotes viral membrane binding in vitro. (A) ER-localized SV40 immunoprecipitated from infected CV-1 cells, along with the indicated amounts of purified SV40 (100, 200, and 300 ng of VP1) and purified recombinant BiP (5, 10, 20, and 30 ng), was separated by SDS-PAGE. The BiP band was visualized by silver staining and the VP1 band by Coomassie staining. The amounts of VP1 and BiP in ER-localized SV40 were quantified with ImageJ (NIH) using purified SV40 and BiP as standards in order to calculate the numbers of BiP molecules per viral particle. (B) ER-localized SV40 processed as described for panel A was incubated without ATP (i.e., BiP-bound SV40) or with ATP (2 mM) to generate BiP-less SV40. Following reprecipitation of the virus, the samples were subjected to SDS-PAGE and silver staining. (C) BiP-bound or BiP-less SV40 processed as described for panel B was incubated with liposomes labeled with avidin via a biotinylated phospholipid. Following reprecipitation of the virus, the samples were subjected to SDS-PAGE and analyzed by silver staining for the biotin-avidin complex and by immunoblotting with anti-VP1 antibodies.

FIG 2

Grp170 induces release of ER-localized SV40 from BiP in vitro. (A) Coomassie staining of C-terminally FLAG-tagged GFP (GFP-FLAG), C-terminally FLAG-tagged Grp170 (Grp170-FLAG), and N-terminally FLAG-tagged Sil1 (FLAG-Sil1) purified from 293T cells. (B) ER-localized SV40 immunoprecipitated from infected CV-1 cells was incubated without ATP or with ATP (0.2 μM) and the indicated recombinant protein (250 nM). Following reprecipitation of the virus, the samples were subjected to SDS-PAGE and silver staining. The black line indicates that intervening lanes have been spliced out. (C) The experiment was performed as described for panel B, except the immunoprecipitate was incubated with Grp170-FLAG in the absence of ATP where indicated. (D) Coomassie staining of purified C-terminally FLAG-tagged BiP (BiP-FLAG). (E) BiP-FLAG (300 nM) was incubated with the indicated recombinant protein (300 nM) and immunoprecipitated with an anti-BiP antibody. The immunoprecipitates were subjected to SDS-PAGE followed by immunoblotting with an anti-FLAG antibody. Input samples were also analyzed by immunoblotting with an anti-FLAG antibody. (F) Coomassie staining of purified N-terminally FLAG-tagged BiP (FLAG-BiP). (G) FLAG-BiP was incubated with [α-³²P]ATP to form the radiolabeled ADP-BiP complex. The ADP-BiP complex was then incubated with the indicated proteins in the absence or presence of unlabeled ATP. ADP release from BiP was analyzed by TLC. (H) BiP-less ER-localized SV40 was prepared as described for panel B. The ability of BiP-bound or BiP-less ER-localized SV40 to bind to liposomes was assessed as described for Fig. 1C.

FIG 3

Grp170 promotes SV40 ER-to-cytosol membrane transport. (A) Infected CV-1 cells transfected with the indicated siRNA were harvested and were subjected to the ER-to-cytosol membrane transport assay as described by Inoue and Tsai (14). Cytosol, membrane, and Triton X-100 fractions were subjected to SDS-PAGE followed by immunoblotting with the indicated antibodies. (B) The VP1 band intensity in the cytosolic fraction represented in panel A was quantified with ImageJ (NIH). Data represent means ± standard deviations (SD) of the results of at least 3 independent experiments. (C) Reverse transcription-PCR (RT-PCR) analysis of the unspliced (u) and spliced (s) forms of the XBP1 mRNA from cells transfected with the indicated siRNA or treated with DTT. (D) CV-1 cells initially transfected with scrambled or Grp170 siRNA no. 1 were subsequently transfected with BiP-S, infected with SV40 for 18 h, and harvested. Whole-cell extracts (WCEs) were incubated with S-protein-conjugated beads, and the isolated proteins subjected to SDS–PAGE followed by immunoblotting with anti-VP1 and anti-BiP antibodies. AP, affinity purification.

FIG 4

Grp170 supports polyomavirus infection. (A) Large T-antigen-positive nuclei were scored in SV40- or human BK PyV-infected CV-1 cells transfected with the indicated siRNA. Data represent means ± SD of the results of at least 3 independent experiments. A Student two-tailed t test was used for the SV40 infection data. (B) The experiment was performed as described for Fig. 2G, except that G41L Grp170-FLAG was used. Purified G41L Grp170-FLAG results are also shown. (C) Grp170-FLAG or G41L Grp170-FLAG was immunoprecipitated (IP), and the samples were subjected to immunoblotting with the indicated antibodies. (D) Luciferase was incubated with BSA, Grp170-FLAG, or G41L Grp170-FLAG at 42°C for 30 min and centrifuged. The resulting supernatant and pellet fractions were subjected to SDS-PAGE and analyzed by Coomassie staining. The black line indicates that intervening lanes have been spliced out. (E) CV-1 cells initially transfected with scrambled or Grp170 siRNA no. 1 were subsequently transfected with GFP-FLAG, Grp170-FLAG, G41L Grp170-FLAG, or FLAG-Sil1. Cells were then infected with SV40 and subjected to immunofluorescence analyses using anti-FLAG and anti-T-antigen antibodies. FLAG and T-antigen doubly positive cells were counted and analyzed as described for panel A. Data represent means ± SD of the results of at least 3 independent experiments. A Student two-tailed t test was used. (F) WCEs derived from cells processed as described for panel E were analyzed by SDS-PAGE and immunoblotted with the indicated antibodies. (G) COS-7 cells transfected with the indicated DNA construct were infected with SV40 for 12 h and subjected to the ER-to-cytosol membrane transport assay as described for Fig. 3A. The cytosol fraction was subjected to SDS-PAGE, followed by immunoblotting with the indicated antibodies.

FIG 5

SV40 triggers Grp170 to reorganize into foci in the ER. (A) Cells transfected with FLAG-BiP were mock infected or infected with SV40 for 16 h, fixed, and subjected to immunofluorescence staining with anti-FLAG and BAP31 antibodies. The bar represents 20 μm. (B) Cells were processed as described for panel A, except Grp170-FLAG was used. (C) Cells transfected with scrambled or Grp170 siRNA no. 1 were processed as described for panel A, except endogenous (endo) BiP antibodies were used.