Figure 1.
(A) NIH-3T3 cells were infected with MCMV at an MOI of 5 or treated with tunicamycin (Tun) for 4 h. Xbp1 mRNA transcripts were amplified by RT-PCR, digested with PstI, and separated on an ethidium bromide-stained agarose gel. The spliced transcript, Xbp1s, lacks the PstI site and migrates slower than the digested unspliced transcript, Xbp1u. (B) NIH-3T3 cells were treated as described for panel A. Xbp1s and Xbp1u transcripts were quantified by real-time RT-PCR. Changes in the Xbp1s/Xbp1u ratio relative to untreated cells are plotted as bar diagram showing means ±SEM of four replicates. (C) NIH-3T3 cells were infected as above and treated in addition with Tun for the last 4 h before harvest. Xbp1 transcripts were analyzed as in A. (D) NIH-3T3 cells were treated as in C, and Xbp1 transcripts were quantified as in B. (E) NIH-3T3 cells were MCMV-infected and treated with thapsigargin (Tg) for the last 4 h before harvest. Xbp1 transcripts were quantified as in B. (F) NIH-3T3 cells were MCMV-infected and treated with Tun for the last 4 h before harvest. Nuclear protein extracts were analyzed by immunoblot for the presence of XBP1s protein. Heterochromatin protein 1α (HP1α) was used as a loading control. (G) NIH-3T3 cells were infected with MCMV at an MOI of 5 and treated with Tun for 4 h. ERdj4 transcripts were quantified by real-time RT-PCR. Results are shown as fold induction relative to untreated cells (means ±SEM of four replicates).
Figure 2.
(A) NIH-3T3 cells stably expressing TEV-HA-tagged IRE1 were mock infected or infected with MCMV at an MOI of 5. Whole cell lysates (WCL) were applied to an anti-HA sepharose matrix. IRE1 and interacting proteins were eluted by TEV protease digestion, separated by SDS-PAGE, and silver stained. Specific bands (arrow heads) were excised and analyzed by protein mass spectrometry. (B) 293A cells were cotransfected with expression plasmids for IRE1-HA and Flag-tagged M50, m144, or Calnexin (CNX), respectively. IRE1 was subjected to immunoprecipitation (IP) with an anti-HA antibody. IRE1 and coexpressed proteins were detected by immunoblot in IP samples and WCL using anti-HA and anti-Flag antibodies, respectively. (C) 293A cells were cotransfected with expression plasmids for IRE1-HA and Flag-tagged M50 or m144, respectively. M50 and m144 were precipitated with an anti-Flag antibody. IRE1 and coexpressed proteins were detected in IP samples and WCL as described above. (D) 10.1 fibroblasts transduced with a retroviral vector expressing myc-tagged IRE1 were infected with an MCMV expressing HA-tagged M50 or m41 at an MOI of 4. At the indicated time points IRE1 was immunoprecipitated with an anti-myc antibody, and HA-tagged proteins were detected by immunoblot. (E) NIH-3T3 cells were infected with the same viruses as in D. After 24 h, M50 and m41 proteins were immunoprecipitated with an anti-HA antibody. IRE1 was detected in IP samples and WCL using an IRE1-specific antibody.
Figure 3.
Intracellular localization of IRE1 and M50.
(A) NIH-3T3 cells were cotransfected with expression plasmids for IRE1-HA and Flag-tagged M50 or UL56 respectively. 24 h post transfection, cells were fixed and subjected to immunofluorescence staining using HA- and Flag-specific antibodies. Cell nuclei were stained with Draq5. The Pearson correlation coefficient (PC) was determined for transfected cells. (B) 10.1 cells stably expressing IRE1-3xmyc were infected with MCMV-M50HA. At 16, 18, and 20 hpi cells were fixed and subjected to immunofluorescence staining using myc- and HA-specific antibodies. (C) 10.1 cells were infected with wt MCMV or MCMV-M50HA. Cells were fixed 20 hpi and stained with the same anti-HA antibody as in B and an antibody against the viral IE1 protein.
Figure 4.
M50 expression reduces IRE1 protein levels.
(A) NIH-3T3 cells were cotransfected with plasmids encoding IRE1-HA (1 µg) and Flag-tagged M50 or m144 (0.5, 1, or 2 µg). After 24 h, cell lysates were analyzed by immunoblot using protein- or tag-specific antibodies. (B) NIH-3T3 cells were cotransfected with plasmids encoding M50 (2 or 3 µg) and wildtype IRE1 or the K599A kinase-dead IRE1 mutant (1 µg). Cells were analyzed by immunoblot as described above. (C) 10.1 cells transduced with an IRE1-HA-expressing retroviral vector were infected with MCMV at an MOI of 5. Cells were harvested at the indicated time points, and IRE1, M50, and actin levels were determined by immunoblot using protein- or tag-specific antibodies. (D) NIH-3T3 fibroblasts were infected with MCMV at an MOI of 5. RNA was isolated at the indicated time points, and Ire1 transcripts were quantified by real-time RT-PCR. Means ±SEM of three replicates are shown relative to uninfected cells. (E) 293T cells were cotransfected with expression plasmids for IRE1-HA and M50. After pulse-chase labeling with [35S]methionine, IRE1 was immunoprecipitated with an anti-HA antibody and analyzed by autoradiography. (F) Signals in blot E were quantified by densitometry relative to the 0 h chase value.
Figure 5.
Identification of the region required for IRE1 binding and degradation.
(A) Schematic representation of the mutant M50 proteins used in the following experiments. Proline-rich (P) sequence, transmembrane (TM) domain, and the peptide recognized by the M50-specific antibody (ab) are indicated. Numbers on the right indicate amino acid positions. The HSV-1 UL56 protein was used as an unrelated control protein. 56TM is an M50 mutant containing the TM domain of HSV-1 UL56. (B) NIH-3T3 cells were cotransfected with plasmids coding for IRE1-HA (1 µg) and the proteins shown in panel A (2 µg). After 24 h, IRE1 levels were analyzed by immunoblot using an anti-HA antibody. M50 mutants and UL56 were detected with M50- and Flag-specific antibodies. (C) 293A cells were cotransfected with expression plasmids for IRE1-HA and full-length (fl.) or mutant M50. IRE1 was immunoprecipitated (IP) with an anti-HA antibody, and coprecipitating M50 proteins were detected by immunoblot using an M50-specific antibody. The same proteins were detected in whole cell lysates (WCL). LC, antibody light chain.
Figure 6.
M50 is required for IRE1 downregulation and inhibition of Xbp1 splicing during MCMV infection.
(A) 10.1 fibroblasts stably expressing myc-tagged IRE1 were infected with an MCMV M50 deletion mutant (ΔM50) or the parental control virus at an MOI of 3. Cells were harvested at 0, 24, and 48 hpi. IRE1 and M50 expression was determined by immunoblot. The viral immediate-early 1 (IE1), the viral late protein M55/gB, and cellular β-actin were used as infection and loading controls, respectively. (B) Normal 10.1 fibroblasts were infected as described above and treated for 4 h with Tun. Spliced and unspliced Xbp1 transcripts were quantified by real-time RT-PCR. Changes in the spliced/unspliced ratio relative to untreated cells are plotted as bar diagram showing means ±SEM of four replicates. (C) ERdj4 transcripts were quantified in the same cell by real-time RT-PCR. Results are shown as fold induction relative to untreated cells (means ±SEM of three replicates). Significance was determined using the Student's t-test. *, p<0.5; **, p<0.01; ***, p<0.001; ns, not significant.
Figure 7.
Modulation of the IRE1-XBP1 pathway by HCMV UL50.
(A) 293A cells were cotransfected with plasmids expressing HA-tagged murine or human IRE1 and Flag-tagged M50, UL56, NEMO, or UL50 respectively. Cell lysates were harvested 24 h after transfection. IRE1 was immunoprecipitated (IP) with an anti-HA antibody. IRE1 and coexpressed proteins were detected by immunoblot in IP samples and whole cell lysates (WCL) using anti-HA and anti-Flag antibodies, respectively. (B) HFF cells were cotransfected with plasmids encoding IRE1-HA (1 µg) and Flag-tagged HCMV UL50 or HSV-1 UL56 (2 µg). After 24 h, IRE1 levels were determined by immunoblot using an anti-HA antibody. UL50 and UL56 were detected with an anti-Flag antibody. (C) MRC-5 cells transduced with a retroviral vector expressing HA-tagged human IRE1 were infected with HCMV at an MOI of 3. At the indicated time points cells were harvested, and IRE1 levels were determined by immunoblot. HCMV IE1 and IE2 and β-actin were detected as infection and loading controls, respectively. (D) MRC-5 cells were infected with HCMV at an MOI of 3 and treated with Tun for the last 4 h before harvest. Spliced and unspliced XBP1 transcripts were quantified by real-time RT-PCR. Changes in the spliced/unspliced ratio are shown relative to untreated cells (means ±SEM of three replicates).
Figure 8.
IRE1 functions and inhibition by M50 and UL50.
Accumulation of unfolded proteins in the ER leads to recruitment of chaperones such as BiP and activation of ER stress sensors such as IRE1. (A) IRE1 dimerizes, autophosphorylates itself, and activates an endoribonuclease activity, which mediates Xbp1 mRNA splicing. The XBP1s protein activates transcription of ERAD genes such as ERdj4. (B) MCMV M50 and HCMV UL50 interact with IRE1 and induce IRE1 degradation, thereby inhibiting the IRE1-XBP1 pathway shown in A. (C) Activated IRE1 can also cleave certain glycoprotein (gp)-encoding mRNAs and microRNAs. (D) Recruitment of TRAF2 by activated IRE1 can lead to JNK or caspase-12 activation and subsequent induction of apoptosis.