Human cytomegalovirus specifically controls the levels of the endoplasmic reticulum chaperone BiP/GRP78, which is required for virion assembly

Abstract

The endoplasmic reticulum (ER) chaperone BiP/GRP78 regulates ER function and the unfolded protein response (UPR). Human cytomegalovirus infection of human fibroblasts induces the UPR but modifies it to benefit viral replication. BiP/GRP78 protein levels are tightly regulated during infection, rising after 36 h postinfection (hpi), peaking at 60 hpi, and decreasing thereafter. To determine the effects of this regulation on viral replication, BiP/GRP78 was depleted using the SubAB subtilase cytotoxin, which rapidly and specifically cleaves BiP/GRP78. Toxin treatment of infected cells for 12-h periods beginning at 36, 48, 60, and 84 hpi caused complete loss of BiP but had little effect on viral protein synthesis. However, progeny virion formation was significantly inhibited, suggesting that BiP/GRP78 is important for virion formation. Electron microscopic analysis showed that infected cells were resistant to the toxin and showed none of the cytotoxic effects seen in uninfected cells. However, all viral activity in the cytoplasm ceased, with nucleocapsids remaining in the nucleus or concentrated in the cytoplasmic space just outside of the outer nuclear membrane. These data suggest that one effect of the controlled expression of BiP/GRP78 in infected cells is to aid in cytoplasmic virion assembly and egress.

FIG. 1.

HCMV temporally regulates BiP/GRP78 protein levels. Proteins were harvested from HCMV-infected HFFs at the indicated times postinfection or from uninfected cells treated for 24 h with thapsigargin (Thaps). Thirty micrograms of extracted protein from each sample was analyzed by Western blot analysis as described in Materials and Methods. BiP-C and BiP-N, antibodies directed to the C terminus and N terminus of BiP, respectively.

FIG. 2.

Depletion of BiP by SubAB toxin does not alter the steady-state levels of viral proteins. Proteins were harvested from HCMV-infected HFFs that were either left untreated (no treatment) or treated with WT or Mut toxin. The times (hours postinfection) of toxin addition and protein harvest are given. Proteins were analyzed by Western blot analysis as described in Materials and Methods. BCP, the BiP/GRP78 C-terminal cleavage product.

FIG. 3.

Depletion of BiP/GRP78 inhibits production of infectious HCMV virions. (A) HFFs were infected with HCMV, and infectious virions were harvested at the indicated times postinfection (normal growth curve). Virus was also harvested at 48, 72, and 86 hpi from infected HFFs that had been treated with either SubAB (toxin) or SubA_A272B (mutant toxin) at 2 hpi. Viral titers were determined using the TCID₅₀ method. (B) Growth curves were performed as described for panel A, except that SubAB was added 12 h before the harvesting of virus at 48, 60, 72, or 96 hpi.

FIG. 4.

HCMV infection protects HFFs from the cytotoxic effects of BiP/GRP78 depletion by SubAB; however, viral activity in the cytoplasm is inhibited. Mock-infected (A to C) and HCMV-infected (D to F) HFFs were treated with either SubA_A272B (mutant toxin) (A and D) or SubAB (WT toxin) (B, C, E, and F) at 84 hpi and were harvested at 96 hpi for analysis by EM. Arrows point to nucleocapsids in the nucleus.

FIG. 5.

Depletion of BiP/GRP78 by shRNA has the same effect as depletion by SubAB toxin. HFFs were infected with HCMV 24 h after infection with lentivirus vectors expressing shRNA against GFP (control shRNA) (A and B) or BiP/GRP78 (C to F). The cells were harvested at 96 h post-HCMV infection for analysis by EM. White arrows indicate the presence of nucleocapsids in the nucleus.

FIG. 6.

Nucleocapsids accumulate outside of the nuclear envelope in BiP/GRP78-depleted cells. HFFs were infected with HCMV, treated with either WT toxin (A, B, and C) or Mut toxin (D and E) at 84 hpi, and harvested at 96 hpi for analysis by EM. White arrows in panels A to C show accumulation of virion particles outside of the nuclear membrane. White arrows in panels D and E indicate the nuclear membrane.