Article Figures & Data
Figures
- FIG. 1.
A pUL83-deficient virus exhibits a growth defect following infection at a low multiplicity of infection. (A) At a relatively high input multiplicity, normal levels of viral proteins accumulated after infection with 83STOP virus. Human fibroblasts were infected at a multiplicity of 5 PFU/cell with either wild-type (WT) or 83STOP (83S) virus, and viral protein expression was assayed by Western blotting using antibodies to the indicated proteins. Tubulin was assayed as a loading control. (B) At a relatively low input multiplicity, 83STOP virus displayed a growth defect. Fibroblasts were infected at a multiplicity of 0.05 PFU/cell. Cell-free supernatants were harvested on the indicated days postinfection (dpi), and the amount of HCMV present was determined by a 50% tissue culture infective dose (TCID50) assay of fibroblasts. (C) At a relatively low input multiplicity, UL83STOP virus failed to accumulate normal levels of an immediate-early protein. Fibroblasts were infected at a multiplicity of 0.05 PFU/cell, and IE1 protein expression was analyzed by Western blotting and quantified using a phosphoimager. (D) UL83Stop virions contain amounts of pUL82 similar to those of wild-type virions. Equivalent infectious units of the wild type and UL83Stop were assayed for pUL82 content by Western blotting. The filter was stripped and reprobed with antibody to pUL83 to confirm its absence in UL83Stop virions.
- FIG. 2.
pUL83 is important for complete activation of the MIEP during the immediate-early phase of the replication cycle. (A) pUL83 stimulates IE1 accumulation. Fibroblasts were infected at a multiplicity of 1 or 5 PFU/cell with either WT or 83STOP (83S) virus. Cell lysates were analyzed by Western blotting for IE1 and IE2 expression at 6 and 24 hpi. The results are representative of two independent experiments. (B) pUL83 stimulates the accumulation of an immediate-early RNA. Fibroblasts were infected as described in the legend for panel A, and RNA was isolated and assayed by real-time PCR to determine the levels of IE1 and IE2 transcripts. Levels of virus RNA are presented relative to the levels of the cellular GAPDH RNA. (C) pUL83 alone stimulates MIEP activity outside the context of an infection. U2OS cells were transfected with an MIEP-luciferase reporter construct together with increasing amounts of a pUL83 expression vector. The amount of transfected DNA was kept constant by the addition of empty expression vector.
- FIG. 3.
Identification of UL83 binding partners in the context of infection. (A) Diagram showing the UL83 locus in BADinUL83TAP and BADinUL83GFP. (B) BADinUL83GFP grew normally. HFFs were infected with BADinUL83GFP at a multiplicity of 0.1, and the amount of HCMV in cell-free supernatants was determined by a TCID50 assay at the indicated times postinfection. (C) pUL83TAP was localized normally during infection. pUL83 localization was determined during infection with BADwt (top) or BADinUL83TAP (bottom). For unmodified pUL83, anti-UL83 monoclonal antibody was used for immunofluorescence, while anti-protein A was used to visualize pUL83TAP. The white bar indicates 10 μm. (D) Fibroblasts were infected at a multiplicity of 3 PFU/cell with the indicated viruses and harvested at 48 or 72 hpi. pUL83TAP, pUL83GFP, and associated proteins were isolated on magnetic beads, resolved on 4 to 12% SDS-PAGE gradient gels, stained with Coomassie blue, and analyzed by MALDI MS and MS/MS. Identified proteins are indicated.
- FIG. 4.
Confirmation of the pUL83-pUL25 interaction. (A) pUL25 and pUL83 substantially colocalize within infected cells. Fibroblasts infected with BADinUL83GFP at a multiplicity of 0.5 PFU/cell were fixed and stained with antibody specific for pUL25 (red) to test for colocalization with pUL83GFP (green). The nucleus was stained with DAPI to provide context, and the white bar indicates 10 μm. (B) pUL83 interacts with pUL25 during infection. HFFs were mock infected or infected with wild-type virus at a multiplicity of 3 PFU/cell, and pUL25-specific immune complexes were isolated by immunoprecipitation (IP) 72 h later. Lysates were also immunoprecipitated with antibody to IE1 as a specificity control. The presence of pUL83 in pUL25-specific immune complexes was determined by Western blotting (top), and total pUL83 and pUL25 in cell lysates were monitored as controls (bottom).
- FIG. 5.
Confirmation of the pUL83-IFI16 interaction. (A) IFI16 and pUL83 were partially colocalized within infected cells. Fibroblasts infected with wild-type HCMV at a multiplicity of 0.5 PFU/cell were fixed and stained with antibodies specific for UL83 and IFI16 to test for colocalization. The nucleus and Golgi were monitored to provide context, and the white bar indicates 10 μm. (B) IFI16 coprecipitates with antibody to pUL83. Fibroblasts were mock infected (M) or infected with wild-type HCMV (WT) at a multiplicity of 3 PFU/cell and harvested 72 h later. pUL83-specific immune complexes were isolated by immunoprecipitation (IP), and the presence of IFI16 in those complexes was determined by Western blotting using antibodies specific to IFI16. Lysates were assayed by Western blotting for the presence of the indicated proteins as controls. (C) pUL83 coprecipitates with antibody to IFI16. IFI16-specific immune complexes were isolated by immunoprecipitation from mock-infected fibroblasts or at 72 hpi of fibroblasts with wild-type HCMV at a multiplicity of 3 PFU/cell. Immunoprecipitation was also performed with nonspecific antibody (IgG). The presence of pUL83 in precipitates was confirmed by Western blotting with a pUL83-specific antibody. Lysates were assayed by Western blotting for the presence of the indicated proteins as controls. (D) IFI16 interacts with pUL83 throughout the course of infection. Lysates of cells infected at a multiplicity of 3 PFU/cell were prepared at the indicated times and subjected to immunoprecipitation with antibody to pUL83. Lysates were assayed by Western blotting for the presence of the indicated proteins as controls. (E) IFI16 remained in the nucleus during HCMV infection. Fibroblasts were infected at a multiplicity of 0.5 PFU/cell with a derivative of wild-type HCMV expressing a GFP marker protein (green). Cells were fixed and processed for immunofluorescence using an antibody to IFI16 (red) at the indicated times after infection. Nuclei were stained with DAPI to provide context, and the white bar indicates 10 μm. (F) pUL83 was initially localized to the nucleus but was also in the cytoplasm by 72 hpi. Fibroblasts were infected at a multiplicity of 0.5 PFU/cell with a derivative of wild-type HCMV expressing a GFP marker protein (green). Cells were fixed and processed for immunofluorescence using an antibody to pUL83 (red) at the indicated times after infection. Nuclei were stained with DAPI to provide context, and the white bar indicates 10 μm.
- FIG. 6.
pUL83 and IFI16 were present at the MIEP, and IFI16 required pUL83 for this association. Fibroblasts were infected at a multiplicity of 3 PFU/cell with BADwt (WT) or UL83STOP virus (83S). Prior to harvesting at the indicated times after infection, cells were cross-linked with formaldehyde, and IFI16 and pUL83 immune complexes were isolated by immunoprecipitation. Immunoprecipitations carried out without a primary antibody (Ab) were included as a control. The presence of specific DNA sequences (MIEP or UL69 promoter) in the immunoprecipitates was assessed by qRT-PCR.
- FIG. 7.
IFI16 is required for efficient HCMV replication. (A) Knockdown of IFI16 levels by using shRNA. The level of IFI6 in cells expressing control or IFI16-sepcific shRNA was assayed by Western blotting. Tubulin was monitored as a loading control. (B) Wild-type HCMV exhibits a growth defect in IFI16-deficient cells. Normal fibroblasts (diamonds) and IFI16-deficient fibroblasts (triangles) were infected at a multiplicity of 0.1 PFU/cell. Titers of cell-free virus were determined by TCID50 assay. The experiment was performed in triplicate, and standard errors were determined. (C) Accumulation of immediate-early UL122 RNA in IFI16 knockdown cells. Following infection with wild-type HCMV at a multiplicity of 5 or 0.5 PFU/cell, the level of HCMV UL122 RNA was quantified by qRT-PCR in normal (black bars) and IFI16-deficient cells (white bars). The results are the average of two independent experiments. (D) Growth of 83Stop (83S) virus is sensitive to interferon. Fibroblasts were infected at a multiplicity of 0.1 PFU/cell wild-type HCMV or 83S in the absence or presence of 500 U/ml alpha interferon (IFN). Titers of cell-free virus were determined by TCID50 assay. The experiment was performed in triplicate, and standard errors were determined.
Tables
- TABLE 1.
List of proteins identified in the immunoisolates of TAP- and GFP-tagged pUL83a
↵ a pUL83 was isolated via the protein A or GFP tag at 48 hpi and 72 hpi, respectively. Coisolated proteins were analyzed using MALDI MS and MS/MS analyses. The gi number, number of peptides detected, sequence coverage (%), XProteo score (d′) following database searching using the MS data, and peptide sequences confirmed by MS/MS are indicated for each identified protein. Only the proteins that were confirmed via the fragmentation of at least two peptides were included in the table. # indicates peptides unique to either IFI16 or IFI16b. The proteins classified as possible contaminants indicate proteins that we routinely observe as likely nonspecific associations in isolates from mammalian cells.
