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. 2003 Oct;77(20):11105-13.
doi: 10.1128/jvi.77.20.11105-11113.2003.

Role of PACS-1 in trafficking of human cytomegalovirus glycoprotein B and virus production

Colin M Crump  1 , Chien-Hui Hung, Laurel Thomas, Lei Wan, Gary Thomas
Affiliations

Role of PACS-1 in trafficking of human cytomegalovirus glycoprotein B and virus production

Colin M Crump et al. J Virol. 2003 Oct.

Abstract

The final envelopment of herpesviruses during assembly of new virions is thought to occur by the budding of core viral particles into a late secretory pathway organelle, the trans-Golgi network (TGN), or an associated endosomal compartment. Several herpesvirus envelope glycoproteins have been previously shown to localize to the TGN when expressed independently from other viral proteins. In at least some cases this TGN localization has been shown to be dependent on clusters of acidic residues within their cytoplasmic domains. Similar acidic cluster motifs are found in endogenous membrane proteins that also localize to the TGN. These acidic cluster motifs interact with PACS-1, a connector protein that is required for the trafficking of proteins containing such motifs from endosomes to the TGN. We show here that PACS-1 interacts with the cytoplasmic domain of the HCMV envelope glycoprotein B (gB) and that PACS-1 function is required for normal TGN localization of HCMV gB. Furthermore, inhibition of PACS-1 activity in infected cells leads to a decrease in HCMV titer, whereas an increase in expression of functional PACS-1 leads to an increase in HCMV titer, suggesting that PACS-1 is required for efficient production of HCMV.

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Figures

FIG. 1.
FIG. 1.
HCMV gB interacts with PACS-1 in a phosphorylation-dependent manner. GST-tagged wild-type HCMV gB cytoplasmic domain (with or without [+ or −] CK2 phosphorylation), mutant gB cytoplasmic domains containing S900D or S900A mutations, or GST alone were incubated with TRX-tagged PACS-1FBR. Protein complexes were recovered with glutathione-agarose, and extracts were analyzed for associated TRX-PACS-1FBR by Western blotting with TRX-specific antisera. Interaction signals from separate experiments were quantified by using National Institutes of Health gel analysis software and are shown as mean interactions ± the SD.
FIG.2.
FIG.2.
PACS-1Admut causes mislocalization of HCMV gB to endosomal compartments. FLAG-tagged HCMV gB was expressed in A7 cells by using replication-defective adenovirus vectors at an MOI of 2 for 24 h. Cells were subsequently infected with control vaccinia viruses (a to c) or vaccinia virus expressing PACS-1 (d to f) or PACS-1Admut (g to o) at an MOI of 10 for 5 h. Cells were fixed and developed with specific antisera to the FLAG tag (M1; a, d, g, j, and m) and assessed for colocalization with either TGN46 (b, e, and h), CI-MPR (k), or internalized dextran (n). M1 staining is shown in green, and colocalization markers are shown in red.
FIG. 3.
FIG. 3.
Depletion of endogenous PACS-1 protein by siRNA causes a mislocalization of HCMV gB. U373 cells were transfected with or without PACS-1-specific siRNA. (A) PACS-1 protein levels were assessed by Western blotting 3 days posttransfection with PACS-1-specific antisera. (B) FLAG-tagged HCMV gB was expressed in control (a to c) or PACS-1 siRNA-treated (d to f) cells by using adenovirus vectors at an MOI of 2 for 24 h. Cells were fixed and developed with antisera specific for the FLAG tag (M1; a and d) and TGN46 (b and e). M1 staining is shown in green, and TGN46 staining is shown in red.
FIG.4.
FIG.4.
Stable expression of PACS-1Admut causes gB but not gH to be mislocalized during HCMV infection. (A) Stable U373-PACS-1, PACS-1Admut, or control cell lines were treated with or without 1 μg of doxycycline/ml for 2 days. Protein expression was assessed by Western blotting with PACS-1-specific antisera. (B) Stable U373 cell lines expressing empty vector controls (a to c and j to l), wild-type PACS-1 (d to f and m to o), or PACS-1Admut (g to i and p to r) were infected with HCMV at an MOI of 3 for 3 days in the presence of 1 μg of doxycycline/ml. Cells were fixed and developed with antiserum specific for HCMV gB (R2448; a, d, g, j, m, and p), together with antiserum specific for γ-adaptin (100/3; b, e, and h) or HCMV gH (14-4b; k, n, and q). HCMV gB staining is shown in green, and γ-adaptin or HCMV gH staining is shown in red.
FIG. 5.
FIG. 5.
Inhibition of PACS-1 function causes a reduction in infectious HCMV production. (A) Stable U373 cell lines expressing empty vector controls, PACS-1, or PACS-1Admut were infected with HCMV at an MOI of 3 in the presence of 1 μg of doxycycline/ml. Cell-associated viral samples were harvested at 6 days postinfection, and the infectious HCMV titer was assessed by plaque assay. Titers from triplicate experiments are represented as mean titers ± the SD. (B) HFF cells were transfected with or without PACS-1-specific siRNA. At 1 day posttransfection, cells were infected with HCMV at an MOI of 3. Cell-associated viral samples were harvested at 6 days postinfection, and the infectious HCMV titer was assessed by plaque assay. Titers from triplicate experiments are represented as mean titers ± the SD.
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