Human cytomegalovirus (HCMV) UL82 gene product (pp71) relieves hDaxx-mediated repression of HCMV replication

Abstract

This study examines the role of the cellular protein hDaxx in controlling human cytomegalovirus (HCMV) immediate-early (IE) gene expression and viral replication. Using permissive cell lines that either overexpress hDaxx or are depleted of hDaxx expression by the use of short hairpin RNA, we demonstrate that hDaxx functions as a repressor of HCMV IE gene expression and replication. In addition, we demonstrate that the impaired growth phenotype associated with the UL82 (pp71) deletion mutant is abolished when hDaxx knockdown cells are infected, suggesting that pp71 functions to relieve hDaxx-mediated repression during HCMV infection.

FIG. 1.

Overexpression of hDaxx represses viral replication. (A) Western blot analysis examining expression of hDaxx and GFP in control U373 cells and GFP-hDaxx-overexpressing U373 clones Dx21 and Dx24. Tubulin was included as an internal loading control. (B) Control U373-GFP (black bars) cells or hDaxx-overexpressing cell clones Dx21 (hatched bars) or Dx24 (vertically striped bars) were infected with wild-type virus at an MOI of 5.0, 1.0, or 0.2 PFU/cell. Infectious virus was harvested at 5 days postinfection for cells infected at an MOI of 5.0 PFU/cell and at 7 days postinfection for cells infected at an MOI of 1.0 or 0.2 PFU/cell and quantified by a plaque assay on human foreskin fibroblasts. Asterisks indicate significant differences in viral titers (P < 0.05) between virus produced on hDaxx-overexpressing cells and on control U373-GFP cells. Error bars indicate standard deviations derived from three independent experiments. Control GFP-expressing or hDaxx-overexpressing (Dx21) cells were infected with wild-type virus at an MOI of 0.2 (C) or 1.0 (D) PFU/cell. Cell lysates were prepared at the indicated hours postinfection (hpi) and assayed for IE1 and IE2 expression by Western blotting. Tubulin was included as an internal loading control.

FIG. 2.

Infection of hDaxx knockdown cells abolishes the UL82 deletion mutant growth phenotype. (A) Western blot analysis of hDaxx expression in control cells expressing a scrambled shRNA (SC) and in hDaxx knockdown cell lines KD2, KD4, and KD5. Tubulin was included as an internal loading control. (B) Control (SC) and hDaxx knockdown (KD5) cells were infected with wild-type virus (WT) or the UL82 deletion mutant virus (ΔUL82) at an MOI of 0.2 PFU/cell and examined at 5 days postinfection for GFP-positive cells. Control (SC) and hDaxx knockdown (KD2, KD4, and KD5) cells were infected with either wild-type (black bars) or ΔUL82 (hatched bars) virus at an MOI of 0.2 (C) or 1.0 (D) PFU/cell. Viruses were harvested at 7 and 5 days postinfection, respectively, and infectious virus was quantified by a plaque assay on UL82-complementing cells. Asterisks indicate significant differences in viral titers (P < 0.005) between ΔUL82 virus produced on hDaxx knockdown cells and on control cells. Error bars indicate standard deviations derived from three independent experiments.

FIG. 3.

IE gene expression following infection of hDaxx knockdown cells. Control (SC) or hDaxx knockdown (KD5) cells were infected with either wild-type (WT) or UL82 deletion mutant (ΔUL82) virus at an MOI of 0.2 (A) or 1.0 (B) PFU/cell. Cell lysates were prepared at the indicated hours postinfection (hpi) and assayed for IE1 and IE2 expression by Western blotting. Tubulin was included as an internal loading control.