UL26-deficient human cytomegalovirus produces virions with hypophosphorylated pp28 tegument protein that is unstable within newly infected cells

Abstract

The human cytomegalovirus UL26 open reading frame encodes proteins of 21 and 27 kDa that result from the use of two different in-frame initiation codons. The UL26 protein is a constituent of the virion and thus is delivered to cells upon viral entry. We have characterized a mutant of human cytomegalovirus in which the UL26 open reading frame has been deleted. The UL26 deletion mutant has a profound growth defect, the magnitude of which is dependent on the multiplicity of infection. Two very early defects were discovered. First, even though they were present in normal amounts within mutant virions, the UL99-coded pp28 and UL83-coded pp65 tegument proteins were present in reduced amounts at the earliest times assayed within newly infected cells; second, there was a delay in immediate-early mRNA and protein accumulation. Further analysis revealed that although wild-type levels of the pp28 tegument protein were present in UL26 deletion mutant virions, the protein was hypophosphorylated. We conclude that the UL26 protein influences the normal phosphorylation of at least pp28 in virions and possibly additional tegument proteins. We propose that the hypophosphorylation of tegument proteins causes their destabilization within newly infected cells, perhaps disrupting the normal detegumentation process and leading to a delay in the onset of immediate-early gene expression.

FIG. 1.

Accumulation of pUL26 protein in cells infected with wild-type or pUL26-deficient deletion mutant HCMV. Replicate cultures of fibroblasts were infected at a multiplicity of 3.0 PFU/cell with ADwt, BADsubUL26, or BADrevUL26. Cells were harvested 48 h postinfection and processed for Western blotting using a pUL26-specific monoclonal antibody.

FIG. 2.

Localization of pUL26 protein in cells infected with wild-type or a pUL26-deficient mutant of HCMV. Replicate cultures of fibroblasts cells were infected at a multiplicity of 3.0 PFU/cell with ADwt or BADsubUL26 multiplicity of infection. Cells were fixed at the indicated time points and processed for immunofluorescence using a pUL26-specific monoclonal antibody.

FIG. 3.

Growth characteristics of wild-type and pUL26-deficient viruses. (A) Production of infectious progeny in fibroblast cultures. Replicate cultures were infected at a multiplicity of 3.0 or 0.01 PFU/cell with ADwt (▴), BADsubUL26 (▪), or BADrevUL26 (⧫). Cells were harvested at the indicated times, and infectious viral progeny was quantified by plaque assay on fibroblasts. (B) Mock-infected fibroblasts transduced with a retrovirus expressing pUL26 as well as mock- or ADwt-infected (multiplicity of 3.0 PFU/cell) nontransduced fibroblasts were harvested and processed for Western blotting using a pUL26-specific monoclonal antibody. (C) Production of infectious progeny in fibroblasts expressing pUL26. Replicate cultures of retrovirally transduced fibroblasts expressing pUL26 were infected at a multiplicity of 0.01 PFU/cell with ADwt (▴) or BADsubUL26 (▪). Cells were harvested at the indicated times and assayed for infectious viral progeny by plaque assay on fibroblasts. (D) Accumulation of viral DNA after infection with wild-type or UL26-deficient viruses. Replicate cultures of fibroblasts were infected at a multiplicity of 3.0 or 0.01 PFU/cell with ADwt or BADsubUL26 and harvested at the indicated times. Total cellular DNA was assayed by slot blotting using an HCMV-specific probe.

FIG. 4.

Accumulation of viral proteins and mRNA in cells infected with wild type or a UL26-deficient mutant of HCMV. (A) Replicate cultures of fibroblasts were infected at a multiplicity of 3.0 PFU/cell with ADwt or BADsubUL26. Cells were harvested at the indicated times and processed for Western blotting using antibodies directed toward pIRS1, pTRS1, IE1, pp65, pUL44, or pp28. (B) Replicate cultures of fibroblasts were infected at a multiplicity of 3.0 PFU/cell with ADwt or BADsubUL26. Cells were harvested at the indicated times, and total mRNA was analyzed by Northern blot using a probe specific for IE1.

FIG. 5.

Analysis of the delivery of viral proteins and DNA upon infection. (A) ADwt and BADsubUL26 virions were purified by centrifugation in glycerol tartrate gradients. Equal amounts of virion protein were analyzed by Western blot for minor capsid protein (mCP), pp65 and pp28. (B) Replicate cultures of fibroblasts were infected at a multiplicity of 3.0 PFU/cell with ADwt or a BADsubUL26 for 30 min or 2 h, washed with sodium citrate buffer to inactivate extra-cellular virus followed by PBS, and then harvested. Total cellular DNA along with the equivalent amount of input virus was processed for slot blot assay using an HCMV-specific probe. (C) Replicate cultures of fibroblasts were infected at a multiplicity of 3.0 with ADwt or BADsubUL26 for 30 min, washed with sodium citrate buffer to inactivate extracellular virus, and harvested at various times. Total protein lysates were analyzed by Western blotting for pp65 and pp28 accumulation. (D) Replicate cultures of fibroblasts were infected at a multiplicity of 6.0 PFU/cell with ADwt or BADsubUL26. Cells were fixed 4 h postinfection and processed for immunofluorescence using a pp28- or pp65-specific monoclonal antibody.

FIG. 6.

Analysis of pp28 isoforms present in wild-type and pUL26 deficient mutant virions. (A) ADwt and BADsubUL26 virions were purified by centrifugation glycerol tartrate gradients. Equal amounts of virion protein were analyzed by 2-D gel electrophoresis and immunoblotted for pp28. Altered isoforms identified by ovals and a rectangle. (B) Glycerol tartrate gradient-purified ADwt virions were mock treated or treated with lambda phosphatase. An equal amount of virion protein was analyzed by 2-D gel electrophoresis and immunoblotted for pp28.