Exon 3 of the human cytomegalovirus major immediate-early region is required for efficient viral gene expression and for cellular cyclin modulation

Abstract

The human cytomegalovirus (HCMV) major immediate-early (IE) proteins share an 85-amino-acid N-terminal domain specified by exons 2 and 3 of the major IE region, UL122-123. We have constructed IE Delta30-77, a recombinant virus that lacks the majority of IE exon 3 and consequently expresses smaller forms of both IE1 72- and IE2 86-kDa proteins. The mutant virus is viable but growth impaired at both high and low multiplicities of infection and exhibits a kinetic defect that is not rescued by growth in fibroblasts expressing IE1 72-kDa protein. The kinetics of mutant IE2 protein accumulation in IE Delta30-77 virus-infected cells are approximately normal compared to wild-type virus-infected cells, but the IE Delta30-77 virus is delayed in expression of early viral genes, including UL112-113 and UL44, and does not sustain expression of mutant IE1 protein as the infection progresses. Additionally, cells infected with IE Delta30-77 exhibit altered expression of cellular proteins compared to wild-type HCMV-infected cells. PML is not dispersed but is retained at ND10 sites following infection with IE Delta30-77 mutant virus. While the deletion mutant retains the ability to mediate the stabilization of cyclin B1, cdc6, and geminin in infected cells, its capacity to upregulate the expression of cyclin E has been reduced. These data indicate that the activity of one or both of the HCMV major IE proteins is required in vivo for the modulation of cell cycle proteins observed in cells infected with wild-type HCMV.

FIG. 1.

Construction of the IE Δ30-77 mutant BAC. (A) The HCMV major immediate-early region. To construct the IE Δ30-77 BAC, nucleotides coding for amino acids 30 to 77 of IE1 72 and IE2 86 were removed from pHB5 as described in Materials and Methods. The resulting mutant BAC encodes shorter forms of both IE1 72 and IE2 86 proteins, as the nucleotides near the exon 3 splice junctions have been preserved. A rescued version of the IE Δ30-77 BAC was generated in which the altered region was replaced with wild type sequences. (B) EcoRI digests of the recombinant BACs. A total of 1 μg wild type (pHB5), IE Δ30-77, or rescued IE Δ30-77 BAC DNA was cut with EcoRI, and the resulting digests were separated by field inversion gel electrophoresis on a 1% agarose gel in 0.5× Tris-borate-EDTA. MW, molecular weight.

FIG. 2.

IE Δ30-77 virus replicates with a kinetic defect not rescued by wild-type IE1 72 protein provided in trans. HFF or ihfie1.3 cells were infected with wild-type, IE Δ30-77, or rescued IE Δ30-77 virus with 5 or 0.5 IE⁺ units/cell, as indicated. HFF (A and B) or ihfie1.3 (C and D) were infected at a low (A and C) or high (B and D) MOI, infected cell supernatants were collected at the indicated times, and titers were determined as described in Materials and Methods. Virus aliquots were collected from a culture until all cells in the dish appeared to be dead. The experiment generating a growth curve for a MOI of 5 in HFF was repeated three times, and the results of a representative experiment are shown.

FIG. 3.

Early viral gene expression is delayed following low-multiplicity infection of HFF with IE Δ30-77 recombinant virus. G₀-synchronized HFF were infected with 0.5 IE⁺ units/cell of wild-type, IE Δ30-77, or rescued IE Δ30-77 viruses or mock infected and harvested at the indicated times p.i. Total cellular RNA was analyzed by quantitative real-time RT-PCR as described in Materials and Methods to measure the relative levels of transcripts expressed from the early UL112-113 (A), delayed early UL89 (B), or late R160461 (C) viral loci. Experiments were repeated at least three times, each time with duplicate reactions, and the results of a representative experiment are shown. To ensure that an equal amount of RNA was included in each reaction, samples were analyzed with G6PD-specific primers and probe (D). When mock-infected (Mock) cell RNA was analyzed with UL112-113, UL89, or R160461 probes, the result was below the limit of detection; these values are not indicated on the graphs.

FIG. 4.

The kinetics of viral DNA replication in IE Δ30-77 virus-infected cells follow the increase in viral early gene expression. G₀-synchronized HFF cells were infected with 0.5 IE⁺ units/cell of wild-type, IE Δ30-77, or rescued IE Δ30-77 viruses or mock infected (Mock) and harvested at the indicated times p.i. Viral DNA amplification was measured by quantitative real-time PCR as described in Materials and Methods using primers and probe directed against the unspliced viral UL77 gene.

FIG. 5.

IE and early protein expression is altered in IE Δ30-77 virus-infected cells. G₀-synchronized HFF cells were infected with 0.5 IE⁺ units/cell of wild-type (Wt), IE Δ30-77 (Δ3), or rescued IE Δ30-77 (R) viruses or mock infected (M) and harvested at the indicated times p.i. Equal amounts of cell lysates (shown in micrograms) were separated by SDS-PAGE and transferred to nitrocellulose. IE1 72, IE2 86, and UL44 protein levels were analyzed by Western blotting as described in Materials and Methods. Cellular actin levels were analyzed as a control for protein loading. Asterisks (*) indicate the position of IE2 86 or mutant IE2 86 in lanes containing 24-h-p.i. samples.

FIG. 6.

IE1 72 protein expression is not maintained and viral early protein expression is delayed during high-multiplicity infection with IE Δ30-77 virus. G₀-synchronized HFF cells were infected with 5 IE⁺ units/cell of wild-type (Wt), IE Δ30-77 (Δ3), or rescued IE Δ30-77 (R) viruses or mock infected (M) and harvested at the indicated times p.i. Equal amounts of cell lysates (shown in micrograms) were separated by SDS-PAGE and transferred to nitrocellulose. Immediate-early (IE1 72 and IE2 86, using antibody CH16.0), early (UL112-113 and UL44), and delayed early/late (pp65, major capsid protein [MCP], and pp28) protein levels were analyzed by Western blotting as described in Materials and Methods. Cellular actin levels were analyzed as a control for protein loading. Asterisks (*) indicate the positions of the 84-, 50-, 43-, and 34-kDa proteins encoded by UL112-113. Dashes (—) indicate prominent nonspecific proteins that cross-react with the UL112-113 antibody.

FIG. 7.

IE1 72 and UL44 proteins are not efficiently expressed at the same time in IE Δ30-77 virus-infected cells. G₀-synchronized HFF cells were infected with 5 IE⁺ units/cell of wild-type, IE Δ30-77, or rescued IE Δ30-77 viruses and fixed in 2% paraformaldehyde 24 h p.i. Cells were stained with Hoechst dye to visualize nuclei, anti-IE1 72 monoclonal antibody p63-27, and antibody to UL44, followed by appropriate FITC- and TRITC-conjugated secondary antibodies (Materials and Methods). Arrows indicate the same cells in IE1 72 and UL44 fields. Magnification, ×400.

FIG. 8.

Viral gene expression is delayed in IE Δ30-77 virus-infected cells. G₀-synchronized HFF cells were infected with 5 IE⁺ units/cell of wild-type, IE Δ30-77, or rescued IE Δ30-77 viruses and fixed in 2% paraformaldehyde at the indicated times p.i. Cells were stained with Hoechst dye to visualize nuclei and antibody to pp65 (A) or pp28 (B), followed by appropriate FITC-conjugated secondary antibodies (Materials and Methods). Magnification, ×400. Inset, 200% enlargement of pp65 expression in wild-type, IE Δ30-77, or rescued IE Δ30-77 virus-infected cells. Merge, overlaid viral antigen and Hoechst-stained images.

FIG. 9.

Altered expression of cell cycle regulatory proteins in IE Δ30-77 virus-infected cells. (A) G₀-synchronized HFF cells were mock infected (M) or infected with 10 IE⁺ units/cell of wild-type (Wt), IE Δ30-77 (Δ3), or rescued IE Δ30-77 (R) viruses and harvested at the indicated times p.i. Cells were maintained in media containing 1% serum after 6 h p.i. (B) ihfie1.3 cells were mock infected (M) or infected with 5 IE⁺ units/cell of wild-type (Wt), IE Δ30-77 (Δ3), or rescued IE Δ30-77 (R) viruses and harvested 24 h p.i. Cells were maintained in media containing 10% serum throughout the time course. For both cell types, equal amounts of cell lysates (shown in micrograms) were separated by SDS-PAGE gel electrophoresis and transferred to nitrocellulose. Cyclin B1, cdc6, geminin, cyclin E, and cyclin A protein levels were analyzed by Western blotting as described in Materials and Methods. Cellular actin levels were analyzed as a control for protein loading.

FIG. 10.

PML is not dispersed following infection with IE Δ30-77 virus. G₀-synchronized HFF cells were infected with 5 IE⁺ units/cell of wild-type, IE Δ30-77, or rescued IE Δ30-77 viruses or mock infected (Mock) and fixed in 2% paraformaldehyde 24 h p.i. Cells were stained with Hoechst dye to visualize nuclei, anti-IE1 72 monoclonal antibody p63-27, and antibody to PML, followed by appropriate FITC- and TRITC-conjugated secondary antibodies (Materials and Methods). Magnification, ×1000.