The IE2 60-kilodalton and 40-kilodalton proteins are dispensable for human cytomegalovirus replication but are required for efficient delayed early and late gene expression and production of infectious virus

Abstract

The human cytomegalovirus (HCMV) IE2 86-kDa protein is an essential transactivator of viral and cellular gene expression. Additional proteins of 60 and 40 kDa are expressed from the IE2 gene at late times postinfection and are identical to the C terminus of IE2 86. We have constructed HCMV recombinants that express wild-type full-length IE2 86 but do not express the IE2 40- and 60-kDa proteins. Each of these recombinants is viable, indicating that neither the 60-kDa nor the 40-kDa protein is required for virus replication, either alone or in combination. Cells infected with the IE2 60 and IE2 40 deletion mutants, however, exhibit decreased expression of selected viral genes at late times. In particular, expression of the viral DNA replication factor UL84 is affected by the deletion of IE2 40, and expression of the tegument protein pp65 (ppUL83) is affected by the deletion of both IE2 40 and IE2 60. IE2 60 and IE2 40 are also required for the production of normal levels of infectious virus. Finally, IE2 40 appears to function as a repressor of major immediate-early transcription in the infected cell. These results begin to define functions for the IE2 60- and IE2 40-kDa proteins and indicate that these products contribute both to the expression of selected viral genes and to the overall progression of the infection.

FIG. 1.

Construction of the IE2 40 and IE2 60 deletion mutant viruses. (A) Partial sequence of the wild-type HCMV major IE region from the 3′ end of exon 4 through the 5′ portion of exon 5. Exon sequences are capitalized, and intron sequences are in lowercase. The regions that were changed to eliminate expression of the IE2 40 and IE2 60 proteins are underlined. The codons for the initiating methionines of each protein are boxed. (B) Schematic of the major IE region. The relative locations of the exons and the TATAA boxes and initiating methionines for IE1 72, IE2 86, IE2 60, and IE2 40 are indicated. The sequences that were altered to remove the predicted TATAA boxes for the late RNAs are indicated. For the mutations, nucleotides that are identical to the wild-type sequence are capitalized and altered nucleotides are in lowercase. The diagram is not to scale. MIEP, major immediate-early promoter.

FIG. 2.

Major immediate-early protein expression is altered following infection with IE2 Δ40 and IE2 Δ60 viruses. G₀-synchronized HFF cells were infected with 5 PFU/cell of wild-type (Wt), mutant (Δ40, Δ60, or Δ40+60), and rescued mutant (40 R, 60 R, or 40+60 R) virus or mock infected (M) and harvested at the indicated times p.i. Equal amounts of cell lysates, in micrograms, were separated by SDS-PAGE and transferred to nitrocellulose. IE1 72, IE2 86, IE2 60, and IE2 40 protein levels were analyzed by Western blotting as described in Materials and Methods. Cellular actin levels were analyzed as a control for protein loading. The asterisk indicates that the Δ40 72-h lane is slightly overloaded in the IE2 Western blot (top panel) relative to the IE2 86 and IE1 72 Western blot (middle panel).

FIG. 3.

Deletion of IE2 40 and IE2 60 reduces virus production following high-multiplicity infection. HFF were infected with 5 PFU/cell of wild-type, mutant, or rescued mutant virus. Infected-cell supernatants were collected at the indicated times, and titers were determined as described in Materials and Methods. The two graphs indicate the titers determined in two independent experiments. All plaque assays were performed in duplicate.

FIG. 4.

IE1 72 and IE2 86 RNA levels are altered in deletion mutant virus-infected cells. G₀-synchronized HFF cells were infected with 5 PFU/cell of wild-type virus, mutant viruses (IE2 Δ40, IE2 Δ60, or IE2 Δ40+60), and rescued mutant viruses (IE2 Δ40 R, IE2 Δ60 R, or IE2 Δ40+60 R) or mock infected and harvested at the indicated times p.i. Total RNA was analyzed by quantitative real-time RT-PCR as described in Materials and Methods to measure the relative levels of (A) IE1 72 or (B) IE2 86 transcripts. The values plotted on the graphs are the averages of two or three independent experiments, and range bars indicating the highest and lowest values obtained in the independent experiments are shown. To ensure that an equal amount of RNA was included in each reaction, samples were analyzed with G6PD-specific primers and probe. Values shown in the graphs have been standardized to G6PD levels. When mock-infected cell RNA was analyzed with either one of the TaqMan probes, amplification was near or below the limit of detection. In addition, there was no amplification when the samples were treated with RNase prior to PCR analysis.

FIG. 5.

Early viral protein expression is not altered following infection with IE2 Δ40 and IE2 Δ60 viruses. (A) G₀-synchronized HFF cells were infected with 5 PFU/cell of wild-type (Wt), mutant (Δ40, Δ60, or Δ40+60), and rescued mutant (40 R, 60 R, or 40+60 R) virus or mock infected (M) and harvested at the indicated times p.i. Equal amounts of cell lysates, in micrograms, were separated by SDS-PAGE and transferred to nitrocellulose. UL44 and UL57 protein levels were analyzed by Western blotting as described in Materials and Methods. Cellular actin levels were analyzed as a control for protein loading. (B) G₀-synchronized HFF cells were infected with 5 PFU/cell of wild-type (Wt), IE2 86ΔSX-EGFP, IE2 Δ40+60 (Δ40+60), or rescued IE2 Δ40+60 (40+60 R) virus and harvested at the indicated times p.i. Equal amounts of cell lysates, in micrograms, were separated by SDS-PAGE and transferred to nitrocellulose. UL44 and UL57 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. 6.

UL84 and pp65 protein expression is reduced following infection with IE2 Δ40 and IE2 Δ60 viruses. (A) G₀-synchronized HFF cells were infected with 5 PFU/cell of wild-type (Wt), mutant (Δ40, Δ60, or Δ40+60), and rescued mutant (40 R, 60 R, or 40+60 R) virus or mock infected (M) and harvested at the indicated times p.i. Equal amounts of cell lysates, in micrograms, were separated by SDS-PAGE and transferred to nitrocellulose. UL84 and pp65 protein levels were analyzed by Western blotting as described in Materials and Methods. Cellular actin levels were analyzed as a control for protein loading. (B) G₀-synchronized HFF cells were infected with 5 PFU/cell of wild-type (Wt), IE2 86ΔSX-EGFP, IE2 Δ40+60 (Δ40+60), or rescued IE2 Δ40+60 (40+60 R) virus and harvested at the indicated times p.i. Equal amounts of cell lysates, in micrograms, were separated by SDS-PAGE and transferred to nitrocellulose. UL84, pp65, 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.

FIG. 7.

UL83, but not UL84, RNA levels are altered in deletion mutant virus-infected cells. G₀-synchronized HFF cells were infected with 5 PFU/cell of wild-type virus, IE2 86ΔSX-EGFP virus, mutant viruses (IE2 Δ40, IE2 Δ60, or IE2 Δ40+60), and rescued mutant viruses (IE2 Δ40 R, IE2 Δ60 R, or IE2 Δ40+60 R) or mock infected and harvested at the indicated times p.i. Total RNA was analyzed by quantitative real-time RT-PCR as described in Materials and Methods to measure the relative levels of (A) UL83 or (B) UL84 transcripts. The values plotted on the graphs are the averages of two or three independent experiments, and range bars indicating the highest and lowest values obtained in the independent experiments are shown. To ensure that an equal amount of RNA was included in each reaction, samples were analyzed with G6PD-specific primers and probe. Values shown in the graphs have been standardized to G6PD levels. When mock-infected cell RNA was analyzed with either one of the TaqMan probes, amplification was near or below the limit of detection. In addition, there was no amplification when the samples were treated with RNase prior to PCR analysis.