The major immediate-early gene ie3 of mouse cytomegalovirus is essential for viral growth

Abstract

The significance of the major immediate-early gene ie3 of mouse cytomegalovirus (MCMV) and that of the corresponding ie2 gene of human cytomegalovirus to viral replication are not known. To investigate the function of the MCMV IE3 regulatory protein, we generated two different MCMV recombinants that contained a large deletion in the IE3 open reading frame (ORF). The mutant genomes were constructed by the bacterial artificial chromosome mutagenesis technique, and MCMV ie3 deletion mutants were reconstituted on a mouse fibroblast cell line that expresses the MCMV major immediate-early genes. The ie3 deletion mutants failed to replicate on normal mouse fibroblasts even when a high multiplicity of infection was used. The replication defect was rescued when the IE3 protein was provided in trans by a complementing cell line. A revertant virus in which the IE3 ORF was restored was able to replicate with wild-type kinetics in normal mouse fibroblasts, providing evidence that the defective growth phenotype of the ie3 mutants was due to disruption of the ie3 gene. To characterize the point of restriction in viral replication that is controlled by ie3, we analyzed the pattern of expression of selective early (beta) and late (gamma) genes. While we could detect transcripts for the immediate-early gene ie1 in cells infected with the ie3 mutants, we failed to detect transcripts for representative beta and gamma genes. These data demonstrate that the MCMV transactivator IE3 plays an indispensable role during viral replication in tissue culture, implicating a similar role for the human CMV ie2 gene product. To our knowledge, the ie3 deletion mutants represent the first MCMV recombinants isolated that contain a disruption of an essential gene.

FIG. 1

Construction of ie3-deficient MCMV BAC genomes. The HindIII map of the MCMV genome is shown at the top. The expanded map of the HindIII K and L fragments represents the major IE gene region of MCMV. Coding exons are shown in black, and the first noncoding exon of the ie1/ie3 transcription unit is depicted as an open rectangle. The gray box marks the MCMV enhancer ie1/ie3 promoter. The structure of the ie1 and ie3 transcripts is indicated below line 1 of the expanded map. Starting with the parental MCMV BAC plasmid pSM3fr (line 1), the other BAC plasmids pSM3frdie3 (line 2), pSM3frdie3::GFP (line 3), and pSM3fr-rev (line 4) were generated by successive rounds of homologous recombination in E. coli as described in Materials and Methods. The deletion in the fifth exon of the ie3 gene is marked by the delta (Δ). The cross-hatched box in front of the GFP ORF represents the HCMV MIEP.

FIG. 2

Structural analysis of the ie3-deficient MCMV BAC genomes. Ethidium bromide-stained agarose gel of HindIII-digested BAC plasmids pSM3fr (lane 1), pSM3frdie3 (lane 2), pSM3frdie3::GFP (lane 3), and pSM3fr-rev (lane 4) after separation on a 0.7% agarose gel. The names of the MCMV HindIII fragments (7) and the sizes of the molecular-weight markers are shown in the left and right margin, respectively. New fragments in the BAC plasmids are marked with white arrows.

FIG. 3

Structural analysis of the genomes of the MCMV ie3 mutants. DNA isolated from NIH 3T3-Bam25 cells infected with the parental MCMV (lane 1), the ie3-deficient mutants MCMVdie3 (lane 2), and MCMVdie3::GFP (lane 3), or the revertant virus MCMVrev (lane 4) was subjected to HindIII digestion, separated on a 0.7% agarose gel, and stained with ethidium bromide. Size markers are shown in the right margin, and the names of the HindIII fragments (7) are indicated in the left margin. New fragments in the genomes of the MCMV mutants are marked by white arrows.

FIG. 4

Growth curve analysis of the MCMV ie3 mutants. NIH 3T3 (A, B) or NIH 3T3-Bam25 (C, D) cells were infected at an MOI of 0.05 (A, C) or 2 PFU per cell (B, D) with the parental MCMV, MCMVdie3, and MCMVdie3::GFP and the revertant MCMVrev. At the indicated time points after infection (days p.i. [dpi]), supernatants from the infected cultures were harvested and titered on monolayers of NIH 3T3-Bam25 cells. The limit of detection was 20 PFU/ml. Error bars indicate the standard deviation from three separate cultures.

FIG. 5

Detection of viral transcripts after infection with an MCMV ie3 mutant. NIH 3T3 (lanes 1 through 6) or NIH 3T3-Bam25 (lane 7) cells were infected at an MOI of 0.5 PFU per cell with parental MCMV (lanes 1, 2, and 5) or MCMVdie3::GFP (lanes 3, 4, 6, and 7) in the presence (lanes 2 and 4) or absence (lanes 1, 3, and 5 to 7) of cycloheximide. Whole-cell RNA was harvested at 13 h p.i. (lanes 1 through 4) or 20 h p.i. (lanes 5 through 7), treated with DNase, and reverse transcribed using oligo(dT). PCRs were performed using primer sets specific for ie1, ie3, M54, M55, M115, and HPRT as described in Materials and Methods. Amplified products were separated on 1.5% agarose gels and visualized by ethidium bromide staining. Sizes of the amplified products are indicated by arrows. Specific PCR-amplified products were not detected in control reactions in which the reverse transcriptase was not added during the RNA reverse transcription reaction (data not shown).