Abundant early expression of gpUL4 from a human cytomegalovirus mutant lacking a repressive upstream open reading frame

Abstract

The human cytomegalovirus UL4 gene encodes a 48-kDa glycoprotein, expression of which is repressed at the translational level by a short upstream open reading frame (uORF2) within the UL4 transcript leader. Mutation of the uORF2 initiation codon in the viral genome eliminates ribosomal stalling at the uORF2 termination site, resulting in early and abundant gpUL4 protein synthesis. This mutation does not appear to affect viral replication kinetics in human fibroblasts. These results reveal that the unusual uORF2 inhibitory mechanism is a principal determinant of the abundance and timing of gpUL4 expression but is nonessential for replication in cell culture.

FIG. 1

Construction of recombinant wild-type and mutant CMV. The CMV genome containing unique long (U_L) and unique short (U_S) regions flanked by repeats (ab, b′ a′c′, and ca) is depicted (top) along with the approximate positions of the cosmid fragments used in construction of the recombinant viruses (middle). The ∼9.6 kb SacI/BamHI insert present in pEQ694 (bottom) contains the UL4 gene and flanking regions. Relevant restriction sites are indicated. The pEQ694 sequence corresponding to the 5′ end of the UL4 mRNA contains a BglII linker insertion (bold) into a NaeI site and a mutation of the uORF2 AUG codon to AAG (underlined).

FIG. 2

Analyses of wild-type and mutant CMV genomic structures. DNA purified from HF infected with the indicated viruses was digested with HindIII or with HindIII and BglII After electrophoretic separation, the ethidium bromide staining pattern was photographed (A) and samples were analyzed by Southern blot hybridization (B and C) using either a whole-virus probe (B) or a UL4 probe (C). (D) Diagram of the UL4 probe.

FIG. 3

Kinetics of UL4 gene expression by wild-type and mutant viruses. Extracts of HF were prepared at various times after infection with the indicated viruses. After sodium dodecyl sulfate-polyacrylamide gel electrophoresis, gpUL4 (A) and ppUL44 (C) were detected by immunoblot analysis using rabbit anti-gpUL4 serum or a mouse monoclonal antibody, respectively. (B) RNA samples harvested from HF infected at the same time were analyzed by Northern blot hybridization, using probes specific for UL4 or β-actin. Since all the RNA samples were analyzed on one Northern blot for each probe, the mock-infected-cell lane, shown only in the CMV(Towne) panel, is the control for all panels.

FIG. 4

Mutation of the uORF2 AUG codon eliminates ribosomal stalling at the uORF2 termination site. Extracts of mock-infected HF or HF infected with rTowne-1 or vEQ694-1 were analyzed by toeprint assay using a primer that anneals to the 3′ end of the UL4 transcript leader as described previously (3).

FIG. 5

Kinetics of viral replication. After infection of triplicate dishes of HF with the indicated viruses (multiplicity of infection = 3), the cell medium was collected for determination of titers, and fresh medium was added every 24 h. CMV titers (means ± standard deviations) were determined by plaque assay. Values below the dotted line represent titers of less than ∼3 PFU/ml.