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Viral gene expression during human cytomegalovirus (CMV) infection is regulated at multiple levels, including translation (14). Previous studies of gpUL4 (or gp48), the glycoprotein product of the UL4 gene (8), revealed that its expression is repressed by an unusual translational mechanism that depends on the nascent peptide product of the second of three short upstream open reading frames (uORF2) within the UL4 transcript leader (3, 4, 9, 10, 17). Results from transfection and cell-free translation assays support a model in which the peptide product of uORF2 inhibits translation termination at its own stop codon (3, 4, 6). As a result, ribosomes stall on the mRNA and block ribosomal access to the downstream UL4 cistron.

Clinical isolates of CMV have naturally occurring polymorphisms in uORF2 that change the predicted peptide sequence and, based on transfection assays, are predicted to reduce or eliminate the inhibitory effect of uORF2. In fact, the two such viral isolates analyzed thus far express considerably more gpUL4 protein than strains that have inhibitory uORF2 sequences, even though the levels of UL4 mRNA are similar (1). However, the conclusion that uORF2 controls gpUL4 expression during viral infection based on these observations is limited by the fact that clinical isolates differ at multiple genetic loci in addition to uORF2. Therefore, the present studies were designed to determine the role of uORF2 in controlling gpUL4 expression during infection using an engineered virus that contains precise mutations of the UL4 transcript leader but which is otherwise isogenic with wild-type CMV(Towne).

Construction of uORF2 CMV. To assess the effect of uORF2 on gpUL4 expression when both are expressed from their natural positions within the viral genome, we constructed recombinant CMV in which the UL4 transcript leader was either wild-type or mutant (Fig. 1). The cosmids Tn15, Tn20, Tn23, Tn26, Tn44, Tn45, Tn47, and Tn50 (13) (kindly provided by G. Kemble, Aviron, Mountain View, Calif.) were digested with PacI and then cotransfected into diploid human foreskin fibroblasts (HF) by calcium phosphate coprecipitation (13) to generate a recombinant wild-type CMV (rTowne-1). Two independent mutant viruses that lack uORF2 (uORF2), vEQ694-1 and vEQ694-2, were constructed using the same cosmid fragments except that Tn45 was digested with SpeI in addition to PacI and a SacI/BamHI fragment derived from plasmid pEQ694 was added to the mixture of fragments. pEQ694 was constructed by inserting an ~9.6-kb SacI/BamHI fragment derived from Tn45 into pBS+ (Stratagene, Inc.). The uORF2 AUG codon in this plasmid was changed to AAG, thereby eliminating uORF2 (Fig. 1). In addition, eight nucleotides were inserted into the NaeI site in the UL4 transcript leader in pEQ694, creating a BglII site to facilitate the identification of these mutants. The insertion of this BglII linker did not alter uORF2 inhibitory activity in transient-transfection assays (data not shown). Viruses isolated from transfected cells were plaque purified three times prior to subsequent analyses.

View larger version (12K): [in this window] [in a new window]   FIG. 1.   Construction of recombinant wild-type and mutant CMV. The CMV genome containing unique long (UL) and unique short (US) 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).

View larger version (65K): [in this window] [in a new window]   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.

Abundant early expression of gpUL4 in uORF2-CMV-infected cells. Using the wild-type and recombinant viruses, we evaluated the effects of the mutations on gpUL4 expression. HF were infected with CMV(Towne), rTowne-1, vEQ694-1, or vEQ694-2 at a multiplicity of infection of 3. Cell extracts prepared by sodium dodecyl sulfate lysis every 12 h until 96 h postinfection (p.i.) were analyzed for gpUL4 accumulation by immunoblot assays using gpUL4 polyclonal rabbit serum (1). While little or no gpUL4 was detectable in CMV(Towne)- or rTowne-1-infected cells even at late times, abundant gpUL4 was detectable at 12 h p.i. in vEQ694-1- and vEQ694-2-infected cells (Fig. 3A). The level of accumulated gpUL4 increased by 24 h p.i. and then remained relatively constant in abundance through 96 h p.i. in these cells. The basis for the slight changes in electrophoretic mobility of gpUL4 during infection is unknown, but they may be due to variation in posttranslational modification of this heavily glycosylated protein (8).

View larger version (54K): [in this window] [in a new window]   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 -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.

Mutation of uORF2 eliminates ribosomal stalling on the UL4 mRNA. In previous studies, we utilized the toeprint (or reverse transcription inhibition) assay to detect ribosomal stalling on the UL4 mRNA. This assay is similar to a primer extension reaction but is performed in a crude translation extract such that premature termination occurs when the reverse transcriptase encounters a barrier, such as a ribosome, on the mRNA (11). Previous results demonstrated that cell-free translation of mRNAs containing wild-type but not mutant uORF2 sequences caused ribosomes to stall at the uORF2 termination codon (3, 6). Ribosomal stalling was also detected in CMV(Towne)-infected cells (3).

View larger version (44K): [in this window] [in a new window]   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).

uORF2 CMV has the same growth kinetics as wild-type virus. Our ability to isolate mutant viruses that lack uORF2 and overexpress gpUL4 demonstrates that the uORF2 regulatory mechanism is not essential for viral growth in cell culture. To assess the consequences of the uORF2 mutation for viral growth more carefully, we compared viral production after infection of HF with our wild-type and mutant viruses. As shown in Fig. 5, vEQ694-1, vEQ694-2, and rTowne-1 all produced similar titers of extracellular virus while CMV(Towne) produced slightly less virus at each time point. Although we have not investigated the basis for differences in production between CMV(Towne) and the other viruses, these results might reflect unrecognized genetic differences between CMV(Towne) and viruses that were generated from the cosmids even though the cosmids were derived from an isolate of CMV(Towne) (13). Regardless, these results suggest that the uORF2 inhibitory mechanism does not have a discernible impact on CMV replication kinetics in HF.

View larger version (23K): [in this window] [in a new window]   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.

Discussion. These studies demonstrate that the unusual translational mechanism mediated by uORF2, previously characterized using transient transfection, retrovirus-mediated gene transduction, and cell-free translation assays (1, 3-7, 9, 17), also functions to repress expression of the authentic UL4 gene contained in its natural context in the viral genome. Previous studies also identified polymorphisms within uORF2 that affect the inhibitory function of uORF2 in naturally occurring clinical isolates of CMV (1), suggesting that uORF2 is a principal determinant of gpUL4 expression. However, because clinical isolates are genetically heterogeneous, polymorphisms other than those within uORF2 could also be responsible for differences in gpUL4 expression among isolates. Therefore, this study of wild-type and uORF2 mutants in an isogenic background was necessary to establish the role of uORF2 in governing gpUL4 expression during viral infection. Our comparisons of wild-type and mutant viruses reveal that mutation of the uORF2 AUG codon in the viral genome prevents ribosomes from stalling on the gpUL4 transcript leader and results in early and abundant gpUL4 synthesis.