INTRODUCTION

Top Abstract Introduction Materials and Methods Results Discussion References

Previous investigations into the basis for the scarcity of the human cytomegalovirus (HCMV) gpUL4 protein (gp48) at early times in infection revealed that its expression is controlled by an unusual translational mechanism (2-4, 6). The second of three short upstream open reading frames (uORFs), uORF2, within the 5' leader of the gpUL4 mRNA encodes a 22-codon peptide that mediates inhibition of downstream translation. Ribosomes that translate uORF2 fail to cleave the peptidyl-tRNA bond linking the nascent uORF2 peptide to tRNA^Pro, the tRNA responsible for decoding the last uORF2 codon (3). These results suggest a model in which the nascent uORF2 peptide inhibits downstream translation by blocking translation termination at its own stop codon, thereby causing ribosomes to stall on the mRNA and to block access to the gpUL4 initiation codon by other scanning ribosomes.

Although the precise mechanism by which the uORF2 peptide inhibits translational termination is not yet known, the uORF2 peptide sequence is critical for this activity (2, 3, 6). Mutations that alter carboxy-terminal amino acids release the inhibitory effect of uORF2 on downstream translation and also eliminate both the ribosomal stalling and the blockage of peptidyl-tRNA hydrolysis that occur after translation of wild-type uORF2. In contrast, synonymous mutations of these same codons retain the inhibitory properties of wild-type uORF2. Thus, like a few uORFs in other eukaryotic genes (9, 12, 13, 15, 22), uORF2 acts in a peptide sequence-dependent manner. Comparisons among known sequence-dependent uORFs have not yet revealed any consensus sequences or shared motifs that might help to identify molecular interactions between these uORFs and the cellular translational machinery that are responsible for the inhibitory effects.

In order to gain additional insight into the uORF2-mediated inhibitory mechanism, we extended our investigations by determining the role of codons in the more amino-terminal portion of uORF2. Mutational analyses identified several codons within this region that are essential for the uORF2 inhibitory function. A survey of uORF2 sequences among clinical HCMV isolates revealed that some of these essential codons are polymorphic. Consistent with analyses of engineered mutations, certain polymorphisms eliminate the inhibitory effect of uORF2 on downstream translation, demonstrating that uORF2 is structurally and functionally polymorphic.

	MATERIALS AND METHODS

Top Abstract Introduction Materials and Methods Results Discussion References

Cells, virus, and viral DNA. HCMV (Towne) was grown on primary human fibroblasts (HF) as described previously (6). Frozen suspensions of cells infected with HCMV strains isolated from bone marrow transplant recipients were obtained from the Clinical Virology Laboratory and from B. Torok-Storb of the Fred Hutchinson Cancer Research Center. Clinical isolates C1, C4, and C6 were plaque purified prior to use in infection experiments. To purify viral DNA for sequencing, a sample of ~10 to ~20 µl obtained by scraping a frozen virus-infected cell suspension with a wooden applicator stick was resuspended in either proteinase K buffer (10 mM Tris [pH 7.8], 5 mM EDTA, 0.5% sodium dodecyl sulfate [SDS]) or lysis buffer (50 mM KCl, 10 mM Tris-HCl [pH 8.3], 0.01% gelatin, 0.45% NP-40, 0.45% Tween 20). After proteinase K was added to a final concentration of 100 µg/ml, the reaction mixtures were incubated at 37°C for 1 h and then extracted twice with phenol-chloroform (pH 8.0). Viral DNA was precipitated with ethanol and resuspended in TE buffer (10 mM Tris [pH 8.0], 1 mM EDTA).

Plasmids and sequence analyses. Plasmids pEQ239, pEQ325, pEQ422, pEQ429, and pEQ430 have been described previously (4, 6, 17). A series of -galactosidase (-Gal) expression plasmids (pEQ641 through pEQ648, pEQ700 through pEQ708, and pEQ724 through pEQ729) containing the gpUL4 (gp48) transcript leader with various mutations or deletions in the uORF2 sequence were created by PCR-based mutagenesis with plasmid pEQ422 as a template and with the 5' primers shown in Table 1 and the 3' primer 7 (5'CGAGGTGCTGTTTCTGGT). The amplified products were digested with HindIII and AflII and inserted into pEQ422 that had been digested with HindIII and AflII, thus replacing the uORF2 sequences as depicted in Table 1 and Fig. 1 through 5. The gpUL4 leader sequences in these plasmids were verified by automated sequence analysis with primer 7 and an Amplitaq FS kit (Perkin-Elmer).

Transfection and RNA analyses. -Gal expression plasmids were transfected into primary HF with DEAE-dextran as described previously (1). Twenty-four hours after transfection, cells were infected with HCMV at a multiplicity of infection of 3, and at 48 h postinfection, -Gal expression was measured by adding the fluorogenic substrate 4-methylumbelliferyl--D-galactoside to the cell culture medium. The mean (plus standard deviation) fluorescence values from triplicate 60-mm-diameter dishes minus the mean value for cells transfected with control pEQ430 are plotted. Whole-cell RNA was extracted by guanidinium isothiocyanate solubilization of cells, pooling of lysates from triplicate dishes, and pelleting of RNA through CsCl (8). -Gal mRNA was detected by Northern blot analysis as described previously (7).

Immunoblot assays. HF in 100-mm-diameter dishes were infected in duplicate with HCMV (Towne) or with clinical isolate C1, C4, or C6 at a multiplicity of infection of 0.1. RNA was isolated from one of each set of duplicate plates as described above at 18 days postinfection, a time when all cells displayed viral cytopathic effects. For protein analysis, cells on the second plate were washed twice with phosphate-buffered saline and lysed in 500 µl of 2% SDS at 60°C and the DNA was sheared by multiple passages through a 27-gauge needle. Approximately 40 µg of protein per sample was separated by SDS-polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes (Micron Separations, Inc.), which were immunoblotted with a Western-Light Plus protein detection kit (Tropix). For detection of gpUL4 protein, rabbit polyclonal antibody (gp48MAPC), raised to the multiple antigenic peptide (19) (TTENSRNYYFRREDAN), was used at a 1:10,000 dilution. For ppUL44, a mouse monoclonal antibody (Virusys Corporation, Berwick, Maine) was used at a 1:20,000 dilution.

	RESULTS

Top Abstract Introduction Materials and Methods Results Discussion References

Effect of amino-terminal deletions of uORF2 on downstream translation. To test whether the amino-terminal portion of uORF2 contributes to inhibition of downstream translation, we constructed plasmids that express transcripts containing mutations affecting codons 3 through 12 of uORF2 within the gpUL4 mRNA leader upstream of the -Gal ORF. The translational impact of these uORF2 mutations was assessed by measuring -Gal activity and -Gal RNA levels after transfection of the expression plasmids into HF and subsequent infection with HCMV.

View larger version (15K): [in this window] [in a new window]   FIG. 1.   Effects of deletion of codons 3 through 12 on uORF2 inhibitory activity. (A) Plasmids containing deletions of codon 3 ( 3, pEQ643), codons 3 through 7 ( 3-7, pEQ644), and codons 3 through 12 ( 3-12, pEQ645) of uORF2 were transfected into triplicate dishes of HF. Control plasmids were pEQ422, which contains an optimal-context AUG codon (o/c) but otherwise wild-type uORF2 (wt), and pEQ429, which contains an optimal-context AUG codon and a missense mutation of proline to alanine at codon 22 (P22A). Subsequent to HCMV infection, -Gal activity (B) and lacZ mRNA levels (C) were measured as described in Materials and Methods.

Effects of amino-terminal amino acid substitutions on uORF2-mediated inhibition. The deletion mutations described above alter both the length of the uORF2 peptide and its amino acid content. To distinguish which of these changes was responsible for the reduction of uORF2 inhibitory activity, we replaced codons 3 through 11 in toto with either conservative or nonconservative substitutions. If the length but not a particular sequence of the amino-terminal region is required for the inhibitory mechanism, then both the conservative and nonconservative mutants should retain the inhibitory activity. On the other hand, if amino acid coding sequences within the amino terminus of uORF2 are required for translational inhibition, then conservative mutations may have little or no effect while nonconservative substitutions would be more likely to disrupt uORF2 peptide-mediated inhibition.

View larger version (14K): [in this window] [in a new window]   FIG. 2.   Missense mutations of codons 3 through 11 reduce uORF2 inhibitory activity. (A) Plasmids having conservative (pEQ641) and nonconservative (pEQ642) substitutions in uORF2 and the same control plasmids as those used for the Fig. 1 experiments were transfected into HF. After HCMV infection, -Gal activity (B) and lacZ mRNA levels (C) were measured as described in Materials and Methods. o/c, optimal-context AUG codon; wt, wild-type uORF2.

View larger version (15K): [in this window] [in a new window]   FIG. 3.   At least two codons, one in positions 3 through 5 and one in positions 6 through 8, are required for full uORF2 inhibitory activity. (A) Plasmids containing triple amino acid uORF2 substitutions derived from subsets of the nonconservative missense mutations in pEQ642 (Fig. 2) were transfected into HF. Following infection with HCMV, -Gal activity (B) and lacZ mRNA levels (C) were measured as described in Materials and Methods. o/c, optimal-context AUG codon; wt, wild-type uORF2.

Multiple codons in the amino-terminal region of uORF2 are each necessary for inhibition of downstream translation. The preceding results suggest that specific amino acids within the amino-terminal portion of the uORF2 peptide are necessary for translational inhibition. To precisely define the critical codons, we analyzed expression downstream from uORF2 mutants with single missense mutations in codons 3 through 12 (Fig. 4A). Complete release of the inhibitory signal occurred when either codon 7 or 8 was mutated (pEQ704 or pEQ705, respectively) (Fig. 4B). Mutation at codon 3, 4, 5, or 6 (pEQ700, pEQ701, pEQ702, or pEQ703, respectively) released the inhibitory signal partially. The increase in -Gal expression from the codon 3 mutant was somewhat surprising since deletion of codon 3 (pEQ700) maintained the inhibitory signal (Fig. 1). Mutation of codons 9, 10, and 11 (pEQ706, pEQ707, and pEQ708, respectively) individually maintained most of the repressive affect of uORF2 on downstream translation (Fig. 4D), consistent with the preservation of the inhibitory effect by the triple mutant with mutations in codons 9 through 11 (pEQ648) (Fig. 3B).

View larger version (28K): [in this window] [in a new window]   FIG. 4.   Individual codons within the amino-terminal portion of uORF2 are important for inhibitory activity. Plasmids containing individual missense mutations (A) were transfected into HF in two separate experiments (B and C; D and E). Following infection with HCMV, -Gal activity (B and D) and lacZ mRNA levels (C and E) were measured as described in Materials and Methods. o/c, optimal-context AUG codon; wt, wild-type uORF2.

Survey of uORF2 in HCMV clinical isolates. We previously showed that clinical isolates of HCMV contain sequence polymorphisms in uORF2 (6). While key carboxy-terminal codons, such as proline 21 and proline 22, were consistently highly conserved, some polymorphisms located closer to the amino terminus affected some codons that, based on the results of the above-described analyses, seemed to be necessary for uORF2 function.

Inhibitory activity of naturally occurring uORF2 variants. To test whether polymorphisms found in clinical isolates affect uORF2 function, we cloned the gpUL4 transcript leaders from three previously sequenced clinical isolates, C1, C4, and C6 (6) into lacZ expression plasmids (pEQ448, pEQ449, and pEQ450, respectively) (Fig. 5A) and analyzed -Gal expression in transfection and infection assays. The uORF2 from these clinical isolates had various effects on downstream translation, ranging from full inhibition with the C4 sequence to partial or full release of inhibition with the C1 and C6 uORF2 sequences (Fig. 5B). Since these plasmids contain the wild-type, rather than the optimal-context, AUG codon at the start of uORF2, we used pEQ239, which contains the gpUL4 leader from HCMV (Towne) and has the wild-type AUG context, and pEQ325, which contains a mutation of the uORF2 AUG to AAG (AUG), as controls in this experiment.

View larger version (19K): [in this window] [in a new window]   FIG. 5.   Effects of polymorphisms found in clinical isolates on uORF2 inhibitory activity. (A) Deduced amino acid sequence of clinical isolates C6, C4, and C1. Dashes indicate amino acid identity with HCMV (Towne). Plasmids containing the gpUL4 leader from these clinical isolates (B and C) or containing individual mutations found in isolate C6 (D and E) were transfected into HF. After infection with HCMV, -Gal activity (B and D) and lacZ mRNA levels (C and E) were measured as described in Materials and Methods. o/c, optimal-context AUG codon; wt, wild-type uORF2.

Expression of gpUL4 after infection with clinical isolates. The above-described experiments demonstrated that naturally occurring polymorphisms in some clinical isolates of HCMV eliminate the inhibitory effect of uORF2 in transient-transfection assays. These results predict that gpUL4, the product of the downstream reading frame in the authentic viral mRNA, should be translated more efficiently after infection by these strains. However, since results of transient-transfection assays are not always reliable indicators of regulatory mechanisms, we tested this prediction by analyzing gene expression in cells infected with the C1, C4, C6, and Towne isolates of HCMV (Fig. 6). Substantially more gpUL4 protein was detected by immunoblot assay after C1 and C6 infection than after Towne or C4 infection (Fig. 6A), despite the similar levels of abundance of UL4 mRNA in all samples (Fig. 6A, bottom). To ensure that the differences in gpUL4 protein expression were not due to a generalized lower level of viral protein synthesis in C4- and Towne-infected cells, we also measured the expression of the viral ppUL44 DNA binding protein (Fig. 6B). An immunoblot revealed similar levels of ppUL44 protein in C4-, C6-, and Towne-infected cell extracts and slightly less in C1-infected cell extracts. The greater expression of gpUL4 protein in the C1- and C6-infected cells despite comparable levels of UL4 RNA and ppUL44 protein in all samples supports the conclusion that uORF2 exerts a major impact on gpUL4 expression during viral infection.

View larger version (31K): [in this window] [in a new window]   FIG. 6.   gpUL4 expression after infection with HCMV (Towne) and clinical isolates. HF extracts obtained 18 days after mock infection or after infection with the HCMV isolate C1, C4, C6, or Towne were separated by SDS-polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes. Immunoblots were probed with a polyclonal antiserum to gpUL4 (A) or an anti-ppUL44 monoclonal antibody (B) as described in Materials and Methods. Molecular size markers are indicated on the left of each blot. Whole-cell mRNA was examined by Northern blot analysis with a UL4 probe (A, bottom). The position of the 18S rRNA migration is indicated.

	DISCUSSION

Top Abstract Introduction Materials and Methods Results Discussion References

Translational repression by uORF2 in the HCMV gpUL4 mRNA occurs by a highly unusual mechanism. The nascent uORF2 peptide remains covalently attached to tRNA^Pro, the tRNA that decodes the final uORF2 codon (3). In addition, ribosomes stall at the uORF2 termination codon (2). These observations suggest that the nascent uORF2 peptide interferes with the peptidyl-tRNA hydrolysis reaction that normally occurs when a ribosome encounters a stop codon. Consequently, ribosomes remain at the uORF termination site on the mRNA, creating an obstacle that prevents other ribosomes from scanning to the downstream cistron (Fig. 7).

View larger version (81K): [in this window] [in a new window]   FIG. 7.   Model of uORF2 peptide interactions with ribosomes or ribosome-associated translation factors. When a ribosome reaches the uORF2 termination codon (UAA), the nascent uORF peptide (chain of circles) remains linked to the tRNA decoding the final uORF2 proline codon, CCU. The ribosome stalls at this site, creating a roadblock that obstructs other ribosomes from scanning to the downstream cistron. Changes in uORF2 amino acids that fully (black circles) or partially (dark-gray circles) alleviate uORF2 inhibitory function, do not affect it (white circles), or for which insufficient data are available (light-gray circles) are shown. The repressive effects of uORF2 may be mediated through its interactions with ribosomal components, such as the peptidyl transferase center (PTC) and the peptide exit domain, or translation factors involved in peptidyl-tRNA hydrolysis, such as eukaryotic release factors.

A distinctive feature of this mechanism is its strict dependence on the uORF2 peptide sequence for inhibition of peptidyl-tRNA hydrolysis, ribosomal stalling, and inhibition of downstream translation (2, 3, 6). In the present study, we discovered that sequences located in the amino-terminal region, like those at the carboxy terminus, are also required for translational inhibition. Mutation of certain amino acid positions (e.g., codons 7, 8, and 12) alleviated much or all of the uORF2 repressive activity. Mutations at other positions had less (e.g., codons 3 through 6) or no (e.g., codons 9 through 11) effect on uORF2 inhibitory activity. Since only one amino acid substitution was analyzed for most codons in uORF2, we cannot exclude the possibility that other amino acid substitutions might have different effects. In fact, nonconservative mutations at codons 7 and 8 (Fig. 4) completely eliminated the inhibitory effect of uORF2 while conservative mutations affecting codons 7 and 8 along with codons 3 through 11 (Fig. 2) only partially reduced the uORF2 inhibitory effect. On the other hand, multiple mutations at codons 9 (K9V, K9Q, and K9E), 10 (K10V and K10E), 11 (L11M and L11Q), 16 (T16A and T16I), 21 (P21A and P21S), and 22 (P22A and P22T) (reference 6 and Fig. 4) have been tested and in all these cases alternative mutations affected uORF2 function in a concordant manner. Thus, these transfection data provide a provisional guide to uORF2 codons that are essential and dispensable for inhibition of downstream translation.

Only a few other eukaryotic genes contain uORFs that have been shown to function in a peptide sequence-dependent manner like uORF2. Inhibition of mammalian S-adenosylmethionine decarboxylase translation requires specific amino acids at the three carboxy-terminal codons of the 6-amino-acid uORF (9), while codons 2 and 3 can be altered without affecting the inhibitory effect. Analyses of the uORF sequence requirements in the cases of Saccharomyces cerevisiae CPA1 and its Neurospora crassa homologue arg-2 (12, 22), the mammalian 2-adrenergic receptor (13), and the mammalian retinoic acid receptor 2 (15) are less complete, but at least some codons located near the middle regions of these uORFs are essential for translational inhibition. We now have evidence that codons dispersed throughout uORF2 in the gpUL4 mRNA are required for the inhibitory mechanism. Comparisons of the uORF sequences among these few examples of sequence-dependent uORFs and those shown to act in a sequence-independent manner (5, 10, 14, 21) do not reveal any distinguishing motifs. Thus, sequence data alone are insufficient for accurately predicting which uORFs, among those found in 5 to 10% of cellular genes and in a strikingly high percentage of some subsets such as oncogenes (11), act in a sequence-dependent manner.

Prompted by the finding that some codons that are critical for uORF2 function are ones that are polymorphic among clinical isolates of HCMV, we investigated the structural and functional diversity of uORF2. In the majority of the 19 clinical HCMV isolates studied here, the uORF2 peptide sequence was the same as that found in the laboratory strains Towne and AD169, both of which preserve the inhibitory effect on downstream translation (17). However, in ~15% of the isolates surveyed, polymorphisms that affect critical codons in uORF2 were present, and thus these strains may express gpUL4 at higher levels. Consistent with these predictions, polymorphisms present in C1 and C6 both allowed high levels of -Gal expression while those present in C4 did not (Fig. 5B). Further, with C6, of the four codons that differ from those of uORF2 in the prototypic strain Towne, the S7L polymorphism accounted for the loss of repression by the C6 uORF2 (Fig. 5D) as predicted by analyses of engineered mutations (Fig. 4).

The transfection results led us to evaluate whether gpUL4 translation is increased in cells infected with isolates having noninhibitory uORF2 variants. Indeed, gpUL4 protein is more abundant after infection with C1 and C6 than after infection with HCMV (Towne) or C4 despite similar levels of UL4 RNA (Fig. 6). We cannot exclude the possibility that other genetic differences among isolates somehow contributed to the differences in gpUL4 expression. However, these findings strongly support the hypothesis that uORF2 polymorphisms are major determinants of gpUL4 expression. Studies of isogenic viruses with and without uORF2 will be needed to definitively establish the role of uORF2 in controlling gpUL4 expression during viral infection.

The observed high-level expression of gpUL4 after C1 and C6 infection of cells in culture demonstrates that the uORF2 repressive mechanism is not essential for viral growth in cell culture. Since these and several other isolates predicted to contain uORF2 sequences that allow efficient downstream translation are low-number-passage clinical isolates, it seems likely that the uORF2 inhibitory mechanism is also not essential for viral replication in humans. However, it is possible that the uORF2 polymorphisms that are inactive in HF in cell culture remain active in other cell types in infected patients. In addition, although the isolates were passaged only a few times in culture, some of these polymorphisms may have arisen ex vivo. Determining whether polymorphisms in uORF2 sequence and function affect the pathogenesis or replication efficiency of HCMV during natural infections will require additional studies. The polymorphisms identified here may be useful for typing of HCMV isolates in efforts to discover linkages between viral genetic loci and disease manifestations (16, 20).

Although we do not know the precise molecular interactions between the uORF2 peptide and the cellular translational machinery that repress translation termination, our experiments support the model depicted in Fig. 7. The uORF2 peptide prevents the peptidyl-tRNA hydrolysis reaction during translation termination, resulting in ribosomal stalling and inhibition of downstream translation. Thus, we hypothesize that the nascent uORF2 peptide interacts with a ribosomal constituent or a translation factor involved in the translation termination reaction. The identification of amino acids dispersed throughout the 22 codons of uORF2 that are necessary for inhibiting translation should be useful in guiding attempts to identify the factors with which the nascent uORF2 peptide interacts.