Human Cytomegalovirus Gene UL21a Encodes a Short-Lived Cytoplasmic Protein and Facilitates Virus Replication in Fibroblasts ^▿

Plasmids and antibodies.

pYD-C235 is a pLPCX-derived retroviral vector (Clontech) that expresses a DsRed gene driven by an internal ribosome entry site 2 (IRES2) (36). pYD-C423, pYD-C428, and pYD-C429 were created by amplifying the UL21a, UL99 (encoding pp28), and UL44 coding sequences and cloning the PCR products upstream of the IRES2 of pYD-C235, respectively. pYD-C255 contained a GalK/kanamycin dual-expression cassette that was used for the first step of linear recombination (31) (see below).

To generate a polyclonal antibody to pUL21a, the entire UL21a coding sequence was cloned upstream of a His₆ tag in the expression vector pET-22b (Novagen). The His-tagged fusion protein was produced in Escherichia coli, purified by using Ni-NTA agarose beads (Qiagen), and used as an immunogen to generate the rabbit antisera (Covance). The rabbit antisera were subsequently affinity-purified by using the UL21a recombinant protein (Covance), resulting in the high-titer, highly specific anti-pUL21a polyclonal antibody that was tested for its specific interaction with the immunogen or the virus-produced pUL21a by immunoblot assay. Additional primary antibodies used in the present study included: anti-β-actin (clone AC15; Abcam); anti-p53 (clone OP-03, Calbiochem); anti-UL44 (clone 10D8; Virusys); anti-MCP (anti-major capsid protein) (generous gift from Wade Gibson, John Hopkins University); and anti-IE1, anti-pp65, anti-pUL69, and anti-pp28 (36) (generous gifts from Thomas Shenk, Princeton University).

Cells and viruses.

Primary human newborn foreskin fibroblasts (HFFs), embryonic lung fibroblasts (MRC-5), HeLa cells, and mouse ts20 cells (10) (a generous gift from Harvey Ozer, UMDNJ-New Jersey Medical School) were propagated in Dulbecco modified Eagle medium supplemented with 10% fetal calf serum, nonessential amino acids, and penicillin-streptomycin. For transient expression, HeLa cells were transfected by using Lipofectamine (Invitrogen), and HFFs were electroporated by using Amaxa Technologies (Lonza) with plasmids expressing the proteins of interest according to the manufacturers' instructions.

Various BAC-HCMV clones were constructed to reconstitute recombinant HCMV viruses. The BAC-HCMV clone pAD-GFP was used as the parental clone to produce wild-type virus (ADwt). pAD-GFP carries the full-length genome of HCMV strain AD169 and contains a simian virus 40 (SV40) early promoter-driven green fluorescent protein (GFP) gene in place of the viral US4-US6 region (36, 44). All other recombinant BAC clones were constructed by using a linear recombination protocol in the bacterial strain SW102 (31). A GalK/kanamycin dual marker cassette was amplified by PCR from pYD-C255 with a pair of 70-bp primers that had 5′-terminal 50-bp sequences homologous to the viral sequences of targeted sites (see Table 1 for all primers). The marker cassette was subsequently recombined into pAD-GFP at the locus of interest by linear recombination to generate substitution or insertional mutant BAC-HCMV clones. Resulting transformants were selected on kanamycin-containing LB plates to identify clones carrying the marker cassette. One of the substitution mutant BAC-HCMV clones, pADsubUL21a, carried the marker cassette in place of UL21a. To create a UL21a marker rescue BAC-HCMV clone (pADrevUL21a), the UL21a sequence was amplified by PCR and subsequently recombined into pADsubUL21a to replace the marker cassette by linear recombination. The resulting recombinants were selected on 2-deoxy-galactose (DOG)-containing minimal media plates for the loss of GalK/kanamycin. To create a UL21a clean-deletion BAC-HCMV clone (pADdlUL21a), a pair of overlapping 70-bp primers were generated that together spanned 50 bp both upstream and downstream of the UL21a ORF. The PCR fragment generated from these primers was recombined into pADsubUL21a to replace the marker cassette by linear recombination. To insert the GFP-S tag at the N terminus of the UL21a ORF in the viral genome, the GalK/kanamycin marker was amplified by PCR and inserted immediately downstream of the UL21a start codon by linear recombination in pAD/Cre (44), a BAC clone carrying the full-length AD169 genome without GFP, resulting in the clone ADinGalK/kan-UL21a. The GFP-S tag sequence was amplified from the plasmid pIC113 (9) and recombined into the viral genome of ADinGalK/kan-UL21a, in frame at the N terminus of the UL21a ORF, to replace the marker sequence by linear recombination, resulting in ADinGFP-UL21a. All recombinant BACs were verified by restriction digestion, Southern blotting, PCR, and direct sequencing analysis.

To reconstitute virus, 2 to 4 μg of the BAC-HCMV DNA and 1 μg of the pp71-expression plasmid were transfected into MRC-5 fibroblasts by electroporation (44). Culture medium was changed 24 h later, and virus was harvested by collecting cell-free culture supernatant when the entire monolayer of infected cells was lysed. Alternatively, virus was also produced by collecting cell-free culture supernatant from HFFs infected at a multiplicity of infection (MOI) of 0.005. Virus-containing culture supernatants were then purified by ultracentrifugation through a 20% d-sorbitol cushion at an average relative centrifugal force of 53,000 × g for 1 h, resuspended in Dulbecco modified Eagle medium with 10% fetal calf serum, and saved as viral stocks. HCMV virus titers were determined in duplicate by plaque assay (31), and their DNA content was determined by real-time quantitative PCR (see below).

Analysis of viral growth kinetics.

HFFs or MRC-5 cells were seeded in 12-well plates overnight to produce a subconfluent monolayer. Cells were then inoculated with recombinant HCMV viruses for 1 h at an MOI of 0.001 for multistep growth analysis or an MOI of 1 for single-step growth analysis. The inoculum was removed, infected monolayers were rinsed with phosphate-buffered saline, and fresh medium was replenished. At various times postinfection, cell-free virus was collected by harvesting medium from infected cultures. In addition, infected cells were pelleted and then lysed by a freeze-thaw cycle followed by sonication, cell debris was cleared by low-speed centrifugation, and supernatants were saved as cell-associated virus samples. Titers of each virus sample were determined by plaque assay. Moreover, plaque size was measured by using ImageJ software (National Institutes of Health), and the P value associated with the Student paired t test with a two-tailed distribution was calculated by scoring at least 50 plaques per infection.

Analysis of DNA, RNA, and proteins.

To prepare virion DNA for quantification by real-time quantitative PCR, 100 μl of cell-free virus (or 10 μl of purified virus) was treated with DNase I (30 U; Roche) at 37°C for 30 min to remove contaminating DNA, followed by incubation at 75°C for 20 min to stop the reaction. The samples were then incubated overnight at 55°C in lysis buffer (400 mM NaCl, 10 mM Tris [pH 8.0], 10 mM EDTA, 0.1 mg of proteinase K/ml, 0.2% sodium dodecyl sulfate [SDS]), and DNA was extracted with phenol-chloroform and treated with 20 μg of RNase A/ml for 1 h at 37°C. DNA was extracted again with phenol-chloroform, precipitated with ethanol, and resuspended in nuclease-free water (Ambion). To prepare intracellular DNA from infected cells for quantification, HCMV-infected HFFs were collected at various times postinfection, resuspended in lysis buffer, and incubated at 55°C overnight. DNA was extracted with phenol-chloroform, treated with 100 μg of RNase A/ml for 1 h at 37°C, extracted again with phenol-chloroform, precipitated with ethanol, and resuspended in nuclease-free water.

Viral DNA was quantified by real-time quantitative PCR as previously described (31) by using a TaqMan probe (Applied Biosystems) and primers specific for the HCMV UL54 gene (30). Cellular DNA was quantified with SYBR green PCR Master Mix (Clonetech) and a primer pair specific for the human β-actin gene (5′-CTC CAT CCT GGC CTC GCT GT-3′ and 5′-GCT GTC ACC TTC ACC GTT CC-3′). The accumulation of viral DNA was normalized by dividing UL54 gene equivalents by β-actin equivalents. The accumulation of wild-type viral DNA at 2 h postinfection (hpi) was arbitrarily set as 1.

mRNA transcripts expressed during HCMV infection were analyzed by Northern blotting and rapid amplification of cDNA ends (RACE) as previously described (31, 44). For Northern blot analysis, single-stranded DNA probes were prepared by using PCR-generated templates and a Strip-EZ PCR kit (Ambion) according to the manufacturer's instructions. Primer pairs used to generate the templates were as follows: 5′-ATG GGA GGT AGC CCT GTT CC-3′ and 5′-TTA AAA CTG GTC CCA ATG TTC TT-3′ (for the UL21a probe), 5′-GTA GCC TAC ACT TTG GCC ACC-3′ and 5′-TTA CTG GTC AGC CTT GCT TCT A-3′ (for the UL123 probe), and 5′-GCG CGC CAG TAC TTT AAC ACA G-3′ and 5′-TCA CGA GTT AAA TAA CAT GGA TTG-3′ (for the UL86 probe). Primers used to generate the ³²P-labeled strand-specific antisense probes were as follows: 5′-TCGTCCAGCAGTAGCACCAGCGGATTGG-3′ (for the UL21a probe), 5′-TTA CTG GTC AGC CTT GCT TCT A-3′ (for the UL123 probe), and 5′-TCA CGA GTT AAA TAA CAT GGA TTG-3′ (for the UL86 probe). RACE analysis was performed by using SMART PCR cDNA synthesis kit (Clontech). Gene-specific primers 5′-TCG TCC AGC AGT AGC ACC AGC GGA TTG G-3′ and 5′-ATC TGT GCG CAT GGA CTT TCG GGC TCG C-3′ were used for 5′ and 3′ RACE, respectively (Fig. 1A).

To analyze protein accumulation by immunoblotting, total cell extracts were prepared by lysing phosphate-buffered saline-washed infected cells in SDS-containing sample buffer. Virion proteins were prepared by purifying virions by ultracentrifugation through sorbitol cushion and resuspending in SDS-containing sample buffer. Proteins were resolved by electrophoresis on a SDS-containing polyacrylamide gel, transferred to polyvinylidene difluoride membranes, hybridized with primary antibodies, reacted with horseradish peroxidase-conjugated secondary antibodies, and visualized by using SuperSignal West Pico chemiluminescent substrate (Thermo Scientific) (42).

To analyze intracellular protein localization by immunofluorescence, cells grown on glass coverslips were fixed in 2% paraformaldehyde, permeabilized with 0.1% Triton X-100 for 5 min, incubated with the primary antibody, and subsequently labeled with the secondary antibody. Labeled cells were counterstained with TO-PRO-3 (Molecular Probes) to visualize the nuclei and then mounted on slides with Prolong Gold antifade reagent (Molecular Probes). Images were captured by using Zeiss LSM Image software with a Zeiss LSM 510 META confocal laser scanning microscope (31).

Proteasome inhibition assays.

The protease inhibitors Z-Leu-Leu-Leu-CHO (MG132), N-acetyl-Leu-Leu-Met (ALLM), and N-acetyl-Leu-Leu-Nle-CHO (ALLN; Calbiochem) were resuspended in dimethyl sulfoxide (DMSO; Sigma) at a concentration of 10 mM as stock solutions. In overexpression experiments, protease inhibitors were added to culture medium 48 h after HeLa cells were transfected with expression plasmids, and cell lysates were collected 24 h later for immunoblot analysis. In HCMV infection experiments, MG132 was added to culture medium of infected HFFs 10 h before cell lysates were collected.

UL21a encodes a single transcript with early gene expression kinetics.

No products have been previously identified from the UL21/UL21a gene locus. Since UL21a is highly conserved at the amino acid level among primate CMVs, we hypothesized that this gene encoded a protein product.

We carried out Northern blot analysis to assess potential transcripts arising from the UL21a gene locus during HCMV infection (Fig. 1B). Using a UL21a-specific antisense probe, we detected a single specific transcript of ∼600 bp during wild-type virus infection. The accumulation of this transcript was evident as early as 8 hpi and became more abundant by 24 hpi. The transcript was absent in infection of ADsubUL21a, the recombinant virus in which the entire UL21a putative coding sequence was replaced by a marker sequence (see Fig. 5A), indicating its UL21a-specific origin. The expression kinetics of the transcript was determined by inhibition assays for protein translation and DNA replication. Cycloheximide (CHX), a protein synthesis inhibitor, increased the accumulation of the immediate-early transcript of UL123, whereas it significantly reduced the accumulation of the UL21a transcript at 8 and 24 hpi, ruling out UL21a as an immediate-early gene. Phosphonoacetic acid (PAA), a DNA synthesis inhibitor, blocked the accumulation of the 8-kb transcript of the late gene UL86 (7) but did not decrease production of the UL21a transcript at 48 hpi, ruling out UL21a as a late or leaky late gene. Together with its temporal expression pattern, these results indicate that UL21a is transcribed with early gene kinetics.

To map the ends of the UL21a transcript, we performed both 5′- and 3′-RACE analysis of HCMV-infected HFFs using two UL21a-specific primers that would produce overlapping RACE products (Fig. 1A). Both RACE reactions produced a single band, providing additional evidence that UL21a encodes a single transcript (Fig. 1C). Sequence analysis of RACE products indicated that the UL21a transcript was unspliced and initiated 23 bp upstream of the start codon. All of the three 3′-RACE products terminated at different locations within 30 to 75 bp downstream of the stop codon, suggesting that the transcript can be polyadenylated at multiple sites within this region (Fig. 1A). Notably, 5′-RACE analysis did not detect a product that could encode the N-terminal portion of the putative UL21 ORF (Fig. 1A), even after extending the reaction for an additional 10 cycles (data not shown). Therefore, we mapped the UL21a transcript but found no evidence for the presence of a UL21-specific transcript.

pUL21a is expressed but targeted for degradation in a proteasome-dependent manner during HCMV infection.

To directly detect the protein products encoded by UL21a, we created a rabbit polyclonal antibody against a recombinant protein derived from the UL21a coding sequence. The antisera were affinity purified with the UL21a recombinant protein to generate a specific polyclonal antibody. The antibody was sensitive since it could detect the recombinant protein at the level of 10⁻⁸ nmol when used in immunoblot analysis (data not shown). Importantly, this antibody detected a single protein, termed pUL21a, with an apparent molecular mass of ∼15 kDa in the lysates of cells infected with wild-type virus but not in mock-infected cells or in cells infected with UL21a deletion virus (Fig. 2A). Therefore, pUL21a was encoded by UL21a, was expressed at the predicted size, and was lost in the deletion mutant virus. The accumulation of pUL21a became visible 10 hpi, peaked at 24 to 48 hpi, and then gradually decreased. Despite the strong sensitivity of the antibody, the signal detected in infected cell lysates was weak, even at 24 hpi (Fig. 2A and data not shown), suggesting low levels of pUL21a accumulated during HCMV infection.

We were intrigued by the apparent low levels of pUL21a because the accumulation of the UL21a transcript appeared abundant during infection (Fig. 1B), and thus hypothesized that pUL21a was targeted for degradation. We tested this hypothesis by expressing pUL21a in the presence or absence of protease inhibitors. MG132 is a specific inhibitor of the proteasome, ALLN is a calpain inhibitor that also acts as a weak inhibitor of the proteasome, and ALLM is a calpain inhibitor that does not inhibit the proteasome at the concentrations used here (22). When proteins were expressed from the HCMV immediate-early promoter in HeLa cells in a transient-expression experiment, the proteasome inhibitor MG132 drastically enhanced the accumulation of pUL21a, whereas the calpain inhibitor ALLM had a minimal effect (Fig. 2B). ALLN increased the accumulation of pUL21a to some extent, a finding consistent with its weak inhibitory activity to the proteasome. As a control, all three inhibitors had a minimal effect on the accumulation of another HCMV protein, pp28. The degradation of pUL21a could be markedly inhibited by MG132 at a concentration as low as 0.5 μM (Fig. 2C). At a concentration of 2.5 μM, treatment with MG132 for as little as 5 h had a clear effect in the rescue of pUL21a accumulation (Fig. 2C). Interestingly, when pUL21a accumulation was stabilized, a second, faster-migrating protein band was also detected by the α-pUL21a antibody, suggesting that an additional form of pUL21a might be produced. These results indicate that the proteasome-mediated degradation of pUL21a is independent of other viral factors. Furthermore, such degradation appears to be pUL21a-specific since the viral protein pp28 was stable independent of proteasome inhibition.

We then tested whether pUL21a was also subject to rapid degradation in the context of viral infection. HFFs were infected with ADwt or ADsubUL21a, treated with MG132 or DMSO control at 38 hpi, and analyzed for the accumulation of viral proteins at 48 hpi by immunoblot analysis (Fig. 2D). Again, whereas MG132 had a marginal effect on the accumulation of HCMV proteins IE1 and pUL44, it drastically increased the accumulation of pUL21a during HCMV infection. Therefore, pUL21a is targeted for proteasome-dependent degradation during HCMV infection.

Degradation of pUL21a is unaltered in cells lacking a functional ubiquitin-conjugation system.

The most common mechanism by which proteins are targeted to the 26S proteasome is via polyubiquitination at internal lysine residues and, in some cases, ubiquitination can occur at cysteine, serine, and threonine residues (6, 40) or at the N terminus of the protein (2, 11). The UL21a ORF contains no lysines or cysteines and, furthermore, we could not detect pUL21a-ubiquitin conjugates in cells treated with MG132 (data not shown). We hypothesized that pUL21a degradation was ubiquitin independent, a mechanism that has been recently reported for a limited number of proteins (24, 45). To test this hypothesis, we transiently transfected pUL21a-expressing vector into mouse ts20 cells that carried a temperature-sensitive E1 ubiquitin-activating enzyme (10) and examined the accumulation of pUL21a at both permissive (35°C) and restrictive (39°C) temperatures. In these cells, the ubiquitin pathway was disabled at the restrictive temperature, thus stabilizing proteins, such as p53, that were targeted for the ubiquitin-mediated degradation (10). In this experiment, the ubiquitin pathway was active at 35°C, reducing p53 to the undetectable level and, as anticipated, MG132 treatment stabilized p53 (Fig. 3). At 39°C, p53 was stabilized, and MG132 treatment had no effect on its accumulation, indicating that the ubiquitin conjugation pathway was indeed disabled at this restrictive temperature. In contrast, the pUL21a level was equally low at both temperatures, but the protein level was greatly enhanced by MG132, suggesting that the degradation of pUL21a is proteasome dependent and ubiquitin independent.

pUL21a specifically localizes to the cytoplasm.

Next, we sought to determine the intracellular localization of pUL21a by indirect immunofluorescence. To determine the localization pattern of pUL21a in the absence of any other viral proteins, HeLa or HFF cells were transfected with the plasmid expressing both a CMV immediate-early promoter-driven viral protein (i.e., pUL21a, pp28, or pUL44) and IRES-driven DsRed. The overexpressed pUL21a was detected throughout the cytoplasm by the α-pUL21a antibody but was almost completely excluded from the nucleus of DsRed-positive cells (Fig. 4A and data not shown). The antibody was specific to pUL21a since it did not cross-react with other overexpressed viral proteins (i.e., pp28 and pUL44). We then analyzed the localization of pUL21a during virus infection. Unfortunately, the low level of pUL21a accumulation during infection relative to that in overexpression (data not shown) and perhaps also the low sensitivity of α-pUL21a antibody to the nondenatured protein prevented us from detecting the native pUL21a during infection by immunofluorescence analysis, even in the presence of MG132 (Fig. 4B and data not shown). To circumvent this issue, we created a recombinant HCMV virus, ADinGFP-UL21a, which accumulated a functional, GFP-S tagged variant of pUL21a at much higher levels than the native protein during infection by an unknown mechanism (see Materials and Methods and data not shown). Similar to what was observed in overexpression experiments, the tagged UL21a protein was detected predominantly in the cytoplasm by the α-pUL21a antibody during the entire course of ADinGFP-UL21a infection (Fig. 4B). In a few infected and transfected cells, some pUL21a-specific staining was also observed in the nucleus (data not shown). pUL21a is only 123 aa long with an apparent molecular mass of ∼15 kDa, a size that would allow it to pass through nuclear pores freely. The low level of pUL21a staining in the nucleus of these cells might represent the background level of passive diffusion of the protein through the nuclear pores. Nonetheless, even in these cells, the distribution of pUL21a in the cytoplasm was much more abundant than that in the nucleus. In both overexpression and infection experiments, pUL21a distribution appeared to be diffuse with no apparent colocalization with cytoplasmic organelles.

UL21a facilitates HCMV replication in fibroblasts.

Previously, two genome-wide mutagenic analyses tentatively classified overlapping UL21a/UL21 as augmenting genes because mutations in this locus resulted in attenuated growth of HCMV in fibroblasts (14, 43). Since the transcript and the protein product arising from UL21a have now been identified, we created mutant viruses where UL21a, UL21, and its neighboring genes were individually deleted, and determined whether the UL21a gene was directly required for growth of the virus.

We constructed three deletion mutant BAC-HCMV clones (pADsubUL20, pADsubUL21a, and pADsubUL21.5-UL23) by using a linear recombination-based BAC mutagenesis approach (Fig. 5A) (see Materials and Methods). Each of these clones was derived from the parental BAC clone (pAD-GFP) that carries the genome of the HCMV AD169 strain, expresses an SV40 early promoter-driven GFP gene, and reconstitutes a wild-type control virus ADwt (31, 44). The mutant BAC clones were used to generate mutant viruses, namely, ADsubUL20, ADsubUL21a, and ADsubUL21.5-UL23, respectively, upon transfection into human fibroblasts. In pADsubUL21a or pADsubUL20, the entire coding sequence of UL21a or UL20 was replaced by a GalK/kanamycin marker cassette. In pADsubUL21.5-UL23, the viral genomic sequence encoding the C-terminal half of the UL21.5 ORF (i.e., aa 64 to 103), the miRNA UL22A-1, and the C-terminal half of the UL23 ORF (i.e., aa 121 to 284) was replaced by the marker cassette. In addition, the UL21a gene in ADsubUL21a was subsequently repaired, producing the marker-rescued BAC clone, pADrevUL21a, that was used to generate the marker-rescued virus, ADrevUL21a (Fig. 5A). All recombinant BAC clones were examined for integrity and desired alterations by using EcoRI endonuclease digestion, Southern blotting, PCR, and direct sequencing analysis (data not shown). All other recombinant BAC clones used in the present study were examined with the same level of analysis and were shown to carry the intact viral genome and contain the precise modification at the correct locus.

We then tested the roles of neighboring genes UL20, UL21a/UL21, and UL21.5-UL23, in HCMV growth in HFFs. The first indication of the importance of UL21a in HCMV growth was the observation that the UL21a deletion virus ADsubUL21a generated much smaller plaques and produced progeny virus with titers ∼30-fold lower than those with the wild-type virus (data not shown). On average, the size of plaque produced by ADsubUL21a was no more than 40% of those produced by ADwt at 12 days postinfection (dpi) (Fig. 5B). A multistep growth analysis demonstrated that two independent isolates of UL21a deletion virus were attenuated in growth, producing ∼30-fold less infectious virus than wild-type virus by 16 dpi (Fig. 5C). The marker-rescued virus ADrevUL21a replicated indistinguishably from wild-type virus. Furthermore, ADdlUL21a, a mutant virus in which UL21a was deleted without the insertion of GalK/kanamycin marker sequence, replicated indistinguishably from ADsubUL21a virus (data not shown). In contrast, mutant viruses lacking neighboring genes UL20 (ADsubUL20) or UL21.5-UL23 (ADsubUL21.5-UL23) replicated at wild-type levels, a finding consistent with previous reports (Fig. 5C) (14, 38, 43). These results indicated that UL21a deletion virus is markedly attenuated in growth. This defect is the direct result of loss of UL21a rather than aberrant expression of neighboring genes resulting from the deletion of UL21a or inadvertent second-site mutations generated during mutagenesis.

Under single-step growth condition, the UL21a deletion virus was also attenuated, producing 25-fold less cell-free infectious virus than the wild-type virus at 4 dpi (Fig. 5C). This attenuation was not the result of a defect in the release of infectious virus from host cells because the accumulation of cell-associated infectious virus was similarly reduced in UL21a deletion virus (Fig. 5C).

Intriguingly, we found that the growth defect of UL21a deletion virus was even more pronounced in MRC-5 human embryonic lung fibroblasts (Fig. 5D). As anticipated, ADsubUL20 and ADsubUL21.5-UL23 replicated indistinguishably from ADrevUL21a in MRC-5 fibroblasts. In contrast, at an MOI of 0.001, ADsubUL21a grew at a much reduced rate with delayed kinetics. It produced >2,500-fold less infectious virus than marker-rescued virus ADrevUL21a at 15 dpi, the time when ADrevUL21a reached its peak yield. The growth of ADsubUL21a did not reach its peak until 20 dpi, producing a titer that was still >60-fold lower than the peak yield of ADrevUL21a (Fig. 5D). Thus, the growth of the UL21a deletion virus appeared to be differentially attenuated in HFFs and MRC-5 fibroblasts, which to our knowledge has not previously been reported for any other HCMV mutant viruses.

Since UL21a overlaps with the originally annotated putative gene UL21 (8), it was formally possible that the growth defect of pUL21a deletion virus was the result of the inadvertent deletion of UL21. However, UL21 might not represent a real viral gene because the putative ORF that it encoded was not conserved among primate CMVs and because our analysis found no evidence for the presence of UL21-specific transcripts (Fig. 1). In order to exclude any possible involvement of UL21 in the phenotype of UL21a deletion virus, we took advantage of the recombinant HCMV virus, ADinGFP-UL21a, in which the insertion of GFP at the N terminus of the UL21a ORF fortuitously disrupted the putative UL21 ORF in a manner similar to ADsubUL21a (data not shown) (Fig. 1A). Importantly, ADinGFP-UL21a replicated at wild-type levels despite the expected disruption of UL21 in this virus (data not shown). Collectively, our data provide experimental evidence that the loss of UL21a, not UL21, is directly responsible for the growth defect of ADsubUL21a.

UL21 deletion virus produces progeny with reduced infectivity.

To determine the step of the viral replication cycle that is compromised in the absence of UL21a, we first analyzed viral gene expression and DNA replication in the absence of UL21a. At an MOI of 1, UL21a deletion virus produced representative viral proteins at near wild-type levels (Fig. 6A). The mutant virus had a small decrease (two- to threefold) in viral DNA replication at late times postinfection (Fig. 6B), correlating to a similar decrease in the release of DNase-resistant virion DNA into the supernatants (Fig. 6C). Interestingly, cells infected with the UL21a deletion virus contained significantly more viral DNA (Fig. 6B) and virion proteins (pp65, pp28, and MCP) (Fig. 6A) at 2 hpi, a time prior to viral gene expression. Consistently, ∼10 times more DNA-containing virions were needed for the mutant virus to achieve the same infectivity as the wild-type virus (Fig. 6C). Together, these results suggest that UL21a may have multiple roles; one is to enhance viral DNA replication, and another is to promote the ability of the virus to establish productive infection.

pUL21a is not detected in HCMV virions.

Since UL21a enhanced the infectivity of HCMV virions, it was possible that pUL21a is a tegument or structural protein. However, a proteomics study failed to detect pUL21a in the HCMV virions (37). To confirm this result, supernatants were harvested from ADrevUL21a- or ADsubUL21a-infected cells at 96 hpi, and the virions were partially purified by ultracentrifugation through a sorbitol cushion. These virion samples appeared to be largely free of contamination of cellular debris since they did not contain detectable amounts of the viral protein IE1 or the cellular protein Bip (Fig. 7). On the other hand, the MCP was readily detected and, importantly, pUL69, a protein that is present in the tegument and dense bodies in very low amounts (37, 41), could also be detected in the virion samples of ADrevUL21a (Fig. 7). Under these conditions, however, we could not detect pUL21a in the virion samples. Thus, pUL12a is unlikely to be a component of the HCMV virion.