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School of Biosciences, The University of Birmingham, Edgbaston, Birmingham B15 2TT, UK
Correspondence
Clive Sweet
c.sweet{at}bham.ac.uk
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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1 log10 p.f.u. per tissue) and persisted less well in salivary glands compared with wild-type (wt) and revertant (Rv29.1) virus. In severe combined immunodeficiency mice, Rc29.1 virus showed delayed and reduced replication initially in all tissues (liver, spleen, kidneys, heart, lung and salivary glands). This delayed death until 31 days post-infection (p.i.) compared with wt (23 days p.i.) but at death virus yields were similar to wt. m29 gene transcription was initiated at early times post-infection, while production of a transcript from ORF m29.1 in the presence of cycloheximide indicated that it was an immediate-early gene. ORFs m29.1 and M28 are expressed from a bicistronic message, which is spliced infrequently. However, it is likely that each ORF expresses its own protein, as antiserum derived in rabbits to the m29.1 protein expressed in bacteria from the m29.1 ORF detected only one protein in Western blot analysis of the size predicted for the m29.1 protein. Our results suggest that neither ORF is essential for virus replication but m29.1 is important for optimal viral growth in vitro and in vivo.
Present address: CR UK Institute for Cancer Studies, The University of Birmingham, Vincent Drive, Edgbaston, Birmingham B15 2TT, UK. 
A table showing the GenBank/EMBL/DDBJ accession numbers of the sequences reported in this paper is available with the online version of this paper.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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; Ho, 1991). Murine cytomegalovirus (MCMV) in mice has many disease characteristics similar to that of HCMV in humans. It has a similar tissue tropism, enters the latent state and can be reactivated by immunosuppression (Sweet, 1999). Furthermore, their genomes are linear double-stranded DNA molecules approximately 230 kb in length; HCMV encodes between 156 and 192 predicted open reading frames (ORFs) (Chee et al., 1990; Davison et al., 2003; Dunn et al., 2003; Murphy et al., 2003; Yu et al., 2003; Dolan et al., 2004), while MCMV is predicted to encode >170 ORFs (Brocchieri et al., 2005; Rawlinson et al., 1996; Tang et al., 2006). Seventy-eight of the latter have significant amino acid identity with genes of HCMV (Rawlinson et al., 1996) and the majority of these ORFs are collinear in the two viruses.
Thus, analysis of MCMV homologues of HCMV ORFs are actively being pursued by a number of research groups, while ORFs unique to MCMV with little or no sequence similarity to HCMV have been relatively neglected despite the fact that several of these ORFs (e.g. m04, m06, m138, m144, m145, m152, m155 and m157) have functional HCMV homologues that are involved in immune evasion (Holtappels et al., 2006; Jonjic et al., 2006). Furthermore, these ORFs may also contribute to the species specificity of MCMV.
Preliminary sequencing suggested that the published sequences for m29 and m29.1 may be incorrect and directed our attention to these MCMV unique ORFs. The present studies, involving mutants containing premature stop codon mutations, suggest neither newly described ORF is essential for virus replication but m29.1 has an augmentary function both in vitro and in vivo in immunocompetent and immunodeficient mice. Preliminary characterization of the m29.1 ORF is described.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). Primary mouse embryo fibroblasts (MEF) were prepared from 14-day-old BALB/c mice as described previously (Sammons & Sweet, 1989). MCMV laboratory strains Smith and K181 were provided by C. A. Mims (Department of Microbiology, United Medical and Dental School of Guy's and St Thomas' Hospital, London, UK) (Sammons & Sweet, 1989) and wild-type (wt) MCMV isolates (G4, N1 and K17A) by G. Shellam (Department of Microbiology, University of Western Australia, Australia) (Lyons et al., 1996). Viral titres were quantified in MEF cells by standard plaque assays.
PCR amplification.
The standard PCR reaction used 300 ng DNA in a 50 µl PCR reaction with 2x ReddyMix PCR master mix (Abgene). An initial denaturing step of 2 min at 94 °C was followed by 30 amplification cycles of 94 °C for 45 s, 50–70 °C (depending on primer melting temperature) for 45 s and 72 °C for up to 3 min depending on the length of the expected PCR product.
Sequencing.
PCR products, purified using the GFX PCR DNA and Gel Band Purification kit (GE Healthcare), were sequenced using the Big Dye Terminator Ready Reaction Cycle Sequencing kit (Applied Biosystems) and an ABI 3700 automated sequencer. The entire m29 and m29.1 and partial m30 gene sequences were assembled by comparison to MCMV Smith consensus sequence (Rawlinson et al., 1996) using the NCBI BLAST network service translated using Alltrans and the whole protein sequence for each gene aligned using CLUSTAL W.
Plasmids.
A PCR fragment containing the overlapping m29 and m29.1 sequence (nt 35 241–37 244) (Rawlinson et al., 1996) was cloned into plasmid pCR4Blunt-TOPO (Invitrogen) between EcoRI sites to produce pCR4B-29. Plasmids pRpsl-neo and pKD46 were obtained from Gene Bridges. Plasmid psM3Fr (Wagner et al., 1999) was a gift from U. Koszinowski (Ludwig-Maximilians Universitat Munchen, Pettenkoferstr. 9a, Munchen, Germany). A PCR amplicon of the newly identified m29.1 gene coding sequence was generated and cloned into plasmid pET28a (Novagen) between NcoI and XhoI sites with a C-terminal His-tag to give pET28a-m29.1. The oligonucleotides (restriction enzyme sites are shown in bold) used for amplification were forward (5'-CCGCTCGAGCTAGATGGTGGTGTTTCTCC-3') and reverse primers (5'-CATGCCATGGGCTCGCGTACGGTTATGG-3').
Polyclonal antiserum against m29.1 protein.
The expression vector, pET28a-m29.1, was transformed into Escherichia coli C41 (DE3) to produce m29.1 protein. Protein expression was induced by IPTG. The m29.1 protein was purified using a Ni2+-loaded His-Trap affinity column (GE Healthcare) and supplied to Biogenes to raise polyclonal antibodies.
RT-PCR.
NIH 3T3 cells, treated with or without cycloheximide (200 µg ml–1) or phosphonoacetic acid (300 µg ml–1), were infected with virus at an m.o.i. of 1.0. Total RNA was isolated from cells at different times post-infection using the RNeasy mini kit (Qiagen) and DNA removed using the TURBO DNA-free kit (Ambion) according to the manufacturer's instructions. Reverse transcription was performed on RNA samples using Supercript II reverse transcriptase (Invitrogen) according to the manufacturer's recommendations with the following gene-specific primers (GSP):
m29 forward 5'-ATCCGCATACCGACAGCTTCC-3' (nt 35 750–35 770), m29 reverse 5'-GGATGAAAGCGAAAGTGGCGG-3' (nt 36 457–36 437); m29.1 forward 5'-CTAGATGGTGGTGTTTCTCCTGC-3' (nt 36 029–36 051), m29.1 reverse 5'-GATCTCATGGTCAACTTCGCGG-3' (nt 36 624–36 603); M28 forward 5'-GATCAGATCGCCGTGACTCC-3' (nt 35 194–35 213), M28 reverse 5'-CTGGAAGCTGTCGGTATGCG-3' (nt 35 772–35 753); m29.2 forward 5'-AACAGAGGGATGGAAGCGCC-3' (nt 36 875–36 894), m29.2 reverse 5'-GATCGGGACCTGGATCTCTC-3' (nt 36 996–36 977).
Nucleotide coordinates are given with respect to the published MCMV Smith sequence (U68299 [GenBank] ). The cDNA was then amplified by PCR as described above.
Site-directed mutagenesis.
The introduction of a C to G mutation at nt positions 35 896 and 36 484 in the m29 and m29.1 genes, respectively, for the conversion of the existing tyrosine residue (TAC) to a premature stop codon (TAG) at the 5'-end of the gene was performed using the QuikChange Site-Directed Mutagenesis kit (Stratagene) with plasmid pCR4B-29 according to the manufacturer's instructions. The oligonucleotides (point mutations are shown in bold) were as follows:
m29* forward 5'-TAGAAACGCCCACTAGTCATACGATCGCACG-3' (nt 35 881–35 911), m29* reverse 5'-CGTGCGATCGTATGACTAGTGGGCGTTTCTA-3' (nt 35 911–35 881); m29.1* forward 5'-CGCTAGTATGGAATGCTATCTAGCGTGCACC-3' (nt 36 469–36 499), m29.1* reverse 5'-GGTGCACGCTAGATAGCATTCCATACTAGCG-3' (nt 36 499–36 469).
Nucleotide coordinates are given with respect to the published MCMV Smith sequence (U68299 [GenBank] ). The modified plasmids were named pCR4B-29* and pCR4B-29.1*.
BAC mutagenesis.
A two-step recE/recT homologous recombination was performed to produce the m29 and m29.1 stop codon mutant and revertant to wt viruses. E. coli DH10B containing the MCMV bacterial artificial chromosome (BAC) pSM3fr (chloramphenicol resistance) (Wagner et al., 1999) was transformed with plasmid pKD46, which expresses phage recombinases Red
, Red
and Red
(Yu et al., 2000), and grown to OD600 of 0.1–0.15 on LB medium containing 12 µg chloramphenicol ml–1 and 100 µg carbenicillin ml–1. Following the addition of 5 mM L-arabinose, to induce recE and recT protein expression, cells were further grown to an OD600 of 0.4 and then centrifuged at 2000 g at 5 °C for 10 min. The pellet was resuspended in ice-cold 10 % glycerol and centrifuged again. This was repeated twice and the cell pellet resuspended in an equal volume of ice-cold 10 % glycerol to make competent cells for electroporation. Aliquots (50 µl) were frozen in liquid nitrogen and stored at –80 °C.
A linear DNA fragment (1.3 kb) containing a streptomycin-sensitive and kanamycin-resistant rpsL-neo cassette flanked at the 5'-end with 52 nt upstream of the m29 ORF (nt 35 556–35 607) and at the 3'-end with 51 nt upstream of the m29.1 ORF (nt 36 749–36 799) was amplified by PCR from the pRpsl-neo plasmid template using Extensor Hi-Fidelity PCR master mix (Abgene) and the following primers (sequence from the homology arm is underlined and rpsL-neo sequences are italicized): ETm29 forward (5'-AGGCGACGGAGGTGGGGACGGGCACGGTCGGTTGGATAACCATCTCCGAGAAGGCCTGGTGATGATGGCGGGATC-3') and ETm29 reverse (5'-TCCTCCACGCTCGCGTATAAAATAGGTCTCTGCGAGAGTTGCGCTTCAGACTCAGAAGAACTCGTCAAGAAGGCG-3'). PCR products were digested with DpnI (NEB) to remove any residual template DNA.
Competent cells were thawed once on ice and PCR fragments (100–300 ng in a maximum volume of 10 µl water) were added. Electroporation was performed by using a 1 mm gap ice-cold cuvette and a Bio-Rad Gene Pulser set to 25 µF, 2.3 KV with the pulse controller set at 200 
. SOC medium (1 ml) was added immediately after electroporation. The cells were incubated at 37 °C for 75 min with shaking and then grown at 37 °C overnight on plates containing 12 µg chloramphenicol ml–1 plus 20 µg kanamycin ml–1. Recombinants were selected on their chloramphenicol and kanamycin resistance and streptomycin (80 µg ml–1) susceptibility; integration of the rpsL-neo cassette was confirmed by PCR.
Plasmids pCR4B-29* and pCR4B-29.1* were digested with EcoRI (NEB) to release the linear fragment containing the premature stop codon mutation, which was then used to replace the rpsL-neo cassette as described above. Recombinants were selected on chloramphenicol and streptomycin resistance and kanamycin susceptibility. Recombinant BAC plasmid DNA, isolated using the NucleoBond BAC kit (Macherey-Nagel), was PCR screened and sequenced to confirm the mutated MCMV BAC. The m29 and m29.1 gene-specific mutated BAC MCMVs were transfected into NIH 3T3 cells by using the ExGen500 in vitro transfection reagent (Fermentas) to generate MCMV mutant viruses Rc29 and Rc29.1.
To rescue the m29.1 mutant to wt, a further two-step recE/recT recombination was performed as descried above. Firstly, the rpsL-neo cassette was inserted into the mutant MCMV BAC DNA and secondly, the rpsL-neo cassette was replaced by linear wt DNA. Revertant BAC DNAs were identified by PCR and transfected into NIH 3T3 cells to generate the MCMV revertant virus Rv29.1.
Virus replication in vitro.
MEF cells, seeded into 24-well plates (Corning) at 1x105 cells per well 12 h before virus infection, were inoculated at an m.o.i. of 0.05 (low) or 5.0 (high) p.f.u. per cell. After 1 h incubation, cells were washed twice with PBS and overlaid with growth medium. At 1–7 days post-infection (p.i.), culture fluids were harvested and titrated for the presence of virus.
Virus replication in animals.
Specific-pathogen-free 5-week-old immunocompetent BALB/c mice and 6-week-old immunodeficient CB17 severe combined immunodeficiency (SCID) mice (Charles River) were inoculated intraperitoneally with 104 p.f.u. virus. Infected animals were sacrificed at different times post-infection, salivary glands, lungs, spleens, livers, hearts and kidneys were harvested, homogenized in growth medium, centrifuged and supernatants titrated for the presence of virus.
RNA ligase-mediated rapid amplification of cDNA ends (RLM-RACE).
NIH 3T3 cells were infected with virus at an m.o.i. of 1.0. Total RNA was isolated from cells at 24 h p.i. and DNA removed as described above. RLM-RACE was carried out using the GeneRacer kit (Invitrogen) as recommended by the manufacturer.
For amplification of the 5'-end of the cDNA, the m29.1 GSP1 (5'-ACAGGCGAGTGCGTCGCTATCGT-3') and the GeneRacer 5'-primer supplied with the kit were used and for the 3'-end, the m29.1 GSP2 (5'-GCCTGTCGTGGCTCGGACATGAA-3') and the GeneRacer 3'-primer were used. After gel purification, PCR products were cloned into pGEM-T Easy vector (Promega) and sequenced.
Western blot analysis.
Virus- or mock-infected cells from 75 cm2 tissue culture flasks (Corning) were detached and transferred to a centrifuge tube. This was then centrifuged (6000 g for 10 min at room temperature), cell pellets were lysed in 500 µl ice-cold lysis buffer (1 % Triton X-100, 50 mM Tris/HCl pH 7.5, 150 mM NaCl, 1 mM EDTA, 10 % glycerol, 1 mM PMSF and 1 µg ml–1 each of chymostatin, aprotinin, leupeptin and pepstatin) for 15 min and clarified by centrifugation (15 000 g for 10 min at 4 °C); the supernatants were stored at –80 °C. Equal amounts of protein were separated on SDS-PAGE gels and transferred to a PVDF membrane (Bio-Rad), which was then probed with m29.1 antiserum (dilution of 1 : 300) and incubated with horseradish peroxidase-conjugated anti-rabbit IgG (Sigma) (dilution of 1 : 1000). Proteins were detected using the Opti-4CN substrate kit (Bio-Rad).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). An extra G residue at nt 36 198 changed the predicted m29 and m29.1 ORFs. To confirm this, the MCMV (Birmingham) Smith strain, the K181 (Birmingham) strain and natural wt isolates N1, K17A and G4 were sequenced and all contained the extra G residue (data not shown). The predicted protein of the newly identified m29 ORF is 242 aa in length, 85 aa shorter than that predicted, whereas the m29.1 protein is 210 aa in length, 27 aa longer (Fig. 1b). A single synonymous base change (G to C) was also found at nt 35 944 in all strains examined. Neither of these predicted proteins showed significant similarity to any other herpesvirus proteins. Further analysis of this region also identified a C insertion at nt 37 263 in the MCMV Smith BAC, Smith (Birmingham) strain and isolates N1, K17A and G4 that alters the predicted m30 ORF. This insertion removes the predicted stop codon and the protein continues into the M31 ORF.
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). Non-specific PCR products of 300 and 500 bp were also observed in some mock and test samples. The gene-specific transcript was not expressed in the presence of cycloheximide and was not inhibited by phosphonoacetic acid, indicating that m29 belongs to the early temporal class of genes (Fig. 2d). The m29.1 596 bp gene-specific PCR transcript product was first detected 2 h p.i. and was continuously expressed up to 24 h p.i. (Fig. 2b). Transcription from the m29.1 gene did occur in the presence of cycloheximide, indicating that it is an immediate-early (IE) gene, but its expression was enhanced in the absence of this protein inhibitor (Fig. 2d).
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). It was important to determine whether this ORF was expressed before designing strategies for creating m29 and m29.1 mutants. RT-PCR confirmed this ORF was expressed at 24 h p.i. (Fig. 2c
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RACE analysis of the m29.1 transcript
RACE analysis was performed to examine the nature of the transcript(s) expressed by m29.1. Analysis of the PCR products generated from 5'-RACE revealed one major band of approximately 500 bp, which was cloned. Sequencing of six independent clones revealed that the transcription start site was at nt 36 752, 92 bp upstream from the previously predicted initiator methionine (Rawlinson et al., 1996) (Fig. 3a). Sequencing of a seventh clone revealed that the transcription start site could, with low frequency, also be at nt 36 754, 94 bp upstream from the previously predicted initiator methionine. Both transcription start sites were preceded by an appropriate TATA sequence at nt 36 784 (Fig. 3a).
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Analysis of the MCMV genomic sequence downstream of the m29.1 ORF suggests that the next ORF, designated M28, may be contained within the m29.1 mRNA, since the first AAUAAA polyadenylation signal is located 3' to the M28 ORF (Fig. 3a). Comparison of the sequences of eight independent clones revealed that only one cDNA clone lacked a 123 bp genomic sequence located between the M28 and m29.1 ORFs. Inspection of this sequence revealed splice donor and acceptor sites flanking the 123 nt intron (Fig. 3b). Thus, RACE analysis suggests that the m29.1 ORF produced a 2.4 kb transcript and a low abundance spliced transcript from which a 123 bp intron had been removed.
Production of m29 and m29.1 mutants and revertant
Because of the close proximity and overlapping nature of the ORFs in this region, premature stop codons were introduced using BAC mutagenesis into the m29 and m29.1 ORFs to prevent translation of the particular ORF and produce the Rc29MCMV BAC and the Rc29.1MCMV BAC, respectively, as described in Methods. PCR screening confirmed the correct insertion and orientation of the inserted ORF and sequencing confirmed the presence of the stop codon mutation (data not shown).
To rescue the Rc29.1 mutant to wt, a two-step recE/recT recombination was performed as described above. Firstly, the rpsL-neo cassette was inserted into the mutant Rc29.1MCMV BAC DNA and secondly, the rpsL-neo cassette was replaced by the linear wt DNA sequence to produce the revertant Rv29.1MCMV BAC.
Rc29, Rc29.1 and Rv29.1 viruses were derived following transfection of the respective MCMV BAC plasmid DNA into NIH 3T3 cells and selection of isolated plaques. Viruses containing BACs were then passaged, selecting individual plaques each time, to produce viruses in which the BACs had been excised as confirmed by PCR and sequencing.
m29 and m29.1 gene expression in mutant virus-infected cells
m29 and m29.1 transcripts were expressed by each of the three viruses, Rc29, Rc29.1 and Rv29.1, in NIH 3T3-infected cells (Fig. 4a). Western blot analysis using an anti-m29.1 polyclonal antibody raised in rabbits to bacterial expressed protein detected a protein of approximately 28–30 kDa in wt and Rc29 virus-infected cells, whereas no protein was detected in Rc29.1 virus- or mock-infected cells (Fig. 4b). Unfortunately, no antibody was available to detect the m29 protein as attempts to express this gene in bacterial cells were unsuccessful. Furthermore, immunization of rabbits with a potentially antigenic synthetic peptide (CDRDTPHEQRSGVSG) failed to generate an antibody that recognized m29 protein in Western blots.
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) and has been shown to be expressed in wt virus-infected NIH 3T3 cells (Fig. 2c). The above RACE results suggested that the M28 ORF may be contained within the m29.1 mRNA. To confirm that construction of mutant viruses has not affected ORFs downstream and upstream of the modification, total RNA from Rc29.1- and mock-infected NIH 3T3 cells 24 h p.i. was analysed by RT-PCR. Amplification of a 559 and 121 bp (Fig. 4c) product confirmed the expression of M28 and m29.2 genes, respectively, in Rc29.1-infected cells.
Replication of mutant viruses in tissue culture cells
At an m.o.i. of 5.0, no significant differences were found in growth rates between Rc29 and wt virus (Fig. 5a). At day 2 p.i., viral yields were significantly decreased compared with wt virus (P<0.05) but not at any other time point (Fig. 5a). Similarly, when inoculated at an m.o.i. of 0.05, Rc29 and wt virus produced similar yields at 1 and 2 days p.i., but from 3 to 7 days p.i., Rc29 virus produced a significantly (P<0.05) lower yield than wt virus although these differences were small (1 log10 p.f.u. ml–1) (Fig. 5a).
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). These differences were smaller at later time points but at all times Rc29.1 virus yields were significantly (P<0.05) lower than those of wt and revertant viruses. In contrast, virus yields of wt and revertant Rv29.1 viruses were not significantly different at any time point (Fig. 5b). Similar, but smaller differences were evident at the low m.o.i. (0.05) (Fig. 5b). Interestingly, Rc29.1 virus produced smaller plaques (data not shown), suggesting that this virus had a defect in release or produced fewer particles per cell.
Replication of mutant viruses in immunocompetent animals
In Rc29, Rc29.1, Rv29.1 and wt virus-infected BALB/c mice, inoculated intraperitoneally with 104 p.f.u. of virus, no virus was detected in lungs, spleen, heart, liver or kidneys at any time post-infection. In Rc29 virus-infected mice, virus was first detected 7 days p.i. in salivary glands and peaked at 21 days p.i. (Fig. 6a). No significant difference was found in virus titres between Rc29 and wt virus in this tissue except at days 7 and 10 p.i., when Rc29 virus titres were significantly higher than those of wt virus (P<0.05) (Fig. 6a). In contrast, in Rc29.1 virus-infected mice, virus was not detected until 10 days p.i. in salivary glands and yields peaked at 21 days p.i. (Fig. 6b). Yields were significantly (P<0.05) lower (1 log10 p.f.u. per tissue) than wt at all time points. Unlike wt virus, titres decreased significantly at 28 days p.i. and were undetectable by 35 days p.i. (Fig. 6b). Titres of revertant virus Rv29.1 were similar to those of wt virus (Fig. 6b). These results provide evidence to suggest that m29 and m29.1 ORFs are dispensable for viral replication in vivo but the m29.1 ORF is important for optimal viral growth.
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). In contrast, in Rc29.1 virus-infected mice, virus was not detected until day 10 (salivary gland, spleen, lung and kidney) or day 28 (liver and heart) p.i. and, in all organs, peak titres were again observed 31 days p.i. at the time when these animals became sick and were killed humanely (Fig. 7). Virus yields were significantly lower in salivary glands at day 10, in spleen at days 14 and 21 and in kidneys at day 21 p.i. (P<0.05) (Fig. 7) when compared with wt virus. In all organs, Rc29.1 virus showed delayed replication compared with wt virus although final titres at time of death were similar. Virus isolated from infected mice still contained the relevant mutation.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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) was identified in the m29 and m29.1 ORFs in this study. An extra G residue, at nt 36 198, was found and confirmed in several other strains of MCMV. Insertion of this G shortened the 3'-end of m29 from position 36 727 to 36 474 and extended that of m29.1 from position 36 112 to 36 029. A further C insertion at nt 37 263 was identified in our Smith strain MCMV BAC in the m30 ORF and again confirmed in the MCMV Smith strain (Birmingham) and natural wt isolates (N1, K17A and G4). Insertion of this C residue alters the ORF such that the stop codon in m30 no longer exists and the predicted protein continues into the M31 ORF. A number of sequence discrepancies from the published Smith MCMV sequence have been reported by several other groups. An incorrect G insertion in the m20 ORF at position 20 958 has been observed (Kattenhorn et al., 2004) and removal of this G extended the C terminus of m20 from nt 20 805 to 20 579 on the complementary DNA strand. Similarly, errors in the sequence for M45 (GenBank accession no. DQ978788) and m143 (GenBank accession no. DQ978788) have been reported (W. Brune, personal communication).
The m29 and m29.1 ORFs are overlapping and coded on opposite strands of the viral double-stranded DNA (Rawlinson et al., 1996). The newly predicted m28.2 ORF is encoded within m29, while another newly predicted ORF, m29.2, overlaps m30 and is encoded on the same complementary strand as the m29.1 ORF just upstream of it. Thus, this region is quite complex and to generate mutants in this region care was needed not to affect neighbouring genes. In this study, point mutations that introduced premature stop codons into the m29 and m29.1 ORFs near to 5'-end of each ORF were constructed using BAC mutagenesis.
The Rc29 virus appears to have no phenotype in that replication in cell culture and immunocompetent mice was essentially similar to wt virus. In contrast, replication of Rc29.1 virus was significantly reduced in vitro and in immunocompetent mice. Furthermore, the first appearance of virus in BALB/c mice was delayed and virus was eliminated earlier than with wt or revertant virus. In SCID mice, onset of infection was also delayed and consequently sickness occurred later (31 days p.i.) than with wt virus (23 days p.i.) although virus yields at the time of death were similar. These results suggested that the in vivo attenuation was due to initial poorer replication.
Transcription from the m29 and m29.1 region was first detected at 3 and 2 h p.i., respectively, and expression was detected at all time points up to 24 h p.i. These observations were consistent with those of others (Tang et al., 2006) who showed expression of the m29 and m29.1 ORFs 24 h p.i. using microarray assays. While m29 was found to belong to the early class of gene, m29.1 expression occurred in the presence of cycloheximide and thus was classed as a member of the IE gene family, suggesting that it might be a transcriptional regulator. Other MCMV IE ORFs produce spliced products and to examine whether this was the case for m29.1, RACE analysis of mRNA extracted from wt virus-infected cells was performed. 5'-RACE revealed two transcription start sites at nt 36 752 and 36 754, 92 and 94 bp upstream from the previously predicted initiator methionine (Rawlinson et al., 1996) and an appropriate TATA sequence at position 36 784. 3'-RACE analysis identified an AAUAAA polyadenylation signal at nt 34 359, followed by a poly(A) tag at nt 34 342. Therefore, the transcript from the m29.1 ORF was about 2.4 kb in length. However, two other interesting observations were made from this analysis. Firstly, the MCMV genomic sequence revealed that ORF M28 may be contained within the m29.1 mRNA because the first AAUAAA polyadenylation signal is located 3' to the M28 ORF. Secondly, a low abundance spliced transcript (one of eight clones) was found to be expressed from the m29.1 ORF in which a 123 bp intron had been removed.
Several possibilities may explain how the m29.1 and M28 ORFs are expressed from one mRNA. Leaky scanning usually occurs when the ribosome binds at the next AUG codon and is unlikely to operate in this case. Suppression of termination occurs relatively frequently with viruses, e.g. the alphavirus nsP4 (Li & Rice, 1989) and retrovirus gag-Pol genes (Wills et al., 1994), but read-through of the m29.1 stop codon would produce an m29.1–M28 fusion protein. Western blot analysis showed only a single protein of the size expected for the m29.1 product. Ribosomal frameshift is unlikely for the same reason. Reinitiation is a possibility as this has been observed for the HCMV gpUL4 (gp48) gene (Cao & Geballe, 1996). However, in the latter case the upstream ORF (uORF2) was short, only 22 codons in length, and repressed expression from the downstream ORF as ribosomes stalled on the uORF2 termination codon (Cao & Geballe, 1996). Clearly, m29.1 is an independent ORF and this mechanism is unlikely to explain how the M28 protein is produced. The most likely explanation is that an internal ribosome entry site (IRES) may be located in between the m29.1 and M28 genes producing a bicistronic mRNA. All eukaryotic mRNAs possess a cap structure at the 5'-end that plays a central role in promoting ribosome binding to the mRNA and to control the rate of translation initiation (Gingras et al., 1999; Shatkin, 1976). Ribosomes may also access eukaryotic mRNA by binding to an IRES as first discovered in picornavirus RNA (Jang et al., 1988; Pelletier & Sonenberg, 1988) and the presence of IRESs has also been reported in herpesvirus mRNA (Bieleski & Talbot, 2001). Translation initiation of the M28 protein could be directed by the IRES segment, which is predicted to be located upstream of the M28 initiation codon. IRES sequences are not conserved. Thus, it was not possible to identify an IRES sequence by a DNA database search. A bicistronic luciferase reporter system could be utilized to investigate whether the M28 protein is expressed from the 2.4 kb bicistronic message, utilizing an IRES sequence as described for the Kaposi's sarcoma-associated herpes virus (Bieleski & Talbot, 2001). Interestingly, if the IRES was incorporated into the intron this might be a mechanism of controlling expression of M28 as removal of the intron would prevent expression of M28. It is possible that at IE times post-infection the frequency of spliced transcripts might increase so that m29.1, but not M28, would be expressed, whereas both would be expressed at later time points.
Thus, in conclusion, the functions of the m29 and m29.1 ORFs are unknown. Neither the transcripts nor the protein products coded by these ORFs had been reported previously. This study indicates that m29 belongs to the early class gene family and is dispensable for virus replication both in tissue culture and in animals. However, the m29 ORF product appears to have a minor effect on virus release during replication. In contrast, the m29.1 ORF is an IE gene and could possibly be a transcription factor. However, apart from the kinetic experiments, no further evidence has been shown to support this assumption. A transcript of about 2.4 kb is expressed from the m29.1 ORF, which produces a protein product of about 28–30 kDa. Interestingly, m29.1 produces a read-through bicistronic message, which includes an intron and encompasses the M28 ORF; this product is rarely spliced. The m29.1 ORF is also dispensable for virus replication in both tissue culture and animals in vivo, but the virus grew to significantly lower yields and showed delayed replication due to the lack of the m29.1 protein, which suggests that the m29.1 ORF is required for optimum viral growth.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Bieleski, L. & Talbot, S. J. (2001). Kaposi's sarcoma-associated herpesvirus cyclin open reading frame contains an internal ribosome entry site. J Virol 75, 1864–1869.
Brocchieri, L., Kledal, T. N., Karlin, S. & Mocarski, E. S. (2005). Predicting coding potential from genome sequence: application to betaherpesviruses infecting rats and mice. J Virol 79, 7570–7596.
Cao, J. & Geballe, A. (1996). Coding sequence-dependent ribosomal arrest at termination of translation. Mol Cell Biol 16, 603–608.[Abstract]
Chee, M. S., Bankier, A. T., Beck, S., Bohni, R., Brown, C. M., Cerny, R., Horsnell, T., Hutchison, C. A., Kouzarides, T. & other authors (1990). Analysis of the protein-coding content of the sequence of human cytomegalovirus strain AD169. Curr Top Microbiol Immunol 154, 125–169.[Medline]
Davison, A. J., Dolan, A., Akter, P., Addison, C., Dargan, D. J., Alcendor, D. J., McGeoch, D. J. & Hayward, G. S. (2003). The human cytomegalovirus genome revisited: comparison with the chimpanzee cytomegalovirus genome. J Gen Virol 84, 17–28.
Dolan, A., Cunningham, C., Hector, R. D., Hassan-Walker, A. F., Lee, L., Addison, C., Dargan, D. J., McGeoch, D. J., Gatherer, D. & other authors (2004). Genetic content of wild-type human cytomegalovirus. J Gen Virol 85, 1301–1312.
Dunn, W., Chou, C., Li, H., Hai, R., Patterson, D., Stolc, V., Zhu, H. & Liu, F. (2003). Functional profiling of a human cytomegalovirus genome. Proc Natl Acad Sci U S A 100, 14223–14228.
Gingras, A. C., Raught, B. & Sonenberg, N. (1999). eIF4 initiation factors: effectors of mRNA recruitment to ribosomes and regulators of translation. Annu Rev Biochem 68, 913–963.[CrossRef][Medline]
Ho, M. (1991). Cytomegalovirus: Biology and Infection, 2nd edn. New York: Plenum.
Holtappels, R., Munks, M. W., Podlech, J. & Reddehase, M. J. (2006). CD8 T-cell-based immunotherapy of cytomegalovirus disease in the mouse model of the immunocompromised bone marrow transplant recipient. In Cytomegalovirus Molecular Biology and Immunology, pp. 383–418. Edited by M. J. Reddehase. Norfolk, UK: Caister Academic Press.
Jang, S. K., Krausslich, H. G., Nicklin, M. J., Duke, G. M., Palmenberg, A. C. & Wimmer, E. (1988). A segment of the 5' nontranslated region of encephalomyocarditis virus RNA directs internal entry of ribosomes during in vitro translation. J Virol 62, 2636–2643.
Jonjic, S., Bubic, I. & Krmpotic, A. (2006). Innate immunity to cytomegaloviruses. In Cytomegalovirus Molecular Biology and Immunology, pp. 285–319. Edited by M. J. Reddehase. Norfolk, UK: Caister Academic Press.
Kattenhorn, L. M., Mills, R., Wagner, M., Lomsadze, A., Makeev, V., Borodovsky, M., Ploegh, H. L. & Kessler, B. M. (2004). Identification of proteins associated with murine cytomegalovirus virions. J Virol 78, 11187–11197.
Li, G. P. & Rice, C. M. (1989). Mutagenesis of the in-frame opal termination codon preceding nsP4 of Sindbis virus: studies of translational readthrough and its effect on virus replication. J Virol 63, 1326–1337.
Lyons, P. A., Allan, J. E., Carrello, C., Shellam, G. R. & Scalzo, A. A. (1996). Effect of natural sequence variation at the H-2Ld-restricted CD8+ T cell epitope of the murine cytomegalovirus ie1-encoded pp89 on T cell recognition. J Gen Virol 77, 2615–2623.
Murphy, E., Rigoutsos, I., Shibuya, T. & Shenk, T. E. (2003). Reevaluation of human cytomegalovirus coding potential. Proc Natl Acad Sci U S A 100, 13585–13590.
Pelletier, J. & Sonenberg, N. (1988). Internal initiation of translation of eukaryotic mRNA directed by a sequence derived from poliovirus RNA. Nature 334, 320–325.[CrossRef][Medline]
Rawlinson, W. D., Farrell, H. E. & Barrell, B. G. (1996). Analysis of the complete DNA sequence of murine cytomegalovirus. J Virol 70, 8833–8849.[Abstract]
Sammons, C. C. & Sweet, C. (1989). Isolation and preliminary characterization of temperature-sensitive mutants of mouse cytomegalovirus of differing virulence for 1-week-old mice. J Gen Virol 70, 2373–2381.
Shatkin, A. J. (1976). Capping of eucaryotic mRNAs. Cell 9, 645–653.[CrossRef][Medline]
Sweet, C. (1999). The pathogenicity of cytomegalovirus. FEMS Microbiol Rev 23, 457–482.[CrossRef][Medline]
Sweet, C., Ball, K., Morley, P. J., Guilfoyle, K. & Kirby, M. (2007). Mutations in the temperature-sensitive murine cytomegalovirus (MCMV) mutants tsm5 and tsm30: a study of genes involved in immune evasion, DNA packaging and processing, and DNA replication. J Med Virol 79, 285–299.[CrossRef][Medline]
Tang, Q., Murphy, E. A. & Maul, G. G. (2006). Experimental confirmation of global murine cytomegalovirus open reading frames by transcriptional detection and partial characterization of newly described gene products. J Virol 80, 6873–6882.
Wagner, M., Koszinowski, U. H. & Messerle, M. (1999). Systematic excision of vector sequences from the BAC-cloned herpesvirus genome during virus reconstitution. J Virol 73, 7056–7060.
Wills, N. M., Gesteland, R. F. & Atkins, J. F. (1994). Pseudoknot-dependent read-through of retroviral gag termination codons: importance of sequences in the spacer and loop 2. EMBO J 13, 4137–4144.[Medline]
Yu, D., Ellis, H. M., Lee, E. C., Jenkins, N. A., Copeland, N. G. & Court, D. L. (2000). An efficient recombination system for chromosome engineering in Escherichia coli. Proc Natl Acad Sci U S A 97, 5978–5983.
Yu, D., Silva, M. C. & Shenk, T. (2003). Functional map of human cytomegalovirus AD169 defined by global mutational analysis. Proc Natl Acad Sci U S A 100, 12396–12401.
Received 2 May 2007;
accepted 12 July 2007.
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, a and b), Rc29 (
, a), Rc29.1 (
, b) or Rv29.1 (
, b) viruses at an m.o.i. of either 5.0 (upper panels) or 0.05 p.f.u. (lower panels) per cell. At 1, 2, 3, 4, 5, 6 and 7 days p.i., culture media were collected. Values represent the mean of three replicates±SD.
