- Open Access
Molecular investigation of the 7.2 kb RNA of murine cytomegalovirus
© Schwarz et al.; licensee BioMed Central Ltd. 2013
- Received: 8 October 2013
- Accepted: 22 November 2013
- Published: 2 December 2013
HCMV encodes a stable 5 kb RNA of unknown function that is conserved across cytomegalovirus species. In vivo studies of the MCMV orthologue, a 7.2 kb RNA, demonstrated that viruses that do not express the RNA fail to establish efficient persistent replication in the salivary glands of mice. To gain further insight into the function and properties of this conserved locus, we characterized the MCMV intron in finer detail.
We performed multiple analyses to evaluate transcript expression kinetics, identify transcript termini and promoter elements. The half-lives of intron locus RNAs were quantified by measuring RNA levels following actinomycin D treatment in a qRT-PCR-based assay. We also constructed a series of recombinant viruses to evaluate protein coding potential in the locus and test the role of putative promoter elements. These recombinant viruses were tested in both in vitro and in vivo assays.
We show that the 7.2 kb RNA is expressed with late kinetics during productive infection of mouse fibroblasts. The termini of the precursor RNA that is processed to produce the intron were identified and we demonstrate that the m106 open reading frame, which resides on the spliced mRNA derived from precursor processing, can be translated during infection. Mapping the 5′ end of the primary transcript revealed minimal promoter elements located upstream that contribute to transcript expression. Analysis of recombinant viruses with deletions in the putative promoter elements, however, revealed these elements exert only minor effects on intron expression and viral persistence in vivo. Low transcriptional output by the putative promoter element(s) is compensated by the long half-life of the 7.2 kb RNA of approximately 28.8 hours. Detailed analysis of viral spread prior to the establishment of persistence also showed that the intron is not likely required for efficient spread to the salivary gland, but rather enhances persistent replication in this tissue site.
This data provides a comprehensive transcriptional analysis of the MCMV 7.2 kb intron locus. Our studies indicate that the 7.2 kb RNA is an extremely long-lived RNA, a feature which is likely to be important in its role promoting viral persistence in the salivary gland.
- Non-coding RNA
Human cytomegalovirus (HCMV) is a member of the β-herpesvirus subfamily and a widespread human pathogen[1, 2]. HCMV infections cause life-threatening illness in the immunocompromised, including bone marrow and solid organ transplant recipients, AIDS patients and patients undergoing cancer chemotherapy. In addition, HCMV infection of the fetus is the leading cause of virally induced birth defects, typically presenting as hearing loss and developmental deficits that impact over 40,000 newborns annually in the US[3–5].
HCMV infection of healthy, immunocompetent individuals elicits a robust host immune response that effectively limits virus replication and pathogenesis. Despite this immune response, HCMV has adapted to long-term infection of humans by balancing persistent replication with clearance by the immune response. HCMV persistently replicates in glandular epithelial tissue and eventually establishes a life-long latent infection of the host. Glandular epithelial cells, such as those in the salivary gland, are key sites of HCMV persistent replication in vivo, and contribute significantly to viral transmission. Healthy individuals may secrete virus in saliva, breast milk, and urine for long periods of time following primary infection[2, 3]. In order for HCMV to successfully persist, it has evolved to replicate in cell types where the full replication cycle elicits little to no cytopathic effect, such as glandular epithelial cells and some types of endothelial cells[6–8]. In addition, the ability to persistently replicate in the host likely depends on reduced immune recognition of virus-infected cells at these specialized sites[9, 10]. Few viral determinants that mediate cytomegalovirus persistence have been identified and little is known about the specific molecular functions that facilitate persistence. These include virus-encoded micro-RNAs and a conserved, virus-encoded G-protein-coupled receptor[11–13]. In addition, we previously identified a long, non-coding RNA (lncRNA), expressed by all cytomegaloviruses, that we showed to also be an important viral determinant of persistence.
During lytic replication, HCMV expresses a 5 kb lncRNA of unknown function (also referred to as RNA5.0)[14–16]. We showed that this RNA is dispensable for replication in cultured cells and is a stable intron produced by the processing of a large precursor transcript expressed from the genomic region flanked by UL105 and UL111A. Orthologous loci are present in every β-herpesvirus genome examined thusfar, although there is little conservation of sequence or RNA size between different CMV species. Each locus shares some common features, including a high AT sequence content (~60%), and the presence of many homopolymeric stretches of A or T residues. The consensus splice donor sequence that defines the 5′ end of the RNA produced from each locus is also well conserved.
Cytomegaloviruses exhibit strict species specificity and there is no animal model for HCMV infection. Murine cytomegalovirus (MCMV) infection of the mouse is widely used as an outstanding small-animal model of HCMV infection for several reasons. HCMV and MCMV share similar genomic sequence and organization and undergo similar replication cycles. Like HCMV, MCMV acutely infects multiple tissues in the mouse, persistently replicates in the salivary gland and establishes a lifelong latent infection of the host. Therefore, MCMV infection of the mouse is an excellent surrogate for the study of pathogenesis in vivo. MCMV expresses a 7.2 kb ortholog of the HCMV 5 kb RNA. We have shown that recombinant MCMV that does not express the 7.2 kb RNA replicates normally in cultured fibroblasts, but is unable to progress from the acute to the persistent phase of infection in mice. We identified a short hairpin sequence near the 3′ end of the intron that is required for accumulation of the RNA during infection. Persistent replication in the salivary gland of the mouse depends on the accurate processing and stable retention of the intron, since recombinants with a mutation in the splice donor site or deletion of the hairpin sequence fail to transition to the persistent phase of replication in vivo. The specific molecular function of this conserved RNA is unknown, but we hypothesize that it mediates processes essential for the virus to evade immune surveillance and/or replicate efficiently at sites of viral persistence.
To gain further insight into the function and properties of this conserved locus, we characterized the MCMV intron in finer detail. We confirmed that the intron locus RNAs are expressed with late gene kinetics. The 7.2 kb intron has an unusually long half-life, whereas the spliced mRNA that results from processing of the intron is metabolized with kinetics similar to most cellular mRNAs. Investigation of the promoter sequence that controls expression of the intron locus revealed a minimal promoter sequence contributing to the transcriptional output of the locus. Although there is no evidence for translation of the intron itself, we discovered that the spliced mRNA encodes a small protein that co-localizes with the RNA within the nuclei of infected cells. Importantly, we show that the RNA is not required for trafficking of virus to the salivary gland in vivo, supporting our hypothesis that the 7.2 kb RNA functions to either evade the host response or maintain viral replication at sites of persistence.
The MCMV 7.2 kb intron locus is transcribed with true late kinetics
In high resolution northern blot analyses specific for the intron RNA, we routinely observed a doublet of bands near 7.2 kb: a major species at approximately 8.0 kb and a minor species migrating faster at 7.2 kb (Figure 1A). These observations were made with multiple intron-specific probes (data not shown). We have been unable to ascertain the basis for this difference in size, although we hypothesize it may be due to effects of lariat secondary structure on RNA migration during electrophoresis resulting in slower migration (data not shown). Likewise, we also observed a doublet of closely migrating bands in northern blot analyses of the spliced RNA product of the locus (Figure 1B). We cannot account for the difference in size based on sequencing of the 5′ and 3′ Rapid Amplification of cDNA Ends (RACE) products (see below). It is possible that we did not capture both species in the RACE reactions but we think it is likely the differences in size reflect variability in 3′ end processing and poly-adenylation that we cannot assess (see Discussion).
Location of transcriptional start sites and RNA processing signals
Sequence 5′ to 3′
7.2 kb Intron (a and a’)
Exon2 (b and b’)
Spliced mRNA (c and c’)
7.2 kb Intron
A single 3′ end was identified at nucleotide position 153,872 by sequencing of 3′-RACE products (Figure 2A). This end is located downstream of a putative polyadenylation signal at position 153,898 (Figure 2B). We also examined the polyadenylation status of the spliced RNA by northern blot analysis of oligo(dT)-selected RNA prepared from MCMV-infected cells. The majority of the spliced mRNA from the intron locus was detected in the poly A + fraction of RNA (Figure 2D). 18S rRNA can only be detected in the non-polyadenylated fraction demonstrating that our fractionation protocol efficiently captured polyadenylated mRNA only (Figure 2D lanes A+ and A-). Taken together, our data suggest that a large precursor RNA is transcribed from the intron locus at late times of infection and processed to yield a single 7.2 kb stable intron and a spliced poly-adenylated mRNA consisting of two exons.
The m106 open reading frame on the spliced mRNA is translated during infection
m106 and the 7.2 kb intron localize to the nucleus of infected fibroblasts
Although we previously demonstrated in fractionation studies that the HCMV 5 kb intron localizes to the nuclear compartment of infected cells, the specific sub-nuclear localization of the RNA was not examined. Fluorescent in situ hybridization (FISH) was used to visualize the 7.2 kb intron in infected mouse fibroblasts. The FISH staining revealed an even, granular distribution of the 7.2 kb intron throughout the nuclear compartment of infected fibroblasts (Figure 3D). Co-staining for the 7.2 kb RNA and m106-GFP revealed that the RNA and m106 protein are found co-localizing in the nucleus late during infection (Figure 3D). We also observed some m106-GFP protein was localized to the cytoplasm of infected cells.
The MCMV 7.2 kb intron is highly stable
Analysis of minimal promoter sequences
To determine if reductions in intron and mRNA expression have an effect on the establishment of persistence in vivo, mice were inoculated with a subset of our panel of recombinant viruses and viral yields measured in the salivary gland at 14 days post-infection (Figure 6F). We observed a slight reduction in viral yield in the salivary glands of mice infected with MCMVdel 20 and a ten-fold reduction of viral yield in mice infected with MCMVdel 135. Viral genome quantification corroborated the measure of infectious virus within the salivary gland (Figure 6G). However, despite 5–10 fold reductions of intron and mRNA production, neither promoter deletion mutant fully attenuated persistent replication to the levels observed in mice infected with MCMVdel SD.
Intron locus products do not influence dissemination to the salivary gland
Non-coding RNAs are known to be expressed by nearly all herpesviruses that infect humans, yet the function of these RNAs in viral replication and pathogenesis has been elusive[15, 31–34]. In our current study, we report further characterization of a lncRNA expressed by MCMV, the 7.2 kb RNA. This RNA is the ortholog of the 5.0 kb RNA of HCMV and we have previously shown that it is a virulence factor that promotes viral persistence in vivo. We showed that during productive infection in fibroblasts, the intron locus RNAs are transcribed with true late gene kinetics. These RNAs are derived from a common precursor RNA and are produced as the result of splicing of two exons that flank the intron to create a smaller mRNA and the 7.2 kb intron. We observed a doublet of bands with probes specific for the intron in northern blot analysis at 8.0 and 7.2 kb. While it is formally possible that these represent two different species of intron, we did not detect evidence of alternative splicing of a larger intron from the precursor RNA by sequencing of 5′-RACE products. We think it is likely that the intron remains in the form of a branched lariat after processing and therefore migrates more slowly during electrophoresis. Similar observations have been made for the LAT of HSV-1. We also detected a doublet of bands corresponding to the spliced mRNA in northern blot analysis. Again, sequencing of 5′-RACE products did not reveal any splicing variations that could account for the size differences. The polyadenylation chain lengths could differ for the individual spliced mRNA molecules representing the doublet observed for the mRNA. Identification of the transcriptional start sites rules out the possibility that a cluster of miRNAs mapped near to the 7.2 kb intron splice donor site originate from the same primary transcript. It remains unknown, however, what functional relationship the miRNAs may have with the MCMV 7.2 kb intron locus, if any, during virus replication and pathogenesis.
Identification of the transcriptional start sites of the primary transcript expressed from the intron locus led us to identify putative transcriptional control elements that contribute to regulating transcription of the intron precursor. We identified two TATA box elements located within 135 bp of the TSS. However, using a luciferase reporter system, this region did not confer significant transcriptional activity above background. Transcriptional control elements driving expression of the adjacent M112/113 locus have been characterized and functioned in our assay as expected. We were able to rule out the possibility that the M112/113 promoter controls transcription of the intron locus since function of this promoter is orientation dependent. Intron RNA and mRNA expression levels in cells infected with recombinant virus bearing a deletion spanning the distal and proximal TATA boxes and transcriptional start site were reduced compared to wild-type MCMV. However, the reduction in transcript levels was not nearly as robust as that observed in recombinants bearing mutations that affect intron stability or processing. We hypothesize that most of the variations we observed in the differing effects of promoter mutations on relative intron and mRNA levels can be accounted for by their different half-lives: since the intron is unusually stable, it appears to be less affected by the promoter deletions whereas the mRNA has a shorter half-life and the relative levels of RNA are more sensitive to modest reductions in transcriptional output associated with the promoter deletions.
Our studies did not identify sequence elements that robustly contribute to transcriptional control of this late transcriptional unit. As a late gene, amplification of genome copy number by DNA replication is thought to be critical for robust L transcription. More recently, it has been demonstrated that viral replication and L gene expression also relies on a distinct set of five genes conserved across beta and gamma herpesviruses: UL79, UL87, UL91, UL92, and UL95[37–39]. It is hypothesized that an RNA polymerase II transcriptional complex including one or more of these gene products is assembled to drive transcription of L genes. MCMV homologs of HCMV UL87, UL91, UL92, and UL95 have been annotated, but not tested for transcriptional activating functions[38–40]. M79, the MCMV homolog of HCMV UL79, has been shown to regulate L gene expression, although it does not appear to promote transcription of the intron locus. It is also possible that we did not include the sequences responsible for binding of L gene transactivators within our reporter assay. Clearly, transcriptional regulation of L genes remains largely unexplored and it is likely that this process is significantly different from the activation of immediate early and early transcriptional units.
Post-transcriptional regulation, metabolism and function of lncRNAs is poorly understood in general. We demonstrated that the 7.2 kb intron accumulates as a consequence of a slow decay rate. Different properties contribute to lncRNA stability include GC content, the presence of specific decay elements, and if they are intronic. Given its intronic and AT-rich nature, the MCMV 7.2 kb lncRNA would be predicted to be highly unstable since introns are rapidly degraded on formation and AT-rich sequences do not form strong secondary structures that might protect the RNA from degradation. A stem-loop structure located near the 3′ end of the intron between the polypyrimidine track and putative branch point was identified using the structural prediction software mFold. Deletion of the stem loop does not impact processing of the precursor transcript, but does prevent accumulation of the intron during infection. We hypothesize that the intron remains in the form of a lariat, similar to the Latency Associated Transcript (LAT) of HSV-1, thereby protecting it from degradation[42, 43]. While the sequence and structural determinants of stability remain largely unknown for the 7.2 kb intron, its long half-life accounts for its accumulation and could be a key component of its functionality.
The spliced mRNA produced by processing of the 7.2 kb RNA spans the m106 ORF. Using an epitope-tagging strategy, we showed that this ORF could be translated during MCMV infection. The GFP-tagged protein co-localized to the nucleus of infected cells with the 7.2 kb RNA. This may reflect a related function of the intron and the m106 protein. Recombinant viruses that specifically disrupt m106 expression without impacting intron production will be useful reagents to probe the function for this viral protein. The m106 protein and its orthologues encoded by other CMVs, including UL106 of HCMV, have some unusual properties. Despite not sharing significant sequence homology, all UL106 orthologues are small (<150 amino acids), highly basic, arginine-rich peptides. It is unknown if other UL106 orthologues are expressed during infection, but given the conservation of the genomic organization of the intron locus among CMVs, it is a distinct possibility to be explored.
Production of the 7.2 kb RNA is required for the establishment of persistence in the salivary glands of mice. By analysis of multiple time points between the acute and persistent phases of infection in mice, we showed that recombinant virus lacking the intron appears to disseminate to the salivary gland as efficiently as wild-type MCMV. However, it is unable to maintain a highly productive replication program in the salivary glands as observed at 14 days post infection. In addition, we did not detect infectious MCMVdel SD in any organs at 14 dpi and genome copy number of the mutant virus was substantially reduced in liver and kidney. It is possible that the mechanisms that prevent establishment of intron-mutant virus persistence in the salivary gland may also promote accelerated clearance of that virus from liver and kidney. At this time, the adaptive immune response acts to limit viral replication and it is possible that the 7.2 kb intron is involved in modulating immune surveillance in some way. Some cellular lncRNAs are involved in transcriptional regulatory processes, therefore, a possible mechanism for evading the immune response could be to regulate cellular or viral genes that are involved in this host pathogen relationship[44, 45].
This current study has provided a detailed transcriptional and functional analysis of the MCMV 7.2 kb RNA locus. Mapping the topography of the 7.2 kb RNA locus allows us to understand the genomic elements that not only comprise the locus but also control its expression. The unusual stability of the 7.2 kb RNA compensates for the weak transcriptional output by the putative promoter region observed and may also provide insight towards molecular function within the nucleus of infected cells. Uncovering the stability determinants of the 7.2 kb RNA will therefore allow us to understand the mechanisms that promote its retention within infected cells. Although a function has yet to be determined to the 7.2 kb RNA, this current analysis has provided the framework for investigating its function during viral persistence.
10.1-mouse embryonic fibroblasts were propagated in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% newborn calf serum, 100 U/ml penicillin, and 100 ug/ml streptomycin. Cells were maintained at 37°C with 5% CO2.
Primers used for recombinant virus production
Sequence 5′ to 3′
RACE products were cloned into pGEM-T-Easy (Promega) and sequenced. Reporter constructs were generated by PCR amplifying sequence upstream of the 7.2 kb RNA splice donor site. Primers used to generate the reporter constructs are indicated in Table 1. Amplicons were resolved by gel electrophoresis and gel purified using the Qiaquick Gel Extraction Kit (Qiagen). Amplicons were cloned into pGEM-T-Easy. After insertion into the pGEM-T-Easy plasmid, the inserts were digested from the plasmid using the flanking EcoRI sequences then subcloned into the pGL3 Basic vector at a newly generated EcoRI site using the site directed mutatgenesis kit (Stratagene). Orientation of the cloned insert was determined by sequencing the pGL3 plasmid. The pGL3-SV40 control plasmid and the renilla phRL-TK normalization control plasmid were used in the luciferase assays (Promega).
Mouse fibroblasts were seeded in 24-well dishes and co-transfected with a pGL3 construct (see above) and phRL-TK using polyethylenimine (PEI) at a 6:1 ratio of PEI to plasmid DNA. The plasmids were cotransfected at a 1:1 ratio with 3 × 1010 copies per well of each plasmid. Protein lysates were harvested 48 hours post transfection and assayed following the Duo-Glow Luciferase assay kit (Promega, Madison, WI). Luciferase activity was normalized to renilla activity in each well and the data is expressed as the fold change of luciferase induction relative to the luciferase induction from the pGL3 promoter-less vector.
To determine expression kinetics, 10.1 fibroblasts were pretreated with 100 μg/mL cyclohexamide or 200 μg/mL PAA 1 hour before MCMV infection. Total RNA was harvested from 10.1 fibroblasts at either 24 hours post infection (h p.i.) or 48 h p.i. with TRIzol LS (Life Technologies) according to the manufacturer’s protocol. RNA was resolved on either a 1.4% or 0.7% glyoxal gel for detection of the spliced mRNA or 7.2 kb RNA respectively. Northern blot analysis for intron locus RNAs was carried out as previously described using specific radio-labeled riboprobes.
RNA half-life analysis was performed by infecting fibroblasts with wild-type MCMV at an MOI of 1.0. At 30 hours post infection, 4 ug/mL Actinomycin D was added to infected cells and RNA was harvested over a time course starting at time 0 and ending at 32 hours post treatment. Transcript levels were quantified by qRT-PCR at the different times points relative to RNA at time 0.
5′ and 3′ RACE
Total RNA harvested from mock or WT MCMV infected 10.1 mouse fibroblasts at 48 hours post infection was analyzed by the First Choice RNA ligase-mediated rapid amplification of cDNA ends kit as recommended by the manufacturer (RLM-RACE, Ambion). Amplification products were purified and TA cloned into pGEM-T-Easy (Promega, Madison, WI) and sequenced (Table 1). For 3′ RACE, RNA was reverse transcribed using a poly(A)-adapter. Amplification products were cloned and sequenced. MacVector software was used to align RACE sequences to MCMV reference sequence.
Oligonucleotides 497 and 50 were end radiolabeled and used in primer extension reactions on total RNA from mock- or MCMV-infected cells as previously described (Table 1). Primer extension products were analyzed by denaturing 10% urea-polyacrylamide gel electrophoresis followed by phosphorimager analysis.
qRT-PCR and qPCR
Total RNA was DNase treated and reverse transcribed using the Quantitect Reverse Transcription kit (Qiagen). Quantitative PCR was performed using the LightCycler 480 Probes Master Mix (Roche) along with IDT hydrolysis probes specific for the intron locus RNAs and selected housekeeping genes (Table 1). Ct values were determined using the Basic Relative Quantification analysis module of the LightCycler 480 (Roche) software. Primer-probe efficiencies were determined by three biological replicates of 10-fold dilutions. The 18S rRNA was used as a reference gene and the relative target levels were quantified by a delta-delta CT method, the Pfaffl method, that incorporates the calculated primer-probe efficiencies (Table 1).
Cells were trypsinized, centrifuged, and collected in PBS. Cells were lysed in RIPA buffer (150 mM NaCl, 1% v/v Nonidet P-40, 0.5% w/v deoxycholate, 0.1% w/v SDS, 5 mM EDTA, 50 mM Tris; pH 8.0) containing protease inhibitor cocktail (Roche). The cell lysate was briefly sonicated to facilitate nuclear protein release and insoluble debris was centrifuged. GFP tagged m106 protein was immunoblotted using a rabbit polyclonal antibody and detected with a fluorescently conjugated secondary antibody using the SuperSignal West Pico Chemiluminescent Substrate (Thermo Scientific). HP1, heterochromatin associated protein 1, was detected similarly as a loading control (Santa Cruz).
FISH and Immunofluorescence
Fluorescently labeled RNA probes antisense to the 7.2 kb intron were generated using the FISH Tag kit (Table 1) (Invitrogen). Briefly, probes were in vitro transcribed from linearized pGEM-T-Easy constructs using an amino allyl modified base in which an alexa flour can be chemically attached to. Following in vitro transcription of the probes, the DNA templates are digested using DNase I and the amino modified RNA is purified over a column then ethanol precipitated. The purified probes are fluorescently labeled according to the manufacturer’s instructions then column purified and subsequent ethanol precipitation. Cells were fixed for 20 minutes in 4% paraformaldehyde, 10% acetic acid in 1x PBS. The fixation was quenched for 20 minutes in PBS with 0.1 M glycine. Cells were washed twice with PBS then permeabilized with 70% ethanol overnight at 4°C. Cells were rehydrated by washing twice with 50% formamide/2x SSC. The probe was denatured by heating at 65°C for 10 minutes in probe buffer then cells were incubated overnight with the denatured probe at 37°C. The following day, cells were washed twice with 0.1X SSC/50% formamide at 50°C then washed once with PBST. Immunoflourescence of m106-GFP was carried out as previously described.
All animal procedures were approved by the Institutional Animal Care and Use Committee of the University of Colorado Denver. BALB/c mice were inoculated by intraperitoneal injection with 5×106 pfu of tissue culture derived wild type or recombinant MCMV in 300 ul DMEM. At designated times mice were sacrificed and liver, spleen, lungs, kidneys, and salivary glands were removed and weighed. Part of the tissue was homogenized and titrated on mouse fibroblasts. The remaining tissue was processed for DNA isolation in order to quantify viral genomes using the DNeasy Blood and Tissue kit according to the manufacturer’s protocol (Qiagen). 250 ng of DNA was analyzed by qPCR for each sample.
TMS, L-AMV, CGA, and CAK participated in the design and execution of the study. TMS and CAK drafted the manuscript. All authors read and approved the final manuscript.
We would like to thank Mario Santiago for the mice used in our in vivo analysis and Linda Van Dyk for helpful discussion.
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