In this study, we identified 27 small RNAs expressed from discrete foci scattered throughout the MCMV genome. There is strong evidence that these RNAs are bona fide miRNAs encoded by MCMV. First, all RNAs were cloned from pools of small RNAs isolated and fractionated from cells infected by MCMV (Table ). Second, the genomic regions directly surrounding all cloned small RNAs were found to fold into typical short hairpin structures by in silico analysis (Fig. ). Third, all mature miRNAs and, in most cases, their predicted pre-miRNAs were detected by Northern blotting exclusively in MCMV-infected cells (Fig. and ). Finally, for some miRNAs, both arms of the predicted pre-miRNAs were cloned. MCMV miRNAs accumulate to very high levels, increasing over time throughout fibroblast infection. At late infection time points, MCMV miRNAs become even more abundant than some of the most abundant host miRNAs, suggesting that they might well eventually outcompete cellular miRNAs. In adenovirus-infected cells, the virus-associated RNAs, which are expressed and accumulate at extremely high levels, saturate the export and processing machinery of cellular miRNAs (2
). Similarly, expression of viral miRNAs in lytic MCMV infection stages might perturb, at least to some extent, some of the fine-tuned regulation orchestrated by cellular miRNAs.
The mature form of a number of MCMV miRNAs was already detectable at 4 to 8 hpi, indicating that they are expressed with early or even immediate-early kinetics (Fig. ). Nonetheless, mature MCMV miRNAs were not detectable in cells treated with cycloheximide for 24 h, indicating that de novo synthesis of viral proteins is likely required for their production. However, for a number of miRNAs, specific signals with sizes very similar to those of the predicted pre-miRNAs were detectable by Northern blotting in cycloheximide-treated cells, which is consistent with immediate-early expression of these pre-miRNAs. A similar observation was made for HCMV miRNA miR-UL36-1 (16
), but not for any other HCMV miRNA. Both the HCMV UL36 transcript and the UL36 pre-miRNA are expressed with immediate-early kinetics. Similar to our observation, the mature miR-UL36-1 is not detectable in cycloheximide-treated cells, whereas the pre-miRNA accumulated to levels higher than those in nontreated cells. Thus, our results extend this initial observation to other viral miRNAs. Grey et al. (16
) suggested that prolonged cycloheximide treatment could deplete the cells of factors required for processing of mature miRNAs, such as Dicer. Our results are in favor of this hypothesis, as we noticed a reduction in the levels of some cellular mature miRNAs in cycloheximide-treated cells (data not shown). However, this cannot explain the accumulation of viral pre-miRNAs, as they also require processing by the cellular factor Drosha. This contradiction might be explained by the fact that the half-life of the Drosha-containing complex may be higher than that of the Dicer-containing complex. It will be of interest to confirm this hypothesis by analyzing Dicer and Drosha expression upon cycloheximide treatment.
Interestingly, not all miRNAs encoded by MCMV followed the same pattern of accumulation. Indeed, the accumulation of both arms of miR-M23-1 was inversely correlated to that of all other miRNAs. We observed that the cloning frequency of miR-M23-1-5p and -3p dropped significantly from 24 to 72 hpi and that both sequences seemed to undergo 3′ polyuridylation (Fig. ). Additionally, three other miRNAs, miR-m21-1, miR-m22-1, and miR-M23-2, showed the addition of one extra U at their 3′ end (Table ). The addition of nontemplated adenosine and uridine has been also reported for some cellular miRNAs, although to a more moderate extent (24
). In plants, miRNAs are known to be protected at their 3′ end by a methyl group added by the methyltransferase HEN1 (27
). HEN1 homologues have been identified in mouse and Drosophila
), but they act on Piwi-interacting RNAs, not miRNAs. In Arabidopsis hen1
mutants, miRNAs are not protected and undergo polyuridylation followed by degradation. Similarly, we hypothesize that the polyuridylation of miR-M23-1-5p and -3p might constitute a prerequisite for their subsequent degradation, which would readily explain the reduction in clone numbers in the libraries over time. At present, it is unclear whether this process reflects a deliberate viral strategy, for instance, to fine-tune the accumulation of some of its miRNAs, or whether it represents a host-directed defense response targeted at pathogenic small RNAs. Nonetheless, this finding might shed light on a novel mechanism whereby miRNAs accumulation might be controlled in animal cells.
About 40% of all clones from the MCMV miRNA library represented a single miRNA, miR-m01-4, which is derived from a pre-miRNA located in the 3′-UTR of the m01 transcript. Additionally, miR-m01-4 accumulates to detectable levels in vivo (Fig. ). To test whether this miRNA is dispensable for viral growth in fibroblasts, we created a knockout MCMV mutant for miR-m01-4. Despite the high abundance of miR-m01-4 in lytically infected fibroblasts, the deletion mutant grew as the wild-type virus did, even under multistep growth conditions. Previously, we created a deletion mutant involving the predicted coding regions of m01 to m16, thereby also deleting miR-m01-1, miR-m01-2, and miR-m01-3, but not miR-m01-4 (M. Popa and Z. Ruzsics, unpublished data). This virus also grew like wild-type virus on MEFs. Together with the results we obtained with the miR-m01-4 mutant, this indicates that, despite their abundance, all four miRNAs derived from the m01 transcript are dispensable for efficient MCMV replication in fibroblasts in vitro.
In a recent study, the m01 transcript was hardly detectable by microarray and reverse transcription-PCR analysis (48
), and a protein product corresponding to the m01 ORF has not been identified yet. We found four pre-miRNAs to be expressed from the m01 locus with three of them (miR-m01-2, -3, and -4) clustering within 450 bp, thereby significantly interfering with the detection of the m01 transcript by PCR. Using PCR primers located just 5′ of pre-miR-m01-2, we were able to detect high levels of m01 transcripts comparable to those of IE1 at 24, 48, and 72 hpi (Fig. ). Thus, we cannot exclude the possibility that m01 may exert functions other than solely encoding for viral miRNAs. However, the high abundance of miR-m01-4, miR-m01-2, and miR-m01-3 in MCMV-infected NIH 3T3 fibroblasts (together they contribute almost 50% of clones in our miRNA libraries) indicates that a large number of these transcripts are utilized for miRNA synthesis. This is reminiscent of observations made with other viruses, e.g., the BART transcript of EBV or the intronic cluster of miRNAs in KSHV (36
). The observation that the m01 transcript is so abundant in infected cells supports the finding that miR-m01-4 and the other m01-derived miRNAs were cloned so often. However, it raises the intriguing possibility that some viral miRNAs are processed nonspecifically, similar to the observation made with adenovirus virus-associated RNAs (3
). Thus, there could be some miRNAs expressed from the viral genome that are of little use to the virus, and some that may be more ancient and confer a significant advantage. This is illustrated by the fact that in EBV, the strain B95.8 is deleted of a large number of miRNAs (17
) without being especially affected compared to a nondeleted strain. The analysis of the phenotype of our deletion mutants in vivo will help sort out the functionally important miRNAs from the others.
Two additional deletion mutants were generated and characterized in vitro; one mutant was deleted of both miR-M23-2 and miR-m21-1, due to their genomic location, and the other was deleted of miR-M44-1. Although we confirmed that these mutants readily lacked expression of the aforementioned miRNAs (Fig. ), their replication phenotype was not clearly affected (Fig. ). A slight reduction in virus titers could be observed, but we cannot rule out the possibility that this is due to perturbation of expression of the neighboring genes. The absence of a phenotype in fibroblasts in vitro does not preclude a function in vivo, as MCMV infects a number of different cell types and encounters an array of antiviral defense mechanisms. For instance, mutation of the SV40 miR-S1 had no effect on single-step virus growth but had important implications in enhancing the recognition of infected cells by cytotoxic T cells because early gene transcripts are normally rapidly cleared by the action of miR-S1 (47
). Likewise, genes located in the vicinity of m01 (m02, m04, and m06) have all been implicated in interfering with the host immune response in vivo. Therefore, the biological function of this and the other MCMV miRNAs requires additional studies that should include immune response analyses. Interestingly, we observed a strong correlation of viral miRNA expression and titers of infectious virus in the livers, lungs, and spleens of infected animals at 3 and 5 days postinfection (Fig. ). Thereby, we provide the first evidence that miRNA expression is also an important feature of productive viral infection in vivo, and the use of the mutants that we generated will prove useful to assess their role.
Few MCMV miRNAs are located in transcripts with a well-defined function. The only genes that have an assigned role are M44, which encodes a DNA binding protein, a cofactor of DNA polymerase (28
); and M55, which encodes glycoprotein B (39
). M23 is a member of the US22 family, but its precise function is unknown (31
). In addition, none of the miRNAs identified here show significant conservation with HCMV miRNAs (16
), which might be explained by the fact that the two viruses are evolutionary distant. More puzzling, however, is the fact that, with one exception, their genomic localization is very different in the two viruses. Indeed, only miRNAs in the M23 region are in a region similar to HCMV miR-UL22A-1 (36
). This observation agrees with previous findings that viral miRNAs are often localized in fast-evolving regions of the genome (33
). The lack of conservation between sequence and location of the various miRNAs in cytomegaloviruses strongly suggests that host rather than viral genome regulatory purposes primarily drive herpesvirus miRNA evolution and function.