MHV-68 is an important small animal model with which to study the pathogenesis of γHV infections. By deep sequencing of small RNA, we systematically investigated the MHV-68 miRNA expression profiles in both lytically and persistently infected cells. We identified six novel MHV-68 miRNA genes and analyzed the expression levels of all MHV-68 miRNAs. Furthermore, we also characterized the cellular miRNA expression signatures in MHV-68-infected versus noninfected NIH 3T3 fibroblasts and in TPA-treated versus nontreated S11 cells. We found that mmu-mir-15b and mmu-mir-16 are highly upregulated upon MHV-68 infection of NIH 3T3 cells, indicating a potential role of cellular miRNAs during MHV-68 infection.
MHV-68 miRNAs were first identified by traditional small-RNA cloning and sequencing (32
). Nine MHV-68 miRNA genes were characterized and experimentally validated, and five more hairpins were predicted. With the more-sensitive deep-sequencing approach, we were able to confirm the existence of all previously described MHV-68 miRNAs and almost all of the star miRNAs, which have not been reported before. Moreover, we identified, for the first time, the five predicted miRNA genes, together with one unpredicted miRNA gene. Notably, all MHV-68 miRNAs, except for mghv-mir-M1-11, reside immediately following the unique vtRNA sequences in the MHV-68 genome. A transcript containing mghv-mir-M1-11 might be generated by read-through transcription beyond the T clusters at which RNA polymerase III usually terminates transcription (6
Almost all MHV-68 miRNAs were detectable in the two infected cell lines we have sequenced (Table ). Overall, the persistently infected S11 cells showed much higher MHV-68 miRNA expression levels than the lytically infected NIH 3T3 cells. mghv-mir-M1-1, mghv-mir-M1-8, and mghv-mir-M1-9 were the three most abundant miRNAs in both cell lines. mghv-mir-M1-5* was more abundant than mghv-mir-M1-5 in infected NIH 3T3 cells, while in S11 cells, mghv-mir-M1-5 was expressed at a higher level. Such different MHV-68 miRNA expression patterns might be suggestive of distinct functions during lytic and persistent/latent infections. By Northern blotting, we could demonstrate the expression of three (mghv-mir-M1-10, -12, and -14) of the six novel MHV-68 miRNAs. Although mghv-mir-M1-13 was found with a comparable read number in the deep-sequencing data sets, it was hardly detectable by Northern blotting. This might be due to its low GC content. However, we were able to demonstrate the expression of mghv-mir-M1-13 by a more-sensitive stem-loop qRT-PCR. This is consistent with a recent report demonstrating that reverse ligation-mediated RT-PCR (RLM-RT-PCR) is able to detect mature MHV-68 miRNAs with at least 100-fold higher sensitivity than Northern blotting (13
). We did not attempt to detect the expression of mghv-mir-M1-11 and -15 by either Northern blotting or stem-loop qRT-PCR because of their extremely low read numbers (1 and 2 reads, respectively) in the deep sequencing.
The MHV-68 miRNAs are among the minority of miRNAs that are under the regulation of RNA polymerase III. It has been shown recently that the generation of the MHV-68 miRNAs is dependent on the A/B boxes in the vtRNA sequences and on the presence of tRNase Z and Dicer, but not on Drosha (4
). By infection of Dicer-deficient MEFs, we confirmed that MHV-68 miRNAs are processed by a Dicer-dependent pathway. Importantly, while Bogerd et al. (4
) showed Dicer dependency by small interfering RNA (siRNA)-mediated knockdown of Dicer in human (HeLa) cells transfected with an MHV-68 miRNA expression plasmid, we demonstrated it by infection of authentic host cells (Dicer−/−
MEFs) with MHV-68.
The vtRNAs were identified and characterized more than 10 years ago and were shown not to be aminoacylated (5
). However, their function still remains unknown. We speculate that the vtRNA sequences remained as remnants during evolution and now serve as promoter sequences for the generation of the MHV-68 miRNAs. How the differential expression of the MHV-68 miRNAs is regulated, and the significance of the existence of the vtRNA-miRNA-miRNA structures, will be interesting to investigate.
The MHV-68 miRNAs were shown to be efficiently associated with the RISC by immunoprecipitation using a monoclonal anti-mouse Ago2 antibody, implying that they are functional. However, the functions of the MHV-68 miRNAs still remain unknown. By matching the seed sequences of the MHV-68 miRNAs, we found that they are predicted to target both viral and cellular proteins (data not shown). The abundance of the MHV-68 miRNAs might also imply that they could function by occupying the cellular RISC machinery, thus interfering with the normal cellular miRNA pathways. The attenuated but nonlethal phenotype of an MHV-68 mutant virus lacking the first 9.5 kbp of the genome, including all vtRNAs and miRNAs, indicated that the vtRNAs and miRNAs are not absolutely essential for lytic replication or for the establishment and maintenance of latency (11
Apart from the MHV-68 miRNAs, the deep-sequencing data also allowed us to analyze the expression signature of the cellular miRNAs in uninfected versus infected NIH 3T3 cells. mmu-mir-15b and mmu-mir-16 were shown to be upregulated after infection, and this observation was validated by Northern blotting. Interestingly, in our previous study (49
), hsa-mir-15a and hsa-mir-16 were found to be upregulated in EBV-infected nasopharyngeal carcinoma samples compared to their expression in healthy control tissues, and the tumor suppressor BRCA-1 was validated as one of the target genes of hsa-mir-15a and hsa-mir-16. Here we could also show that upregulation of mmu-mir-16 and mmu-mir-15b correlated with lower BRCA-1 expression in infected NIH 3T3 cells than in uninfected cells. Notably, other authors reported that NIH 3T3 cells, expressing an RNA transcript that is antisense to the BRCA-1 mRNA and that thus inhibited the expression of BRCA-1 protein, showed accelerated and anchorage-independent growth and tumorigenicity in nude mice (36
Taken together, we have performed deep sequencing of small RNAs expressed in lytically infected NIH 3T3 cells and in persistently infected S11 cells in order to more completely define the miRNA coding potential of MHV-68. Our analysis identified 6 novel MHV-68 miRNAs and additionally provided the first comprehensive overview of both MHV-68 and cellular miRNA expression in infected cells. Our data will aid in the full exploration of the functions of γHV miRNAs.