Herpesvirus of Turkeys (Meleagridis Herpesvirus 1) Encodes a Functional MicroRNA-221 Homolog with High Sequence Conservation

Herpesviruses account for most of the known virus-encoded miRNAs. Herpesvirus of turkey (HVT), a non-pathogenic avian herpesvirus used as an avian vaccine and viral vector, encodes 28 mature miRNAs. This included HVT-miR-H14-3p that showed almost identical sequence to gga-miR-221, suggesting that it is pirated from the avian host. Although the functional homolog between the two miRNAs has been proposed based on the sequence similarity, the direct experimental evidence is still lacking. In this report, we provide the evidence for the first time that HVT-miR-H14-3p is indeed a gga-miR-221 homolog through modulating the expression of p27 Kip1 , a known target of miR-221 by binding to its 3’UTR. We also created an HVT-miR-H14-3p deletion virus and show that this miRNA is not essential for in vitro replication.

Herpesvirus of turkey (HVT), known also as Meleagridis herpesvirus 1, is one of the most widely used Marek's disease vaccine and recombinant vaccine vector used against avian diseases. We and others have previously reported the identification of 28 mature miRNAs encoded by HVT [14] [16]. Some of these miR-NAs such as HVT-miR-H5, shared the "seed" sequence with MDV1-miR-M9 [17] and MDV2-miR-M28 [15], demonstrating that viruses can encode potentially functional orthologs of both host and virus-encoded miRNAs. However with all these miRNA orthologs, the conservation of the sequences is restricted mostly to the functionally important 6/7 nucleotide seed region, suggestive of a selection pressure to maintain this sequence. Base pairing outside the seed region also influences many miRNA: target interactions although predicting these interactions has remained difficult. A virus-encoded miRNA homolog that retains sequence identity both with the seed and non-seed regions would be valuable for delineating the functions of miRNA regions. HVT-miR-H14-3p [14], also named as HVT-miR6 [18], is the first example of such a miRNA, which shows match in 21/23 nucleotides with the host encoded gga-miR-221 (Figure 1(a)) [14]. Although it has been suggested that novel miRNAs can arise de novo from existing hairpin structures [19], the high degree of sequence homology with gga-miR-221 strongly suggested that HVT-miR-H14-3p was snatched by the virus from the host. Moreover, demonstration of partial sequence conservation between the downstream flanking region of HVT-miR-H14-3p in the HVT genome and the gga-miR-221 locus on chromosome 1 of the chicken genome, supports this conclusion [14]. Since all the other known viral miRNA homologs show conservation only in the seed sequences, it is surprising that HVT-miR-H14-3p Y. X. Yao et al. Relative signal intensities of the p27 Western blot band were quantified using Image Quant and normalized against the corresponding signal from the tubulin band. The signal from un-infected cells was set as 1.
is maintained almost in its entirety by the virus. This would probably suggest that the non-seed region of HVT-miR-H14-3p also may have a functional role, or there may be structural constraints that require the miRNA to be maintained in its entirety. Alternatively, the acquisition of gga-miR-221 by the virus may only be a recent event, and further virus evolution could lead to emergence of a more conventional miRNA homolog with restricted sequence conservation.
Extensive studies on the cellular miRNAs miR-221/222 have demonstrated their major roles in cell cycle and cancer [20] [21], including their roles in regulating the expression of cell cycle regulatory proteins such as the cyclin-dependent kinase (cdk) inhibitor p27 Kip1 [22]- [27] and p57 Kip2 [20] [22] [26]. We have demonstrated that miR-221/222 is expressed at high levels in MDV-transformed chicken T-cell line MSB-1 [17] [28]. We also showed that these miRNAs can modulate the expression of chicken p27 Kip1 through the specific sequence in the 3'UTR of the chicken p27 Kip1 transcript [27]. The functional role of these miRNAs in modulating p27 Kip1 protein expression was also confirmed through overexpression studies and by using antagomiRs [27]. On the basis of these studies, we hypothesized that the HVT-miR-H14-3p homolog will have a modulatory role on the expression of the miR-221/222 targets such as the p27 Kip1 .
To determine if HVT-miR-H14-3p can also target p27 Kip1 , we generated a HVT-miR-H14-3p expression plasmid similar to the miR-221 expression construct reported previously [27], by annealing complementary DNA oligonucleotides HVT-H14-F and HVT-H14-R (Table 1) and cloned into RCAS vector. The ability of HVT-miR-H14-3p to target p27 Kip1 was first assessed by co-transfection of DF-1 cells with the HVT-miR-H14-3p expression plasmid along with the reporter construct that featured the 3'UTR in its native form (p27-3'UTR-WT) or a mutant construct with three base-pair mutations in each of the two predicted miRNA binding sites (p27-3'UTR-dbMT) fused to the 3'UTR of the renilla luciferase in psiCHECKTM-2 [27]. The miR-221 expression construct and a negative control miRNA (miR-NS) [27] were also included as positive and negative controls respectively. The luciferase expression was assayed 48 hours after transfection using the Dual-Glo Luciferase Assay System (Promega), and the relative expression of renilla luciferase normalised to the levels of firefly luciferase was determined. For each sample, values from four replicates representative of three independent experiments were used in the analysis. Co-transfection of HVT-miR-H14-3p expression vector with p27-3'UTR-WT resulted in 55% knockdown of renilla luciferase activity, and 30% reduction was seen by miR-221 expression plasmid (Figure 1(b)). Thus, the reporter assay demonstrated that HVT-miR-H14-3p, similar to miR-221, can target p27 Kip1 . We next measured the reduction of p27 Kip1 protein level by HVT-miR-H14-3p and miR-221 in DF-1 cells to further confirm that HVT-miR-H14-3p and miR-221 are functional orthologs. To maximize the effect of HVT-miR-H14-3p and miR-221 on the target p27 Kip1 , we used the replication-competent avian retrovirus RCASBP-B-CN-EGFP [27] vector (a generous gift from Dr. Jon Gilthorpe, Kings College London) to generate HVT-miR-H14-3p and miR-221 expression vectors. As replication competent vectors, these constructs allow expression of high levels of miRNAs from the chicken U6 promoter [27] and virus-infected cells can be tracked by the marker EGFP expression. The transfected Table 1. Sequence of the oligonucleotide primers used (homologous sequences used for recombination are underlined).

Primers
Sequences ( Having demonstrated that miR-221 homolog is functional in modulating the expression of at least one of the target protein, p27 Kip1 , we wanted to examine whether it is either essential for replication or can provide any functional advantages for the virus. For this, we generated HVT-miR-H14-3p deletion mutants of HVT by mutagenesis of pHVT3 [29] using standard procedures [30] [31] in SW105 strain of E. coli (kindly provided by Dr. N. Copeland, NCI Frederick, MD). The two copies of the HVT-miR-H14-3p were deleted sequentially in two steps, first by inserting Kan R cassette amplified by PCR using primers HVT-H14-KD-F and HVT-H14-KD-R. After flipping out the Kan R cassette, the second copy was deleted by the insertion of a galK cassette amplified by the primers HVT-miR-galK-F and HVT-miR-galK-R. The construct from which both copies of the HVT-miR-H14-3p deleted was named as pHVT3-H14-00. The galK cassette in the construct was also used as a negative selection marker for restoration of HVT-miR-H14-3p in the revertant construct pHVT3-H14-R0.
The accuracy of the deletions was checked by PCR using oligonucleotide primers (Table 1)