**4. Viral orthologs of host miRNAs**

Two classes of virus-encoded miRNAs are grouped: viral specific miRNAs and orthologs of host miRNAs. Similar to some viral regulatory proteins, several viral miRNAs have evolved to mimic host effectors. The "seed" region of a miRNA (~nucleotides 2–8 at the 5′ end) plays a key role in directing RISC to its mRNA targets. Binding with perfect seed complementary to the target transcript takes ~60% of regulation by a particular miRNA [45]. Some of viral miRNAs share seed sequences with host miRNAs and at least three viruses: BLV, MDV-1, and Kaposi's Sarcoma-associated Herpesvirus (KSHV) have been shown to regulate transcripts via the same target docking sites as their counterpart host miRNAs [46–48]. Such viral miRNAs could potentially regulate hundreds of transcripts having target sites for a particular host miRNA. Such regulatory networks could affect specific functions such as inhibiting apoptosis.

KSHV and MDV-1 express two distinct miRNAs, which are functional ortholog of host miR-155, a conserved cellular miRNA that is required for the rapid expansion of B and T cells after antigenic stimulation and highly expressed in activated myeloid and lymphoid cells [31, 46–48]. Interestingly, in both MDV-1 and KSHV-induced tumors, there is downregulation of endogenous levels of miR-155 [38, 47, 49], although the mechanisms for such downregulation is not fully understood. It is intriguing to know why these two viruses chose to express their own miR-155 orthologs, while suppressing the host-encoded miRNA with potentially identical functions. The direct role of miR-155 on oncogenesis [50, 51] and induction of cancer [47, 52] have been shown by a number of studies. Furthermore, it has been shown that upregulation of miR-155 is associated with lymphocyte transformation by reticuloendotheliosis virus strain T (REV-T) [53, 54] and EBV [55]. It is therefore striking that mdv1-miR-M4-5p is highly expressed in MDV-1 transformed cells derived from MDV-1-induced T-cell lymphomas [31, 46]. Moreover, lymphoma induction in infected birds is greatly reduced with seed region mutagenesis or deletion of mdv1-miR-M4-5p, suggesting the importance of mdv1-miR-M4-5p in the induction of tumors [34, 35]. The fact that transformation ability of the miR-M4-deleted MDV-1 virus is partly restored by introduction of chicken miR-155, suggests that viral miRNAs can play a major role in enhancing the oncogenic potential of a herpesvirus *in vivo*. However, the oncogenicity of vvMDV strain GX0101 with mdv1-miR-M4 deletion has significantly decreased but was not totally abolished [35]. This report, coupled with the finding that HVT with mdv1-miR-M4 inserted in the genome failed to induce tumors [32], suggested that other factors are also required for viral transformation. Moreover, our recent studies have shown that mdv1-miR-M4 is not essential for maintaining the proliferation of transformed cell lines suggesting that it has probably a more significant role in the initiation of neoplastic transformation [56].

miR-H14-3p, one of the HVT-encoded miRNAs, showed close sequence identity with perfect match of the 21/23 nucleotides including identical seed sequence to the chicken gga-miR-221, suggesting that it is a virus-encoded ortholog [27]. Indeed, the proposed ortholog status of the two miRNAs are supported by the evidence that HVT-miR-H14-3p also modulates the expression of p27Kip1 (cyclin-dependent kinase inhibitor 1B), a known target of miR-221 [57]. Compared to other known viral miRNA orthologs, where only the seed sequences are identical, the full length mature miRNA sequences of miR-H14-3p and gga-miR-221 are almost identical, strongly suggesting that hvt-miR-H14-3p is most likely to have been acquired from the host genome. This is the first example of virus-encoded miRNA showing such close and extended sequence identity with a host miRNA. Furthermore, partial sequence conservation has been observed between the gga-miR-221 locus on chromosome 1 of the chicken genome and the downstream flanking region of hvt-miR-H14-3p in the HVT genome, suggesting hvt-miR-H14-3p is "pirated" by the virus from the host, despite of the fact that herpesviruses have frequently pirated and subverted host genes to their own purposes. Interestingly, p27Kip1, the target shared by the two miRNAs, is a regulator of the cell cycle G1 to S phase transition. MDV-1-induced tumorigenesis may also involve a similar mechanism as miR-221-mediated repression of p27 in cancer progression [58–60]. In order to support viral genome replication and to promote growth of infected cells for additional viral production, the downregulation of p27 could move the cell cycle to the S phase [32]. The sequence homology is also observed between mdv1-miR-M31 and miR-221 in the seed region [31], although it is limited to the minimal miRNA seed region at nucleotide positions 2–7. Although targeting of p27 by miR-M31-3p has not been experimentally validated, the finding that miR-M31 deleted virus reduced the mortality and gross tumor incidence of infected chickens significantly has implied that miR-M31 may act as potential oncogene by targeting p27 [61].

**37**

*Role of Virus-Encoded microRNAs in Avian Viral Diseases*

gga-miR-29 is known to function both as an oncogene and a tumor suppressor depending on the context [62]. Seed sequence homology has been observed between mdv2-miR-M21 and miR-29 suggesting the potential functional ortholog between the two miRNAs. In fact, blv-miR-B4, a BLV-encoded miRNA, has been shown to be a functional ortholog of host miR-29 [12]. Furthermore, RLCVencoded miR-rL1-6-3p and EBV-encoded miR-BART1-3p also share seed sequence with miR-29. Thus, a picture is emerging wherein several virus-encoded miRNAs

It is estimated that ~8% of avian virus-encoded miRNAs possess identical heptameric seed sequence with host miRNAs, thus could potentially act as functional orthologs [7]. However, based on low abundance, untested biogenesis, and unknown functional relevance, it is not clear, whether all of the currently annotated viral or host miRNAs are bona fide miRNAs, thus some seed matches between host and viral miRNAs could arise by chance. Therefore, any proposed functional orthologs between the viral and host miRNAs require further experimental

Although over 500 virus-encoded miRNAs have been identified, an in depth functional study is lacking for most. However, it is clear that virus-encoded miR-NAs can target both cellular and viral mRNAs and this has been confirmed by several studies analyzing the mRNA targetome of the virus-infected cells [64]. The best characterized viral miRNA functions in supporting viral replication include regulating the latent-lytic switch; evading the immune response; promoting cell survival, proliferation, and/or differentiation. All of these functions should be particularly important during persistent infections. As viral miRNAs and proteins work synergistically to promote a cellular environment favorable to the completion of the viral life cycle, modulation of the host cell environment is achieved by multiple and partly redundant mechanisms. The identified targets of avian herpesvirus-encoded

Compared to identification of cellular targets, identifying viral targets of viral miRNAs is more straightforward as viral genomes encode fewer candidate mRNAs. Known examples of viral targets include transcripts, which are antisense to the viral miRNA precursor and transcripts elsewhere with imperfect matches. Perhaps, the former one is the most straightforward examples of determining viral miRNA function as the perfect matching between the miRNA and its target would be predicted to result in a siRNA-like cleavage of the target mRNA if both are co-expressed. For example, iltv-miR-I5 that is antisense to ICP4 cleaves ICP4 mRNA [65]. ICP4, an immediate early viral transactivator, plays a key role in the induction of lytic replication. The ICP4 targeting by viral miRNAs is thought to mediate lytic/latent switch and render the latent state more robust [66]. Apart from iltv-miR-I5, iltv-miR-I6 also maps antisense to the ICP4 gene. However, reporter assay on inhibition of luciferase activity by iltv-miR-I6 was not significant [65]. It has been proposed that this is due to the blockage of accessibility to the binding region following in silico folding of RNA containing the targets for iltv-miR-I5 and iltv-miR-I6. This is consistent with the previous finding that target RNA folding is essential for the efficacy of designed siRNAs [67, 68]. Sequences antisense to known miRNA stem-loop structures may fold into stem-loop structures themselves at an increased propensity.

**5. Target identification of avian herpesvirus miRNAs**

*DOI: http://dx.doi.org/10.5772/intechopen.89688*

are likely to target host pathways [63].

miRNAs are summarized in **Table 1**.

**5.1 Viral targets of viral miRNAs**

validation.

*Non-Coding RNAs*

KSHV and MDV-1 express two distinct miRNAs, which are functional ortholog of host miR-155, a conserved cellular miRNA that is required for the rapid expansion of B and T cells after antigenic stimulation and highly expressed in activated myeloid and lymphoid cells [31, 46–48]. Interestingly, in both MDV-1 and KSHV-induced tumors, there is downregulation of endogenous levels of miR-155 [38, 47, 49], although the mechanisms for such downregulation is not fully understood. It is intriguing to know why these two viruses chose to express their own miR-155 orthologs, while suppressing the host-encoded miRNA with potentially identical functions. The direct role of miR-155 on oncogenesis [50, 51] and induction of cancer [47, 52] have been shown by a number of studies. Furthermore, it has been shown that upregulation of miR-155 is associated with lymphocyte transformation by reticuloendotheliosis virus strain T (REV-T) [53, 54] and EBV [55]. It is therefore striking that mdv1-miR-M4-5p is highly expressed in MDV-1 transformed cells derived from MDV-1-induced T-cell lymphomas [31, 46]. Moreover, lymphoma induction in infected birds is greatly reduced with seed region mutagenesis or deletion of mdv1-miR-M4-5p, suggesting the importance of mdv1-miR-M4-5p in the induction of tumors [34, 35]. The fact that transformation ability of the miR-M4-deleted MDV-1 virus is partly restored by introduction of chicken miR-155, suggests that viral miRNAs can play a major role in enhancing the oncogenic potential of a herpesvirus *in vivo*. However, the oncogenicity of vvMDV strain GX0101 with mdv1-miR-M4 deletion has significantly decreased but was not totally abolished [35]. This report, coupled with the finding that HVT with mdv1-miR-M4 inserted in the genome failed to induce tumors [32], suggested that other factors are also required for viral transformation. Moreover, our recent studies have shown that mdv1-miR-M4 is not essential for maintaining the proliferation of transformed cell lines suggesting that it has probably a more significant role in the initiation of neoplastic transformation [56]. miR-H14-3p, one of the HVT-encoded miRNAs, showed close sequence identity with perfect match of the 21/23 nucleotides including identical seed sequence to the chicken gga-miR-221, suggesting that it is a virus-encoded ortholog [27]. Indeed, the proposed ortholog status of the two miRNAs are supported by the evidence that HVT-miR-H14-3p also modulates the expression of p27Kip1 (cyclin-dependent kinase inhibitor 1B), a known target of miR-221 [57]. Compared to other known viral miRNA orthologs, where only the seed sequences are identical, the full length mature miRNA sequences of miR-H14-3p and gga-miR-221 are almost identical, strongly suggesting that hvt-miR-H14-3p is most likely to have been acquired from the host genome. This is the first example of virus-encoded miRNA showing such close and extended sequence identity with a host miRNA. Furthermore, partial sequence conservation has been observed between the gga-miR-221 locus on chromosome 1 of the chicken genome and the downstream flanking region of hvt-miR-H14-3p in the HVT genome, suggesting hvt-miR-H14-3p is "pirated" by the virus from the host, despite of the fact that herpesviruses have frequently pirated and subverted host genes to their own purposes. Interestingly, p27Kip1, the target shared by the two miRNAs, is a regulator of the cell cycle G1 to S phase transition. MDV-1-induced tumorigenesis may also involve a similar mechanism as miR-221-mediated repression of p27 in cancer progression [58–60]. In order to support viral genome replication and to promote growth of infected cells for additional viral production, the downregulation of p27 could move the cell cycle to the S phase [32]. The sequence homology is also observed between mdv1-miR-M31 and miR-221 in the seed region [31], although it is limited to the minimal miRNA seed region at nucleotide positions 2–7. Although targeting of p27 by miR-M31-3p has not been experimentally validated, the finding that miR-M31 deleted virus reduced the mortality and gross tumor incidence of infected chickens significantly has implied

**36**

that miR-M31 may act as potential oncogene by targeting p27 [61].

gga-miR-29 is known to function both as an oncogene and a tumor suppressor depending on the context [62]. Seed sequence homology has been observed between mdv2-miR-M21 and miR-29 suggesting the potential functional ortholog between the two miRNAs. In fact, blv-miR-B4, a BLV-encoded miRNA, has been shown to be a functional ortholog of host miR-29 [12]. Furthermore, RLCVencoded miR-rL1-6-3p and EBV-encoded miR-BART1-3p also share seed sequence with miR-29. Thus, a picture is emerging wherein several virus-encoded miRNAs are likely to target host pathways [63].

It is estimated that ~8% of avian virus-encoded miRNAs possess identical heptameric seed sequence with host miRNAs, thus could potentially act as functional orthologs [7]. However, based on low abundance, untested biogenesis, and unknown functional relevance, it is not clear, whether all of the currently annotated viral or host miRNAs are bona fide miRNAs, thus some seed matches between host and viral miRNAs could arise by chance. Therefore, any proposed functional orthologs between the viral and host miRNAs require further experimental validation.

## **5. Target identification of avian herpesvirus miRNAs**

Although over 500 virus-encoded miRNAs have been identified, an in depth functional study is lacking for most. However, it is clear that virus-encoded miR-NAs can target both cellular and viral mRNAs and this has been confirmed by several studies analyzing the mRNA targetome of the virus-infected cells [64]. The best characterized viral miRNA functions in supporting viral replication include regulating the latent-lytic switch; evading the immune response; promoting cell survival, proliferation, and/or differentiation. All of these functions should be particularly important during persistent infections. As viral miRNAs and proteins work synergistically to promote a cellular environment favorable to the completion of the viral life cycle, modulation of the host cell environment is achieved by multiple and partly redundant mechanisms. The identified targets of avian herpesvirus-encoded miRNAs are summarized in **Table 1**.

#### **5.1 Viral targets of viral miRNAs**

Compared to identification of cellular targets, identifying viral targets of viral miRNAs is more straightforward as viral genomes encode fewer candidate mRNAs. Known examples of viral targets include transcripts, which are antisense to the viral miRNA precursor and transcripts elsewhere with imperfect matches. Perhaps, the former one is the most straightforward examples of determining viral miRNA function as the perfect matching between the miRNA and its target would be predicted to result in a siRNA-like cleavage of the target mRNA if both are co-expressed. For example, iltv-miR-I5 that is antisense to ICP4 cleaves ICP4 mRNA [65]. ICP4, an immediate early viral transactivator, plays a key role in the induction of lytic replication. The ICP4 targeting by viral miRNAs is thought to mediate lytic/latent switch and render the latent state more robust [66]. Apart from iltv-miR-I5, iltv-miR-I6 also maps antisense to the ICP4 gene. However, reporter assay on inhibition of luciferase activity by iltv-miR-I6 was not significant [65]. It has been proposed that this is due to the blockage of accessibility to the binding region following in silico folding of RNA containing the targets for iltv-miR-I5 and iltv-miR-I6. This is consistent with the previous finding that target RNA folding is essential for the efficacy of designed siRNAs [67, 68]. Sequences antisense to known miRNA stem-loop structures may fold into stem-loop structures themselves at an increased propensity.


#### **Table 1.**

*Avian virus encoded miRNAs and proposed functions highlighted in this review.*

This strategy could allow a virus to express both the viral gene and encode miRNAs antisense to mRNA transcripts lying on the other strand.

Analysis of miRNA binding clusters on the MDV-1 genome by PAR-CLIP identified very strong binding clusters near the 5′ end of the genome. These clusters essentially are antisense to the MDV-1 miRNAs miR-M2, miR-M3, miR-M4, and miR-M12, which are members of Meq-cluster, adjacent to each other, highly expressed and all located antisense to an MDV-1 mRNA encoding the viral R-LORF8 protein [64]. Analysis of indicator constructs in co-transfected 293 T cells or transduced MSB-1 cells confirmed that R-LORF8 is indeed targeted by all four miRNAs. Interestingly, expression of R-LORF8 gene in a form lacking the viral miRNA binding sites caused a substantial reduction in MSB-1 cell growth, suggesting that dysregulated expression of R-LORF8 can exert a deleterious effect in MDV-1 transformed T cells. The molecular basis for this effect is not clear as the function of R-LORF8 in the viral life cycle is currently unknown. The same pattern is observed in MDV-2, where viral miRNAs (miR-M24, M25, M26, M27, M28, and M29) are transcribed from the DNA strand located antisense to R-LORF2. In addition, there are also some other miRNAs encoded by MDV-1, MDV-2, HVT, and DEV, which are antisense to certain viral transcripts [24–28], the possible regulatory roles of those miRNAs against their antisense mRNA transcripts require to be verified individually.

**39**

tumorigenesis.

during infection [71, 72].

**5.2 Cellular targets of viral miRNAs**

*Role of Virus-Encoded microRNAs in Avian Viral Diseases*

As described above, mdv1-miR-M4-5p is a functional ortholog of cellular miR-155. mdv1-miR-M4-5p is also the first avian herpesvirus miRNA known to target both viral and cellular mRNAs. In addition to the cellular targets shared with miR-155, mdv1-miR-M4-5p and mdv1-miR-M4-3p also target viral UL28 and UL32 proteins, respectively. This provided the first evidence of late viral gene targeting by herpesviral miRNA [69]. Rather than 3′UTR, both target sequences are located in the coding region. UL28 and UL32 homologs, which are required for the cleavage and packaging of virion DNA in human herpesvirus 1 (HHV-1) have been found in all subfamilies of mammalian and avian herpesviruses. However, the role of UL28 and UL32 in MDV-1 packaging has not been demonstrated. As UL28 and UL32 are involved in the later stages of MDV replication, it is possible that mdv1-miR-M4 contributes in maintaining MDV-1 latency by downregulating the production of UL28 and UL32 and impairing late MDV morphogenesis and reactivation.

Following the observation of the increased viral pathogenicity or oncogenicity of infected chickens when mdv1-miR-M11 is deleted from Mid-cluster, the potential mechanisms mediated by miR-M11 has been investigated [61]. Meq gene has been predicated as candidate target gene by bioinformatics approach with RNAhybrid. Indeed, Meq as miR-M11 target has been confirmed by dual luciferase assay and qRT-PCR showing downregulation of Meq transcript level following virus infection. It has been suggested that the viral miRNAs in the Meq- or Mid-clusters cooperate with each other for establishing, maintaining latency, and/or triggering

ICP4 and ICP27, two MDV immediate-early (IE) genes, have been identified as potential targets for mdv1-miR-M7-5p by both bioinformatics prediction and subsequent experimental validation [70]. Indeed, this is reflected by a negative correlation between an increase in ICP27 expression and the decreased expression of mdv1-miR-M7-5p during virus reactivation. This is consistent with the early finding that miR-M7-5p is at extremely low levels in MDV-infected CEF but highly expressed in MSB-1 cells [25]. MDV miRNAs produced from LAT-cluster may contribute to the latency by targeting two IE genes. These findings further support the view that herpesvirus miRNAs play a key role in controlling the lytic/latent switch

Viruses such as herpesviruses with latent infection need to keep the host cells alive long enough. Thus, viral miRNAs can promote virus replication through prolonging cell survival and evading immune recognition. Among avian virusencoded miRNAs, the targets of mdv1-miR-M4-5p are most extensively studied due to its critical role in virus induced oncogenesis. As viral orthologs of miR-155, both kshv-miR-K12-11 and mdv1-miR-M4 potentially target the same group of transcription factors as gga-miR-155 including Pu.1, CEBPβ, HIVEP2, BCL2L13, PDCD6, MAP3K7IP2, GPM6B, RREB1, and c-Myb [46, 69]. Subsequent analysis of >1000 cellular mRNAs targeted by MDV-1 miRNAs in MSB-1 cells using photoactivatable ribonucleoside-enhanced cross-linking and immunoprecipitation (PAR-CLIP) resulted in the identification of 73 mRNA 3′UTR targets for miR-M4-5p, of which nine (C1orf103, CSNK1A1, LATS2, MAP3K14, MYB, NR1D2, RORA, RPS6KA3, and WEE1) had previously been identified as targets for miR-155 or kshv-miR-K12-11 in EBV-transformed human B cells and KSHV-transformed human B cells, respectively [64]. Indicator analysis of these 3′ UTRs gave data consistent with the hypothesis that these mRNAs are indeed targets for both miR-M4 in MSB1 and miR-155 in human B-cell lymphomas. Four additional mRNA targets (*FCHSD2, JARID2, PBEF1*, and RAP2A) were also conserved between miR-M4 in MSB-1 and

*DOI: http://dx.doi.org/10.5772/intechopen.89688*

#### *Role of Virus-Encoded microRNAs in Avian Viral Diseases DOI: http://dx.doi.org/10.5772/intechopen.89688*

*Non-Coding RNAs*

**38**

**Table 1.**

This strategy could allow a virus to express both the viral gene and encode miRNAs

Analysis of miRNA binding clusters on the MDV-1 genome by PAR-CLIP identified very strong binding clusters near the 5′ end of the genome. These clusters essentially are antisense to the MDV-1 miRNAs miR-M2, miR-M3, miR-M4, and miR-M12, which are members of Meq-cluster, adjacent to each other, highly expressed and all located antisense to an MDV-1 mRNA encoding the viral R-LORF8 protein [64]. Analysis of indicator constructs in co-transfected 293 T cells or transduced MSB-1 cells confirmed that R-LORF8 is indeed targeted by all four miRNAs. Interestingly, expression of R-LORF8 gene in a form lacking the viral miRNA binding sites caused a substantial reduction in MSB-1 cell growth, suggesting that dysregulated expression of R-LORF8 can exert a deleterious effect in MDV-1 transformed T cells. The molecular basis for this effect is not clear as the function of R-LORF8 in the viral life cycle is currently unknown. The same pattern is observed in MDV-2, where viral miRNAs (miR-M24, M25, M26, M27, M28, and M29) are transcribed from the DNA strand located antisense to R-LORF2. In addition, there are also some other miRNAs encoded by MDV-1, MDV-2, HVT, and DEV, which are antisense to certain viral transcripts [24–28], the possible regulatory roles of those miRNAs against their antisense mRNA transcripts require to be verified individually.

antisense to mRNA transcripts lying on the other strand.

*Avian virus encoded miRNAs and proposed functions highlighted in this review.*

As described above, mdv1-miR-M4-5p is a functional ortholog of cellular miR-155. mdv1-miR-M4-5p is also the first avian herpesvirus miRNA known to target both viral and cellular mRNAs. In addition to the cellular targets shared with miR-155, mdv1-miR-M4-5p and mdv1-miR-M4-3p also target viral UL28 and UL32 proteins, respectively. This provided the first evidence of late viral gene targeting by herpesviral miRNA [69]. Rather than 3′UTR, both target sequences are located in the coding region. UL28 and UL32 homologs, which are required for the cleavage and packaging of virion DNA in human herpesvirus 1 (HHV-1) have been found in all subfamilies of mammalian and avian herpesviruses. However, the role of UL28 and UL32 in MDV-1 packaging has not been demonstrated. As UL28 and UL32 are involved in the later stages of MDV replication, it is possible that mdv1-miR-M4 contributes in maintaining MDV-1 latency by downregulating the production of UL28 and UL32 and impairing late MDV morphogenesis and reactivation.

Following the observation of the increased viral pathogenicity or oncogenicity of infected chickens when mdv1-miR-M11 is deleted from Mid-cluster, the potential mechanisms mediated by miR-M11 has been investigated [61]. Meq gene has been predicated as candidate target gene by bioinformatics approach with RNAhybrid. Indeed, Meq as miR-M11 target has been confirmed by dual luciferase assay and qRT-PCR showing downregulation of Meq transcript level following virus infection. It has been suggested that the viral miRNAs in the Meq- or Mid-clusters cooperate with each other for establishing, maintaining latency, and/or triggering tumorigenesis.

ICP4 and ICP27, two MDV immediate-early (IE) genes, have been identified as potential targets for mdv1-miR-M7-5p by both bioinformatics prediction and subsequent experimental validation [70]. Indeed, this is reflected by a negative correlation between an increase in ICP27 expression and the decreased expression of mdv1-miR-M7-5p during virus reactivation. This is consistent with the early finding that miR-M7-5p is at extremely low levels in MDV-infected CEF but highly expressed in MSB-1 cells [25]. MDV miRNAs produced from LAT-cluster may contribute to the latency by targeting two IE genes. These findings further support the view that herpesvirus miRNAs play a key role in controlling the lytic/latent switch during infection [71, 72].

## **5.2 Cellular targets of viral miRNAs**

Viruses such as herpesviruses with latent infection need to keep the host cells alive long enough. Thus, viral miRNAs can promote virus replication through prolonging cell survival and evading immune recognition. Among avian virusencoded miRNAs, the targets of mdv1-miR-M4-5p are most extensively studied due to its critical role in virus induced oncogenesis. As viral orthologs of miR-155, both kshv-miR-K12-11 and mdv1-miR-M4 potentially target the same group of transcription factors as gga-miR-155 including Pu.1, CEBPβ, HIVEP2, BCL2L13, PDCD6, MAP3K7IP2, GPM6B, RREB1, and c-Myb [46, 69]. Subsequent analysis of >1000 cellular mRNAs targeted by MDV-1 miRNAs in MSB-1 cells using photoactivatable ribonucleoside-enhanced cross-linking and immunoprecipitation (PAR-CLIP) resulted in the identification of 73 mRNA 3′UTR targets for miR-M4-5p, of which nine (C1orf103, CSNK1A1, LATS2, MAP3K14, MYB, NR1D2, RORA, RPS6KA3, and WEE1) had previously been identified as targets for miR-155 or kshv-miR-K12-11 in EBV-transformed human B cells and KSHV-transformed human B cells, respectively [64]. Indicator analysis of these 3′ UTRs gave data consistent with the hypothesis that these mRNAs are indeed targets for both miR-M4 in MSB1 and miR-155 in human B-cell lymphomas. Four additional mRNA targets (*FCHSD2, JARID2, PBEF1*, and RAP2A) were also conserved between miR-M4 in MSB-1 and

miR-155 in EBV-transformed B cells. Among these mRNA targets, JARID2, a cell cycle regulator and part of a histone methyltransferase complex, has been shown to promote apoptosis and decrease cell survival when ectopically expressed [53]. *WEE1* encodes a kinase that blocks cell-cycle progression and has been associated with inflammation and cancer [73]. MYB, a transcription factor, is involved in the regulation of hematopoiesis and tumorigenesis. Another interesting shared gene target for miR-155/miR-M4/miR-K12-11 is large tumor suppressor (LATS), which inhibits cell proliferation and promotes apoptosis by inhibiting YAP transcriptional activity through Hippo pathway. Being evolutionarily conserved from avian to mammal, loss of LATS expression is observed in many human cancers, including acute lymphoblastic leukemia [74]. These observations suggest a potential selective advantage for MDV-1 in inducing downregulation of LATS. Taken together, these observations provide additional evidence for the impact of miR-155 and its orthologs on pathways regulating lymphocyte activation, differentiation, apoptosis, proliferation, immune tolerance, and tumorigenesis [7].

In addition to the candidate mRNA targets described above, latent TGF-β binding protein 1 (*LTBP1*) has also been identified as bona fide host mRNA targets for miR-M4 [75]. Inhibition of *LTBP1* expression by miR-M4-5p induced a significant decrease of TGF-β1 secretion and activation, with suppression of TGF- β signaling and upregulation of c-Myc expression. Interestingly, miR-155 has been shown to suppress TGF-β signaling through targeting SMAD2 and SMAD5 in human diseases [76, 77]. The KSHV-encoded miR-K12-11 also inhibits TGF-β signaling through downregulation of SMAD5 [78]. Thus, these findings indicate that dysregulation of the TGF-β signaling pathway by miR-155 and its viral orthologs may be a common feature shared by oncogenic herpesviruses. Interestingly, MDV1-encoded miRNA miR-M3 also suppress TGF-β signaling through targeting host gene SMAD2, and has been shown to suppress drug-induced apoptosis in cell culture [79], thus adding another layer of confidence to the conclusion that suppression of TGF-β signaling pathway is indeed involved in MDV-induced oncogenesis.

Another identified cellular target of miR-M4 is chicken heterogeneous nuclear ribonucleoprotein AB (hnRNPAB). HnRNPAB, a member of the hnRNP family proteins, plays important roles in both normal biological processes and cancer development. Downregulation of hnRNPAB expression promotes proliferation of both CEF and chicken fibroblast cell line DF-1 [80]. Hence, downregulation of hnRNPAB by miR-M4-5p may be one of the important strategies for MDV-1 to trigger the development of MD lymphomas.

PAR-CLIP identified a list of 1104 cellular mRNAs targeted by MDV-1 miRNAs and a list of 1183 mRNAs targeted by MDV-2 miRNAs in MSB1, of which 419 mRNA targets were shared. Analysis of those cellular mRNAs identified several that were targeted by five or more different viral miRNAs in their 3′ UTRs, suggestive of a possibly important role in restricting innate antiviral immunity. IL-18, a proinflammatory cytokine induced upon infection by several different viruses, is able to stimulate IFN-γ production from T cells [81]. Surprisingly, chicken IL-18 gene contains seven viral miRNA target sites in its 3′ UTR [64]. Indeed, MSB1 cell growth is highly sensitive to inhibition by ectopic IL-18 expression. Whether this is due to induction of chicken IFN-γ expression remains to be determined. Thus, in addition to express viral interleukin-8 (vIL-8), which is required for disease progression and tumor development, this data suggest a second way in which MDV manipulates the host cell immune response.

Taken together, all of the data above suggests that latent/oncogenic viruses may proactively create a cellular environment beneficial to viral latency and oncogenesis through viral miRNA targeting cellular factors involved in antiviral processes including apoptosis. Surely, more targets of miRNAs encoded by avian viruses will

**41**

**7. Conclusions**

*Role of Virus-Encoded microRNAs in Avian Viral Diseases*

be identified with the advances in high-throughput technologies. We should be able to understand the role played by these small and highly effective modulators of gene expression once more targets of viral miRNAs are discovered and an integrated approach of demonstrating the functions and molecular pathways is developed.

Despite, several possible cancer-related genes have been identified as their targets of viral miRNAs, the direct *in vivo* role of those miRNAs such as KSHVencoded miRNAs in neoplastic diseases is difficult to demonstrate in the absence of suitable models. However, excellent disease models of infection by MDV in natural avian host allow examination of the oncogenic potential of virus-encoded miRNAs. As described above, it has been shown that the deletion of the Meq-clustered miR-NAs from the viral genome by BAC mutagenesis abolished the oncogenicity of the virus. miR-M4 is essential for the virus in inducing tumor as the mutant virus with miR-M4 deletion or seed sequence mutation almost lost its oncogenicity, providing evidence for a direct *in vivo* role of a viral miRNA in tumor induction [34]. In a separate study, deletions of the Meq-cluster or miR-M4 alone from the viral genome of vvMDV strain GX0101 strongly decreased its oncogenicity [35]. Subsequent work demonstrated that except for mdv1-miR-M4, most of the other Meq-clustered miRNAs also play critical roles in MDV oncogenesis as both mortality and gross tumor incidence of birds infected with the mutant viruses have reduced significantly for the corresponding virus with single miRNA deletion [82]. These studies provide further information for understanding the molecular determinants that

Having demonstrated the critical roles of Meq-clustered miRNAs in MD pathogenesis and/or tumorigenesis, the role of miRNAs in Mid-cluster, which is transcribed by the same promoter as the Meq-cluster in the latent phase of the infection has been investigated by infection of miRNA deleted virus in animal model [61]. The role of miR-M31-3p acting as oncogene and miR-M11-5p acting as tumor suppressor in MD lymphomagenesis have been proposed based on the decreased mortality and gross tumor incidence by miR-M31 deletion mutant and increased viral pathogenicity or oncogenicity of infected chickens when miR-M11 is deleted.

Recent advances in sequencing technology have led to the identification of a number of miRNAs encoded by avian viruses. Given their ability to target cellular and viral transcripts, and the lack of immune response, miRNAs represent an ideal mechanism of gene regulation during viral infection, latency, and persistence. Similar to host miRNAs, the determination of biologically relevant activities of the reported viral miRNAs is the next step forward. Although the function of most viral miRNAs is to be determined, evidence so far does support the view that viral miRNAs are key regulators in virus biology. By targeting key viral lytic genes directly or modulating cellular regulatory pathways indirectly, the virus-encoded miRNAs could contribute significantly toward switching between lytic and latent infections, thereby regulating viral pathogenesis *in vivo*. Whether a small or large number of critical miRNA-target

interactions have functional significance is a key question to be answered.

It is crucial to define the miRNA targets for both understanding observed phenotypes and providing clues to their functional role. Genome wide biochemical screening are becoming more powerful approaches for the generation of reliable

*DOI: http://dx.doi.org/10.5772/intechopen.89688*

**6. Role of viral miRNAs in pathogenesis**

trigger the development of tumors by oncogenic MDVs.

*Non-Coding RNAs*

miR-155 in EBV-transformed B cells. Among these mRNA targets, JARID2, a cell cycle regulator and part of a histone methyltransferase complex, has been shown to promote apoptosis and decrease cell survival when ectopically expressed [53]. *WEE1* encodes a kinase that blocks cell-cycle progression and has been associated with inflammation and cancer [73]. MYB, a transcription factor, is involved in the regulation of hematopoiesis and tumorigenesis. Another interesting shared gene target for miR-155/miR-M4/miR-K12-11 is large tumor suppressor (LATS), which inhibits cell proliferation and promotes apoptosis by inhibiting YAP transcriptional activity through Hippo pathway. Being evolutionarily conserved from avian to mammal, loss of LATS expression is observed in many human cancers, including acute lymphoblastic leukemia [74]. These observations suggest a potential selective advantage for MDV-1 in inducing downregulation of LATS. Taken together, these observations provide additional evidence for the impact of miR-155 and its orthologs on pathways regulating lymphocyte activation, differentiation, apoptosis,

In addition to the candidate mRNA targets described above, latent TGF-β binding protein 1 (*LTBP1*) has also been identified as bona fide host mRNA targets for miR-M4 [75]. Inhibition of *LTBP1* expression by miR-M4-5p induced a significant decrease of TGF-β1 secretion and activation, with suppression of TGF- β signaling and upregulation of c-Myc expression. Interestingly, miR-155 has been shown to suppress TGF-β signaling through targeting SMAD2 and SMAD5 in human diseases [76, 77]. The KSHV-encoded miR-K12-11 also inhibits TGF-β signaling through downregulation of SMAD5 [78]. Thus, these findings indicate that dysregulation of the TGF-β signaling pathway by miR-155 and its viral orthologs may be a common feature shared by oncogenic herpesviruses. Interestingly, MDV1-encoded miRNA miR-M3 also suppress TGF-β signaling through targeting host gene SMAD2, and has been shown to suppress drug-induced apoptosis in cell culture [79], thus adding another layer of confidence to the conclusion that suppression of TGF-β signaling

Another identified cellular target of miR-M4 is chicken heterogeneous nuclear ribonucleoprotein AB (hnRNPAB). HnRNPAB, a member of the hnRNP family proteins, plays important roles in both normal biological processes and cancer development. Downregulation of hnRNPAB expression promotes proliferation of both CEF and chicken fibroblast cell line DF-1 [80]. Hence, downregulation of hnRNPAB by miR-M4-5p may be one of the important strategies for MDV-1 to trigger the devel-

PAR-CLIP identified a list of 1104 cellular mRNAs targeted by MDV-1 miRNAs and a list of 1183 mRNAs targeted by MDV-2 miRNAs in MSB1, of which 419 mRNA targets were shared. Analysis of those cellular mRNAs identified several that were targeted by five or more different viral miRNAs in their 3′ UTRs, suggestive of a possibly important role in restricting innate antiviral immunity. IL-18, a proinflammatory cytokine induced upon infection by several different viruses, is able to stimulate IFN-γ production from T cells [81]. Surprisingly, chicken IL-18 gene contains seven viral miRNA target sites in its 3′ UTR [64]. Indeed, MSB1 cell growth is highly sensitive to inhibition by ectopic IL-18 expression. Whether this is due to induction of chicken IFN-γ expression remains to be determined. Thus, in addition to express viral interleukin-8 (vIL-8), which is required for disease progression and tumor development, this data suggest a second way in which MDV manipulates the

Taken together, all of the data above suggests that latent/oncogenic viruses may proactively create a cellular environment beneficial to viral latency and oncogenesis through viral miRNA targeting cellular factors involved in antiviral processes including apoptosis. Surely, more targets of miRNAs encoded by avian viruses will

proliferation, immune tolerance, and tumorigenesis [7].

pathway is indeed involved in MDV-induced oncogenesis.

opment of MD lymphomas.

host cell immune response.

**40**

be identified with the advances in high-throughput technologies. We should be able to understand the role played by these small and highly effective modulators of gene expression once more targets of viral miRNAs are discovered and an integrated approach of demonstrating the functions and molecular pathways is developed.

## **6. Role of viral miRNAs in pathogenesis**

Despite, several possible cancer-related genes have been identified as their targets of viral miRNAs, the direct *in vivo* role of those miRNAs such as KSHVencoded miRNAs in neoplastic diseases is difficult to demonstrate in the absence of suitable models. However, excellent disease models of infection by MDV in natural avian host allow examination of the oncogenic potential of virus-encoded miRNAs. As described above, it has been shown that the deletion of the Meq-clustered miR-NAs from the viral genome by BAC mutagenesis abolished the oncogenicity of the virus. miR-M4 is essential for the virus in inducing tumor as the mutant virus with miR-M4 deletion or seed sequence mutation almost lost its oncogenicity, providing evidence for a direct *in vivo* role of a viral miRNA in tumor induction [34]. In a separate study, deletions of the Meq-cluster or miR-M4 alone from the viral genome of vvMDV strain GX0101 strongly decreased its oncogenicity [35]. Subsequent work demonstrated that except for mdv1-miR-M4, most of the other Meq-clustered miRNAs also play critical roles in MDV oncogenesis as both mortality and gross tumor incidence of birds infected with the mutant viruses have reduced significantly for the corresponding virus with single miRNA deletion [82]. These studies provide further information for understanding the molecular determinants that trigger the development of tumors by oncogenic MDVs.

Having demonstrated the critical roles of Meq-clustered miRNAs in MD pathogenesis and/or tumorigenesis, the role of miRNAs in Mid-cluster, which is transcribed by the same promoter as the Meq-cluster in the latent phase of the infection has been investigated by infection of miRNA deleted virus in animal model [61]. The role of miR-M31-3p acting as oncogene and miR-M11-5p acting as tumor suppressor in MD lymphomagenesis have been proposed based on the decreased mortality and gross tumor incidence by miR-M31 deletion mutant and increased viral pathogenicity or oncogenicity of infected chickens when miR-M11 is deleted.

#### **7. Conclusions**

Recent advances in sequencing technology have led to the identification of a number of miRNAs encoded by avian viruses. Given their ability to target cellular and viral transcripts, and the lack of immune response, miRNAs represent an ideal mechanism of gene regulation during viral infection, latency, and persistence. Similar to host miRNAs, the determination of biologically relevant activities of the reported viral miRNAs is the next step forward. Although the function of most viral miRNAs is to be determined, evidence so far does support the view that viral miRNAs are key regulators in virus biology. By targeting key viral lytic genes directly or modulating cellular regulatory pathways indirectly, the virus-encoded miRNAs could contribute significantly toward switching between lytic and latent infections, thereby regulating viral pathogenesis *in vivo*. Whether a small or large number of critical miRNA-target interactions have functional significance is a key question to be answered.

It is crucial to define the miRNA targets for both understanding observed phenotypes and providing clues to their functional role. Genome wide biochemical screening are becoming more powerful approaches for the generation of reliable

and accurate target information of viral miRNAs although target identification using target prediction software provided an initial strategy. Several techniques such as RIP-CHIP (RNA-binding protein immunoprecipitation microarray), HITS-CLIP (high-throughput sequencing cross-linking and immunoprecipitation), PAR-CLIP and proteomics analysis have all contributed large amount of data on potential targets of virus-encoded miRNAs [47, 83–89] although only PAR-CLIP has been used for avian herpesvirus miRNA targetome identification [64]. Cross-linking, ligation and sequencing of hybrids (CLASH) technology, another biochemical screen for miRNA targets, promises to generate the most accurate target information to date, leading the way in the generation of high confidence target datasets which will be invaluable for future studies [90, 91]. These new technologies allow unprecedented and largely unbiased views into miRNAs-mediated regulation of gene expression in virus-infected cells. Undoubtedly, further studies using different approaches and technologies are required toward the clear definition of miRNAs targetome and their functional relevance in viral infection, latency, reactivation, and pathogenesis.
