Cellular targets

therapeutic perspectives, in particular when considering herpesviruses infections. Indeed, it is plausible that blocking the viral miRNAs could be used as a strategy to force these viruses to enter into the lytic stage, and combined to the use of drugs blocking their replication, it could thus eventually allow to purge the latent reservoir.

Furthermore, the control of latency by virus-encoded miRNAs is not only achieved by the targeting of viral genes. Indeed, several KSHV-encoded miRNAs have been shown to target cellular genes, which results in enhanced latency (Lei et al., 2010; Liang et al., 2011; Lu et al., 2010) (reviewed in (Grundhoff and Sullivan, 2011)) such as demonstrated with a deletion mutant of most of the viral miRNAs for which viral lytic replication was significantly enhanced as a result of reduced NF-#B activity (Lei et al., 2010), and similar to some viral proteins expressed during latency, viral miRNAs also modulate the immune response through the targeting of cellular transcripts.

and ULBP3 could also be involved in escaping detection by the adaptive immune system. In addition, other targets involved in the recognition of infected cells by the innate immune system, such as critical factors of toll-like receptor (TLR) signaling (IRAK1 and MYD88), or in the attraction of specialised immune cells implied in the control of viral infection, such as chemokines (CXCL-16, RANTES and CXCL-11), have been described to be repressed by viral miRNAs expressed by different herpesviruses (Table 5).

In addition to regulating immune response, viral miRNAs have also been implicated in the regulation of cell death and apoptosis. This aspect will be further developed in the Results section.

Seto et al., in a study using engineered EBV miRNA-mutants, have analysed the phenotypic changes observed upon primary B cells infection, in comparison with WT EBV. They could show that the miRNAs encoded by EBV’s BHRF1 locus prevented spontaneous apoptosis induced by EBV infection, but also that these miRNAs are implicated in a strong induction of B-cell proliferation during the early phase of infection (Seto et al., 2010). Notably, this study was the first to definitely show that EBV miRNAs contribute to its transforming capacity, and was soon after followed by an independent study by Feederle et al., who confirmed the previous results. Indeed the latter showed that an EBV mutant lacking the 3 BHRF1 miRNAs had its B-cell transforming capacity reduced by more than 20-folds and that infected cells with the mutant virus displayed slower growth, exhibiting a 2-fold reduction in the percentage of cells entering into the S phase. In addition, they observed a stronger expression of EBV’s latent genes and proteins in the mutant virus-infected cells. These observations lead them to suggest that this miRNA cluster can at the same time increase the viral overall load in the infected host (i.e. via an increased proliferation of the infected cells), and reduce the viral antigenic load, two important features for the life-long persistency of latently infected cell into the host (Feederle et al., 2011).

The last category of genes which undergoes repression by virus-encoded miRNAs thus comprise genes involved in cell cycle regulation/oncogenesis, which deregulation is beneficial for the viral lifecycle and that can also have a role in the viral pathogenesis. One of earliest examples is the thrombospondin 1 (THBS1) gene targeting by four of the twelve miRNAs encoded by KSHV, and whose expression had previously been reported to be downregulated in KS lesions. THBS1 is a matricellular protein involved in cell-to-cell adhesion and cell-matrix adhesion (a property that is typically lost in metastasic tumourous cells), and possesses anti-proliferative and anti-angiogenic activity. Its targeting by KSHV

angiogenesis observed in KS lesions. Additionally, it was also described as a strong stimulator of monocytes recruitment to vascular injury and regulator of T-cell migration through extracellular matrix. Its repression is thus of particular interest for the virus, as it may also participate in immune evasion of KSHV-infected cells (Samols et al., 2007). KSHV miRNAs have also been shown to regulate other genes with a function in cell proliferation, such as p21 targeting by miR-K12-1, thus preventing p53-mediated cell cycle arrest in PEL cells (Gottwein and Cullen, 2010), and SMAD5 (similar to mothers against decapentaplegic 5) repression by miR-K12-11, which de-sensitise PEL cells to the cytostatic effect of transforming growth factor beta (TGF-") signaling, thus reinforcing the repercussions of the epigenetic silencing of TGF-" type II receptor mediated by viral LANA (Liu et al., 2012).

Grey et al., using a RISC-IP approach identified many genes involved in cell cycle and tumour progression as being targeted by HCMV miR-US25-1, suggesting a viral-specific function for the miRNA in this pathway. Interestingly, the binding of the miRNA to these gene transcripts occurs in their 5'UTRs, and mediates efficient repression. In addition, they could show that deletion of miR-US25-1 led to an overexpression of G1/S-specific CycE2 (CCNE2). HCMV induces the expression of CCNE1 and CCNE2 upon infection probably to drive resting G0 cells into the G1/S phase, and into which the virus blocks them to create a cellular environment conducive for DNA replication. As overexpression of CycE has been linked to an increase in sensitivity to apoptosis, the virus might thus negatively regulate CCNE2 to generate a fine balance in the protein induction. Following de novo infection, when comparing the WT and mutant virus during time course experiments, the authors observed that miR-US25-1 exerted its repression on CCNE2 only 48h post-infection. This could be linked to the levels of the miRNA, which increase during the progression of the viral infection, but these observations, in addition of an unimpaired viral replication of the mutant virus, thus challenges the hypothesis of an involvement of this regulation in the lytic replicative cycle of virus. Alternatively, it was then proposed that CCNE2 repression, in association of the targeting of genes involved in cell differentiation, could play a role during latency in the aim to manipulate the production of cells generated by the latently infected haematopoietic stem cells to favour certain cell types such as monocytes and macrophages (Grey et al., 2010). Finally as a last example, the recently identified retroviral orthologue of miR-29a, BLV miR-B4, was shown, as its cellular counterpart, to target the tumour suppressor HMG-box transcription factor 1 (HBP1), which is characterised as a cell cycle inhibitor, and whose inhibition could be related to the oncogenic nature of this virus (Kincaid et al., 2012). Modulation by viral miRNAs of the expression of genes involved in cell cycle

arrest and of genes which when repressed lead to cellular proliferation, in addition to benefit to the viral lifecycle, also represents an important advantage for the maintenance and spread of the virus into the host during latency. Indeed, as the latent viral genomes are only passively replicated upon cellular division of the infected cells, the regulation of these genes thus also leads to an increase in the total number of cells bearing the viral genome in the host, which thereby represents a greater virus reservoir. This can thus result in the production of higher virion titers upon lytic reactivation, for a more efficient dissemination into the environment and transmission to new hosts.

Cellular Pathway

Target Function Virus (miRNA) References

Cell-mediated immunity MICB Host stress-induced NKG2D-

ligands

HCMV (UL-112-1), EBV (BART2- 5p), KSHV (K12-7)

(Nachmani et al., 2009; Stern- Ginossar et al., 2007)

ULBP3 BKV (B1-3p), JCV (J1-3p) (Bauman et al., 2011)

CXCL-16 Chemokines MCMV (M23-2) (Dölken et al., 2010b)

RANTES HCMV (UL-148D) (Kim et al., 2012)

CXCL-11 EBV (BHRF1-3) (Xia et al., 2008)

IRAK1, MYD88 TLR Signaling KSHV (K12-1, K12-9) KSHV (K12-5, K12-11)

(Abend et al., 2012)

Cell cycle regulation, oncogenesis

G1/S Cyclin E2 Cyclin HCMV (US25-1) (Grey et al., 2010)

p21 Cell cycle inhibitor KSHV (K12-1) (Gottwein and Cullen, 2010)

SMAD5 Mediator in TGF-" signaling KSHV (K12-11) (Liu et al., 2012) THBS1 Tumour suppressor and anti-

angiogenic factor

KSHV (K12-1, -3-3p, -6-3p, -11) (Samols et al., 2007)

HBP1 Tumour suppressor BLV (B4) (Kincaid et al., 2012)

Cell survival, apoptosis

BACH1 Regulator of transcription KSHV (K12-11) (Gottwein et al., 2007; Skalsky et al., 2007)

BCL6 EBV (BART3, 9, 17-5p) (Martín-Pérez et al., 2012)

TWEAKR TNF weak inducer of apoptosis receptor

KSHV (K12-10a) (Abend et al., 2010)

PUMA Pro-apoptotic factors EBV (BART5) (Choy et al., 2008)

SMAD2 MDV (M3) (Xu et al., 2011)

BclAF1 HCMV (UL112-1) KSHV (K12-5,

-9, -10a, -10b) EBV (BART17-5p)

(Lee et al., 2012; Riley et al., 2012; Ziegelbauer et al., 2009)

Bim EBV (BART Cluster I & II) (Marquitz et al., 2011)

Table 6: Cellular pathways targeted by virus-encoded miRNAs. Adapted from (Tuddenham and Pfeffer, 2013).

Such studies, that have allowed assigning to the currently known viral miRNAs some of their targets and thus that have shed light on viral miRNA functions, have been highly informative in deciphering virus-associated pathologies, particularly cancer. Indeed, miRNA targeting of

innate and adaptive immune response (e.g. that leads to clearance of abnormal cells such as infected cells, but also cancerous cells), is of particular interest when considering the oncogenic viruses encoding miRNAs, such as MDV, BLV, EBV, and KSHV, and is a clear indicator that viral miRNAs actively participate in the onset of tumourigenesis. Nonetheless, the in vivo relevance of such targeting remains to be determined to ultimately confirm the potential involvement of virus-encoded miRNAs in oncogenesis.