Human Herpesvirus-Encoded MicroRNA in Host-Pathogen Interaction

Abstract

Human herpesviruses (HHV) are ubiquitous, linear dsDNA viruses that establish lifelong latency, disrupted by sporadic reactivation. HHV have evolved diverse ingenious mechanisms to evade robust host defenses. Incorporation of unique stem loop sequences that generate v-miRs (v-miRs) exemplifies one such evolutionary adaptation. These noncoding RNAs can control cellular and viral transcriptomes highlighting their significant ability in shaping host-HHV interactions. We summarize recent developments in functional characterization of HHV-encoded miRNAs in shaping the outcome of host-pathogen interaction. V-miRs hijack exosomal routes to gain access into non-infected cells and disrupt antiviral pathways. We propose to exploit tissue and systemic levels of v-miRs as diagnostic and prognostic markers for cancers and immune-mediated diseases. This review delineates the mechanistic role of v-miRs in facilitating viral persistence and tropism by targeting genes associated with cellular (apoptosis, angiogenesis, cell migration, etc.) and viral life cycle (latency, lytic and reactivation). Non-immunogenic dissemination of v-miRs through exosomes confer added advantage to HHV in incessant modulation of host microenvironment. Burgeoning evidences indicate plausible association of v-miRs in various immune-mediated diseases (nasopharyngeal carcinoma, neurological disorders, periodontal diseases, etc.) and herpesvirus-related malignancies indicating their broad-spectrum impact on host cellular pathways. Exploiting v-miRs as reliable diagnostic, prognostic and therapeutic targets will advance the promising outcomes of preclinical discoveries to bedside application.

Graphical Abstract

1. Introduction

Viruses are acellular, submicroscopic, nucleic acid-containing infectious agents that replicate inside host cells including bacteria, plants, and animals. With simple genetic composition, these widespread obligate parasites rely heavily on host protein machinery to facilitate their replication and spread. Viruses have coevolved to adapt inside host species and constantly search for mechanisms that support persistence and spread. With a relatively small genome, strategies that require minimal genetic landscape and confer multifunctional benefits are evolutionarily favorable. In this regard, identification of viral microRNAs (v-miRs), which exhibit features similar to cellular miRNAs are recognized as new virulence factors and have refined our understanding of host-virus interaction.

v-miRs are single-stranded, ~21–24 nucleotides long, noncoding RNAs of viral origin, in particular DNA viruses, that regulate expression of host and viral genes. The v-miRs are processed by host protein machinery that generate cellular miRNAs (Bartel, 2004; Kim, 2015; Grundhoff and Sullivan, 2011). These enigmatic noncoding RNAs have attracted significant attention in shaping the fate of virus interaction with host during disease progression and recent studies have proposed their diagnostic and prognostic value in various pathological conditions including cancers (Grundhoff and Sullivan, 2011; Grey, 2015; Naqvi, 2020). Current miRNA database (miRBase; http://www.mirbase.org/cgi-bin/browse.pl) lists ~530 known v-miRs. These are encoded by herpesviruses, retroviruses, adenoviruses, polyomaviruses, etc. Among these, approximately 81% are of herpesvirus origin where human herpesvirus (HHV) represent ~35% of the known v-miRs suggesting their unique biological requirement by these viruses (Table 1).

Table 1.

Number of precursor and mature miRNAs encoded by human herpesviruses.

Virus Subfamily	Virus Species	Pre-miR	Mature miR
Alpha-herpesviridae	Herpes Simplex Virus 1	18	27
	Herpes Simplex Virus 2	15	26
Beta-herpesviridae	Human Cytomegalovirus	15	26
	Human herpesvirus 6	4	8
Gamma-herpesviridae	Epstein Barr Virus	25	44
	Kaposi’s sarcoma-associated Herpesvirus	13	25

The members of Herpesviridae family are enveloped, double-stranded DNA viruses of 120–240 kbs that infect animals and humans. The greek word ‘herpein’, meaning “to creep” refers to a capstone of their pathogenesis: their ability to establish latent, recurring infections, while the viral genome circulates and persists. Herpesviruses establish lifelong infection, and occasionally reactivate from latency to cause recurrent infections (Naqvi, 2020). Viral persistence inside host relies on evasion of host antiviral responses, primarily suppression of antigen presentation to CD8+ cytotoxic T cells and Natural Killer (NK) cells (Roizman and Sears, 1993). The Herpesviridae family is subdivided into three subfamilies: alphaherpesvirinae, betaherpesvirinae, and gammaherpesvirinae (Table 1). Herpes simplex virus 1 (HSV-1), HSV-2 and Varicella zoster virus (VZV) are members of alphaherpesvirinae, Human Cytomegalovirus (HCMV), HHV-6A, HHV6B and HHV-7 are members of betaherpesvirinae, while gammaherpesvirinae consists of Epstein-Barr Virus (EBV) and Kaposi’s Sarcoma-Associated Herpesvirus (KSHV). Infection with HSV-1 and −2 results in oral or genital herpes lesions, and virus can manifest as encephalitis or meningitis in some subjects (Roizman and Sears, 1993; Tang et al., 2011; Umbach et a., 2008). HCMV infection is a leading viral cause of birth defects and is attributed to life-threatening disease in immunocompromised/immunosuppressed individuals. Similarly, HHV-6 reactivation can result in severe complications in immunocompromised individuals (Tuddenham et al., 2012). Latent infection of gammaherpesviruses may lead to carcinogenesis (Boshoff and Weiss, 2002). EBV is the etiological factor in the development of Hodgkin’s lymphoma, Burkitt’s lymphoma, nasopharyngeal carcinoma (NPC) and gastric cancer (Maeda et al., 2009). KSHV infection is associated with Kaposi’s sarcoma (a cancer commonly observed in AIDS patients), some forms of Multicentric Castleman’s Disease (MCD) and Primary Effusion Lymphoma (PEL) (Wen and Damania et al., 2010; Cesarman et al., 1995). Unlike the gammaherpesvirus, the alpha- and beta-herpesvirus subfamilies can attribute pathogenesis to viral replication and consequent perturbation of immune responses, resulting in the destruction of tissues.

Multiple v-miRs have been identified and characterized in HSV-1 (27), HSV-2 (24), EBV (44), HCMV (26), KSHV (25) and HHV-6B (8) (Table 1). However, to date, no v-miRs are reported for VZV, HHV-6A and HHV-7. The first v-miR was discovered by Pfeffer and colleagues in 2004 from an EBV-infected cell line which is associated with several human tumors such as Burkitt’s lymphoma, Hodgkin’s disease and nasopharyngeal carcinoma (Pfeffer et al., 2004). v-miR-mediated perturbation of host cellular transcriptome may causally contribute to tumorigenic processes (Wen and Damania, 2010). Although betaherpesvirus are not known to be tumorigenic, HCMV-encoded v-miRs can enhance virulence and disease manifestation. HHV-6B, another member of betaherperviruses, encodes for eight mature v-miRs with only limited characterization of biological functions (Tuddenham et al., 2012). A deeper knowledge of the cellular and viral targets of v-miRs is essential to understand the potential diagnostic and therapeutic value in the context of human disease progression.

Here, we summarize the mechanistic role of HHV-encoded v-miRs in viral tropism, life cycle, establishment and pathogenesis by subverting various cellular pathways. We will also highlight the role of exosomes in v-miRs dissemination and diagnostic/prognostic potential of these noncoding RNAs to monitor clinical manifestation and treatment of HHV infections and/or cancers.

2. V-miR biogenesis

Viruses hijack host cellular machinery to perform most of their functions. Similarly, v-miR biogenesis is dependent on host proteins that generate cellular miRNA (Grundhoff et al., 2011, Skalsky et al., 2012; Umbach and Cullen, 2010). To date, there is no report of viral protein required for v-miR biogenesis (Grundhoff and Sullivan, 2011; Cullen, 2013). Transcription of primary v-miR transcripts (pri-miRNAs) is predominantly RNA polymerase II (Pol II) driven; however, some RNA pol III, tRNase Z and other non-canonical pathways are also demonstrated to generate viral pri-miRs (Pfeffer, 2004, Diebel et al., 2010; Bogerd et al., 2010). Pri-miRNAs may contain one or several hairpin loop structures called precursor-miRNAs (pre-miRs) (Fig. 1) (Bartel, 2004, Kim, 2015, Grundhoff and Sullivan, 2011). In this article, we will focus on canonical mode of v-miRs biogenesis.

Fig. 1. Schematic illustrating miRNA biogenesis pathway.

microRNA biogenesis commences with the transcription of primary miRNA (pri-miRNA) transcript by RNA pol II. Pri-miRNA are processed in the nucleus by the DGCR8 and Drosha to generate precursor miRNA (pre-miRNA) which is subsequently exported to the cytoplasm by Exportin 5. In the cytoplasm, pre-miRNAs are processed by Dicer and Ago2 to generate mature, single-stranded miRNAs incorporated into the ribonucleoprotein complex miRNA-induced silencing complex (RISC). The miRNAs regulate post-transcriptional processing of cognate mRNA by base pairing, which leads to translational repression or mRNA degradation.

The dsRNA stem-loop structure within pri-miRNA are recognized by the nuclear protein Drosha, an RNase III-type endonuclease (Bartel, 2004, Kim, 2015, Grundhoff and Sullivan, 2011). In conjunction with its protein cofactor DGCR8 (also known as Pasha), Drosha forms Microprocessor complex that recognizes and cleave stem–loop with 2 nucleotide overhangs at the 3′ terminal. These precursor miRNA (pre-miRNA) are ~65–70 nucleotides in length. Nuclear pore complex protein Exportin 5 (EXP5), which forms a transport complex with GTP-binding protein RAN•GTP and a pre-miRNA, mediates nucleus to cytoplasm transfer of pre-miRNA (Bohnsack et al., 2004). The release of pre-miRNA into cytosol occurs following GTP hydrolysis that causes complex disassembly. Upon export into the cytoplasm, Dicer (RNase III-type endonuclease of about 200 kDa) cleaves pre-miRNA near its terminal loop to generate RNA duplex of ~18–23 nts which interacts with Argonaute (Ago) (Grundhoff and Sullivan, 2011). Only one of the miRNA strand is selectively loaded into an Ago-associated miRNA induced silencing complex (miRISC), while other strand is ejected (Ketting et al., 2001). The mature miRNA guides the miRISC machinery by interaction with the complementary sequences present on the 3′ Untranslated Regions (UTRs) of the cognate transcript; however recent high throughput sequencing have identifed non-canonical mode of v-miR interaction with transcripts that involved coding region and 5’UTR as well (Grey et al., 2010; Gay et al., 2018). The proteins associated with miRISC mediate translational repression of target mRNA or augment mRNA destabilization and decay; both of which reduce translational output of the mRNA (Hammond et al., 2001).

3. Cellular Reservoirs of V-miR and Exosome-Mediated Dissemination

Depending on the virus subfamily, cellular tropism of HHV members vary. For instance, HSV-1 (alphaherpesvirus) target epithelial cells for replication and establish latency in sensory ganglia (Heldwein and Krummenacher et al., 2008). HCMV, a betaherpesvirus, exhibit tropism for epithelial cells as well as monocytes, T cells and NK cells (Schelhaas et al., 2003). HCMV latency is established in monocyte precursors and kidney epithelial cells. In vivo KSHV (gammaherpesvirus) detection is reported in multiple cell types inluding lymphoid cells, monocytes, keratinocytes and oral epithelial cells (Chakraborty et al., 2012; Duus et al., 2004; Chandriani and Ganem, 2007). As a common HHV target cells, epithelial cells and monocytes serve as viral reservoirs expressing v-miRs. In addition, dissemination of v-miRs beyond infected cells can occur via indirect routes.

Exosomes are ubiquitously secreted membrane limited nano-vesicles (20–100 nm in diameter) that contain RNA (including mRNA, lncRNA and miRNAs), proteins, DNA. Exosome from donor cells can fuse and deliver its content to various cell types following uptake by receptor-mediated interaction or through membrane fusion (Han et al., 2016; Pegtela et al., 2010; Gibbings et al., 2009; Valadi et al., 2007). Exosomal pathway provide two-fold benefits to herpesvirus. Firstly, it can spread viral biomolecules (including miRNAs) without activation of antiviral responses. Secondly, functional delivery of exosomal contents allows v-miRs to modulate bystander cell functions, including immune responses, without the need to infect cells. Burgeoning evidences have shown v-miR presence in the exosomes (in vivo, ex vivo or in vitro collected) indicating thier putative role in pathobiology (Han et al., 2016; Chugh et al., 2013; Haneklaus et al., 2012; Naqvi et al., 2018; Kalamvoki et al., 2014). Exosomes isolated from EBV infected B cells or transiently transfected macrophages showed miR-BART-15 (Haneklaus et al., 2012). Interestingly, seed sequence (2–8 nucleotides from the 5′ of miRNA) of miR-BART-15 exhibit homology with cellular miR-223 and post-transcriptionally regulate same target NLRP3, a cytoplasmic pathogenic motif sensor that forms and activates NLRP3 inflammasome. miR-BART-15 in B cell-derived exosomes are functionally active and their delivery into recipient macrophages suppresses NLRP3 and inflammasome formation (Haneklaus et al., 2012). Incorporation of miRNA sequences that are homologs of celluar miRNA and target mutually inclusive set of gene(s) allows virus to rapidly acquire v-miRs with functional and relevant targets to benefit virus. Their dissemination via exosomal route is additionally advantageous to modulate host microenvironment.

KSHV encodes multipe v-miRs that may play critical role in oncogenesis (Cai et al., 2005; Pfeffer et al., 2005). Studies from our lab have shown time-dependent exosomal release of miR-K12-3 in KSHV infected BC-3 cells suggesting that v-miR are targeted to exosomal route and their enrichment in exosomes correlate with viral infection (Naqvi et al., 2018). To evaluate the functional impact of exosomal v-miRs, Chugh et al. collected exosomes from the plasma of control and KSHV infected PEL subjects to assess migration of recipient endothelial cells (HUVEC). Compared to exosomes isolated from control plasma, HUVECs incubated with PEL patients-derived exosomes showed significantly enhanced migration (Chugh et al., 2013). Thus, v-miRs can act as paracrine signaling molecules of viral origin that can perturb cellular function and immune responses of recipient cells. v-miRs, by virtue of altering host transcriptome, can actively and progressively contribute in shaping tissue or cellular microenvironment. Incorporation of multifaceted, non-immunogenic v-miRs by HHV (and other viruses) provide robust mechanisms to perturb cellular pathways in diverse non-permissive cells with a purpose to create a conducive environment for the pathogen.

4. Viral microRNAs shape host-pathogen interaction

Most of the HHV members encode multiple miRNAs suggesting their multifaceted biological functions in shaping host-virus interaction. Interestingly, a comparative in silico analysis from our lab showed that some herpesviruses (EBV, KSHV, HSV-1) have one miRNAs per 3–4 viral transcripts, while in case of human it ranges in the order of one miRNA per 30 transcripts (Naqvi, 2020). Indeed, various v-miRs in different HHV directly target viral transcritps to regulate viral life cycle (Grundhoff and Sullivan, 2011, Grey, 2015; Goodrum et al., 2012). In addition to viral transcripts, v-miRs are evolved to suppress unique subset of cellular trancripts that facilitate viral tropism and persistence. Some v-miR sequences are orthologous to cellular miRNAs to target mutually overlapping set of transcripts (Grundhoff and Sullivan, 2011; Naqvi, 2020). Certain v-miRs are shown to target similar set of genes to reinforce potent suppression of cellular pathways. For instance, activation of apoptotic pathway in virus-infected cell is a highly effective antiviral strategy by host; however, multiple KSHV v-miRs can inhibit this process by targeting caspase 3 (Grundhoff and Sullivan, 2011, Grey, 2015; Goodrum et al., 2012). Thus, v-miRs can regulate both host and viral targets and act as key factors in determining host-pathogen interaction. In the following sections we will thoroughly describe key cellular pathways preferentially targeted by v-miRs and also provide evidences linking their role in deciding the viral life cycle switch.

4.1. V-miR Regulation of Host Cell Cycle and Differentiation

Essentially, it is in the best interest of the virus to keep the host cells alive for a long enough period of time to complete its life cycle. As herpesviruses establish latent infections, the time period in which the host cell is to remain alive is significantly increased (Skalsky and Cullen, 2010). Mulitple v-miRs have been demonstrated to target host genes that are involved in cellular life cycle, proliferation, and survival (Table 2).

Table 2.

List of validated targets (of vmiRs involved in the regulation of Host Cell Cycle and Differentiation

Host Cell Cycle and Differentiation
V-miR	Target	Functional impact	References
HSV1 miR-H4-5p	CDKN2A	Promotes cell proliferation via targeting the cellular cyclin-dependent kinase inhibitor 2A	Zhao et al. (2015)
HSV1 miR-H27	KLHL24	Inhibits transcriptional activation of IE and E genes	Zhao et al. (2015; Wu et al. (2013)
HCMV miR-US24-2-3p	eIF4A1	Promotes latency and control the host cell cycle	Wu et al. (2013)
HCMV miR-US25-1	CCNE2/CD147,BRCC3, E1D1, MAPRE2, H3F3B	Inhibit host cell cycle	Grey et al. (2010); Grey et al. (2007); Wang et al. (2019)
KSV miR-K12-6-3p, KSV miR-K12-1, KSV miR-K12-11, KSV miR-K12-3-3p	THBS1	Inhibition of cell growth and angiogenesis via the activation of TGFβ	Samols et al. (2007)
KSHV miR-K6, KSHV miR-K11	MAF	Endothelial cell reprogramming	Skalsky and Cullen (2010); Hansen et al. (2010)
KSHV miR-K12-1	p21Cip1	Promotes cell cycle progression via targeting the cellular cyclin-dependent kinase inhibitor p21	Gottwein and Cullen (2010)
EBV miR-BART3-5p	DICE1	Cell transformation and proliferation	Lei et al. (2013)
EBV miR-BART7	APC	Cell transformation and proliferation	Wong et al. (2012)
EBV miR-BART19-3p	WIF1/APC	Cell transformation and proliferation	Wong et al. (2012)
EBV miR-BART22	NDRG1	Cell transformation and proliferation	Kanda et al. (2015)

KSHV miRNAs (miR-K12-6-3p, -K12-1, -K12-11 and -K12-3-3p) have been shown to target Thrombospondin 1 (THBS1), which functions in inhibition of cell growth and angiogenesis via the activation of TGFβ (Table 2). THBS1 expression is reduced at both the protein and mRNA levels in cells expressing KSHV miRNAs. This has been proposed as a mechanism to promote cell survival (Samols et al., 2007). Further, KSHV miRNAs can perturb host cell differentiation. Specifically, KSHV-miR-K11 and KSHV-miR-K6 target MAF, a bZIP transcription factor involved in the terminal differentiation of numerous cell types, including endothelial cells. v-miR-mediated inhibition of MAF results in the transcriptional reprogramming of lymphatic endothelial cells. Regulation of differentiation in infected cells has been proposed to play a role in oncogenesis (Skalsky and Cullen, 2010; Hansen et al., 2010). There is evidence that KSHV v-miR also play a role in the promotion of cell cycle progression and cell growth via targeting specific cellular proteins involved in these key pathways. For example, KSHV-miR-K12-1 promotes cell cycle progression via targeting the cellular cyclin-dependent kinase inhibitor, p21 (Gottwein and Cullen, 2010). Furthermore, this v-miR was shown to promote cell survival by activating the NFκB pathway via interference with IκBα (Lei et al., 2010).

HCMV miRNAs have been predicted to target cellular pathways related to cell cycle control. Influencing the cell cycle may allow HCMV to regulate latent and lytic cycles, thereby allowing the virus to establish a lifelong infection. For instance, HCMV-miR-US25-1 is shown to regulate cell cycle (Table 2). This v-miR controls the expression of large repertoire of cellular targets with known functions in cell cycle including cyclin E2 (CCNE2), microtubule-associated proteins, collagenase stimulatory factor (CD147), BRCA1/BRCA2-containing complex, subunit 3 (BRCC3), EP300 interacting factor of differentiation (E1D1), RP/EB family, member 2 (MAPRE2), and histone proteins (H3F3B) (Grey et al., 2007; Grey et a., 2010; Naqvi, 2020). Further involvement of HCMV v-miR in the host cell cycle is evident via the targeting of eukaryotic initiation factor 4A1 (eIF4A1), an essential RNA helicase required for the translational initiation. HCMV-miR-US24-2-3p modulates expression levels of eIF4A1, and thereby can inhibit translation. This may serve as a mechanism for HCMV to promote latency and control the host cell cycle (Wang et al., 2019).

HSV1-encoded miR-H4-5p is yet another example of v-miR promoting the survival and proliferation of latently infected cells (Table 2). This v-miR directly target a cell cycle inhibitor, cyclin-dependent kinase inhibitor 2A (CDKN2A, p16), resulting in cell proliferation and invasion of nerve cells (Zhao et al., 2015). The cellular transcriptional repressor Kelch-like (KLHL24) is targeted by HSV1-miR-H27 to regulate not only the expression of viral immediate early and early genes, but also facilitate inmune evasion, viral replication and proliferation (Wu et al., 2013).

Recent studies have shown that EBV v-miRs play important roles during cell transformation and proliferation (Table 2). EBV encoded miR-BART3-5p targets DICE1 tumor suppressor to promote cellular growth and transformation of host cells (Lei et al., 2013; Wong et al., 2012). Other tumor-suppressor genes with similar roles in host cell proliferation include WIF1 (WNT inhibitory factor 1), targeted by ebv-miR-BART19-3p, and APC (adenomatous polyposis coli) targeted by ebv-miR-BART7 and ebv-miR-BART19-3p. Another tumor-suppressor N-myc downstream regulated gene 1 (NDRG1) is targeted by ebv-miR-BART22 causing the inhibition of differentiation and promotes metastasis of nasopharyngeal carcinoma (NPC) cells (Kanda et al., 2015).

4.2. V-miR Regulation of Viral Life Cycle

The herpesvirus lifecycle consists of both latent and lytic phases. Interestingly, it has been found that distinct v-miR expression patterns exist during these phases. In a latent infection, there is a distinct gene expression profile, which allows the virus to survive and evade the host immune system. Various v-miRs are among these latently expressed genes, and it is believed that they play a role in latency (Sorel and Dewals, 2016). Furthermore, it has been observed that miRNA genes are grouped in portions of the genome associated with latent gene expression (Grundhoff and Sullivan, 2011). V-miRs have been reported to downregulate immediate early (IE) genes and suppress lytic induction. Various v-miRs are antisense to viral transcritps thereby silencing them. For instance, HSV-1 encoded miR-H2, miR-H6 target viral ICP0 and ICP4 RNA, respectively to promote latency by preventing expression of IE or E genes (Umbach et al., 2008; Tang et al., 2008; Tang et al., 2009) (Table 3). Two different HSV1 miRs viz., miR-H3 and miR-H4 downregulate expression of lytic gene ICP34.5 (Umbach et al., 2008; Tang et al., 20080.

Table 3. List of validated vmiR targets (viral and cellular) involved in the regulation of viral lytic and latency cycle.

(c) Cellular gene; (v) Viral gene.

Viral life cycle
V-miR	Target	Functional impact	References
HSV1 miR-H2	ICP0 (v)	Promotes latency by silencing activation of IE or E genes	Umbach et al. (2008); Tang et al. (2008)
HSV1 miR-H3, −4	ICP34.5 (v)	Inhibition of lytic gene expression	Umbach et al. (2008); Tang et al. (2008)
HSV1 miR-H6	ICP4 (v)	Prevent expression of IE or E genes	Umbach et al. (2008); Tang et al. (2009)
HSV1 miR-H27	KLHL24 (c)	Inhibits transcriptional activation of IE and E genes	Wu et al. (2013)
KSHV miR-K12-1-5p	IκBα (c)	Reduces lytic reactivation by modulating NFκB	Lei et al. (2010)
KSHV miR-K12-3	NFIB (c)	Reduces lytic reactivation by modulating NFIB	Lu et al. (2010)
KSHV miR-K12-4	RBL2 (c)	Reduces the expression of viral RTA by modulating its methylation	Lu et al. (2010)
KSHV miR-K12-7; KSHV miR-K12-9	ORF50 (v)	Reduces the expression of RTA by vmiR targeted OFR50, causing latency	Godshalk et al. (2008)
EBV miR-BHRF1-1	RNF4	Promote viral production	Du et al. (2015)
EBV miR-BART6	DICER (c)	Modulates lytic/latent phase infection	Godshalk et al. (2008); Iiaza et al. (2010); Mansouri et al. (2014)

Viruses are solely or predominantly dependent on host machinery for its replication. Cellular transcriptional repressor Kelch-like 24, KLHL24, an important transcription factor that activates viral lytic genes (IE and E), is targeted by HSV1 miR-H27. This inhibits transcriptional activation of lytic phase associated genes (Wu et al., 2013). On the other hand, hsv1-miR-H5 and hsv1-miR-H7 exhibit similar pattern of expression, implicating thier role in HSV-1 latency (Du et al., 2015). A large number of HSV-1 v-miRs with higher expression were detected upon reactivation. Among these is hsv1-miR-H27, supporting its role in efficient replication and proliferation. Besides hsv1-miR-H27, hsv1-miR-H15, hsv1-miR-H17, hsv1-miR-H18, and hsv1-miR-H26 levels were also significantly increased suggesting their involvement in HSV-1 reactivation (Du et al., 2015). Unlike other HHV members, significant homology in miRNA sequences between HSV-2 and HSV-1 have been reported (Du et al., 2015; Sun and Li, 2012). Multiple HSV-2 miRNAs are homologous with HSV-1 miRNAs and have similar functions. For instance, hsv2-miR-H2 and hsv2-miR-H3/4 which target ICP0 and ICP34.5, respectively, perform similar functions in HSV-1 as well. HSV-2 and HSV-1 v-miRs contribute to latency and immune evasion in an almost identical manner (Umbach et a., 2008; Tang et al., 2008; Jurak et al., 2010; Tang et al., 2011). These studies highlight the significance of v-miR sequence conservation and indicate that specific v-miRs are under positive selection pressure to maintain their functions, a feature commonly observed in animals and plant miRNAs.

KSHV establishes a latent infection in various cell types, namely monocytes, dendritic cells, B-lymphocytes, and endothelial cells, contributing to immune evasion and modulation. Kshv-miR-K12-1-5p regulates a viral lytic-latency switch as demonstrated by Lei at al. (2010) where they show v-miR-mediated suppression of IκBα, an inhibitor of NFκB activity (Table 3). Downmodulation of NFκB reduced lytic reactivation and maintained latency in KSHV. Kshv-miR-K12-3 targets the nuclear factor I/B (NFIB) known to act as a replication and transcription (RTA) activator (Lu et al., 2010). The RTA protein is essential for initiation of lytic replication of KSHV therefore, kshv-miR-K12-3 and NFIB binding promotes viral latency. Additionally, kshv-miR-K12-4 repress the retinoblastoma-like protein 2 (RBL2). This protein indirectly maintains the methylation of viral gene (RTA) promoter and consequently, reduces its expression (Hansen et al., 2010). Kshv-miR-K12-7 and kshv-miR-K12-9 target the immediate-early gene ORF50, decreasing the expression of the RTA encoded from this viral gene [Lei et al., 2012]. Similar to other herpesviruses, EBV establishes a lifelong latent infection. Ebv-miR-BART6, plays an important role downregulating Dicer (Table 3), contributing to EBV latency probably by decreasing cellular miRNA levels that can reactivate EBV (Godshalk et al., 2008; Iizasa et al., Mansouri et al., 2014). In addition, EBV miR-BHRF1-1 not only inhibit apoptosis by targeting p53, but in NPC, its absence promotes the maintenance of EBV latent infection and its reactivation could enhance viral replication by targeting RNF4 (Du et al., 2015).

4.3. V-miR Regulation of Apoptosis

Apoptosis is a highly regulated process that occurs in multicellular organisms via one of two main pathways: the intrinsic or the extrinsic pathway. In the intrinsic pathway, apoptosis occurs when a cell senses stress. In contrast, the extrinsic pathway occurs when a cell receives signals from other cells that encourage cell death. Apoptosis is essential for self-regulation and functions as a barrier to cancer (Suffert et al., 2011).

Several studies have demonstrated that v-miRs may play a role in alteration of the host’s normal apoptotic process. Particularly, certain miRNAs of EBV and KSHV have demonstrated an ability to inhibit cell apoptosis through reduction of the expression of pro-apoptotic proteins (Sorel and Dewals, 2016). Multiple v-miRs of both oncogenic gammaherpesviruses have been shown to interfere with apoptosis (Suffert et al., 2011; Banerjee et al., 2016). The EBV encoded v-miRs miR-BHRF1-1, miR-BHRF1-2, miR-BHRF1-3, miR-BART1 and miR-BART2 were discovered in lymphoma cell lines by Pfeffer et al. (2004). miR-BHFR1-1,1-2 and 1–3 impact the regulation of apoptosis. For instance, miR-BHFR1-1 inhibits the apoptosis by targeting of p53 (Table 4) (Suffert et al., 2011). A reduction of apoptosis and potential viral replication is a result of elevated expression of EBV miR-BHFR1-3 during the early stage of EBV infection leading an inhibition of the expression of phosphatase and tensin homolog deleted on chromosome ten (PTEN) (Fig. 2) (Bernhardt et al., 2016). However, during the late phase of infection, miR-BHRF1-2 also inhibits apoptosis by targeting of tumor suppressor PRDM1/Blimp1 and facilitates the growth and development of B-cell lymphoma (Fig. 2) (Ma et al., 2016).

Table 4.

List of validated vmiR targets involved in regulation of the Apoptosis pathway

Apoptosis
Viral miRNA	Target	Function	References
KSHV miR-K12-1; KSHV miR-K12-3; KSHV miR-K12-4	CASP3	Blockade of execution phase of apoptosis	Suffert et al. (2011); Vereide et al. (2014)
KSHV miR K12-11	C-EBPβ	Promote cell survival by reducing p53 expression	Boss et al., (2011); Abend et al. (2010)
KSHV miR-K12-10	TWEAKR	Prevent TWEAK-induced caspase activation	Abend et al. ( 2010)
EBV miR-BHRF1-1	P53	Inhibit the apoptosis by targeting of p53	Suffert et al. (2011)
EBV miR-BHRF1-2	PRDM1/BLIMP1	Inhibition of apoptosis and facilitates the tumor development by targeting PRDM1/Blimp1	Ma et al. (2016)
EBV miR-BHRF1-3	PTEN	Reduced apoptosis by targeting the tumor suppressor, PTEN.	Bernhardt et al. (2016)
EBV miR-BART1, −3, −9, −10, −11	BIM/ BCL2L11	Inhibition of pro-apoptotic signaling	Marquitz et al. (2011)
EBV miR-BART1-3p; EBV miR-BART16	CASP3	Blockade of execution phase of apoptosis	Suffert et al. (2011; Vereide et al. (2014)
EBV miR-BART3-5p	DICE1/CASZ1	Promotes the proliferation of host cells	Lei et al. (2013); Ma et al. (2016)
EBV miR-BART4-5p	BID	Reduces the levels of pro-apoptotic proteins	Ma et al. (2016)
EBV miR-BART5-5p	PUMA	Inhibition of p53 mediated cell death	Choy et al. (2008)
EBV miR-BART6-5p	OCT1	Inhibits apoptosis	Kang et al. (2015)
EBV miR-BART8	STAT1	Reduces the levels of pro-apoptotic proteins	Huang et al. (2014)
EBV miR-BART13-3p	CAPRIN2	Reduces the levels of pro-apoptotic proteins	Riley et al. (2012)
EBV miR-BART16	CREBBP/TOMM22/CASP3/SH2B3	Inhibits apoptosis	Marquitz et al. (2011); Vereide et al. (2014); Kang et al. (2015)
EBV miR-BART20-5p	BAD	Prevents sequestration of pro-apoptotic proteins leading to increased cell survival	Marquitz et al. (2011)
EBV miR-BART22	CASP3/MAP3K5/PAK2/TP53INP1	Inhibits apoptosis	Chen et al. (2017); Kang et al. (2015); Harold et al. (2016)

Fig. 2. V-miRs impair apoptotic pathway by directly targeting associated genes.

Increase in EBV v-miRs during infection process may suppress specific genes involved in apoptosis and thus interfere with the pathway, a beneficial outcome for the pathogen. For instance, EBV miR-BHFR1-1 inhibits the apoptosis by targeting of p53 and EBV miR-BHFR1-3 causes a reduction of apoptosis and induce viral replication by blocking the expression phosphatase and tensin homolog deleted on chromosome ten (PTEN). EBV-miR-BART-20 and EBV-miR-BART-16 interacts with BAX and inhibit cell death. EBV-miR-BART16, EBV-miR-BART22 and, EBV-miR-BART1-3p protect against apoptosis potentially through targeting of caspase 3.

Five different EBV encoded BART miRs (1, 3, 9, 10, 11) were demonstrated as negative regulators of BCL2 interacting mediator of cell death (BIM/ BCL2L11) (Table 4) (Marquitz et al., 2011). Interestingly, this downregulation could not be achieved by individual v-miRs indicating that synergistic miRNA activity is required. Kim et al. (2015), identified reduced expression of BAX, BCL2 antagonist of cell death, in EBV-infected AGS cells (stomach gastric adenocarcinoma cells) compared with EBV-negative AGS cells. They predicted binding sites of five different v-miRs on the 3’UTR of the BAX transcript. Using reporter silencing assays, they showed EBV BART-20, but not other v-miRs, interacts with BAX. Evidently, treatment of EBV-infected cells with the anticancer drugs 5-fluorouracil and docetaxel, did not promote cell death in the presence of ebv-BART-20 (Fig. 2). In yet another example, ebv-BART16 was shown to target TOMM22, a mitochondrial protein that serves as receptor for BAX (Marquitz et al., 2011). However, BART16 and BART1-3p protects against apoptosis potentially through targeting of caspase 3 (Fig. 2) (Suffert et al., 2011; Vereide et al., 2014). miR-BART22 also targets caspase 3 thus blocking apoptosis and is also reported to exert an anti-apoptotic effect by targeting the tumor suppressor, MAP kinase kinase kinase 5 (MAP3K5) (Chen et al., 2017). These findings suggest a critical role of v-miRs in preventing apoptosis by interacting with transcripts of various genes involved in the activation of the intrinsic pathway. Significantly, these proteins determine whether or not a cell commits to apoptosis. This is achieved by regulating the release of mitochondrial protein, cytochrome c. Increased expression of the pro-apoptotic mitochondrial proteins will shift the balance of BAX/BCL2 ratio that favors cell death. Another characterized target of EBV miRNAs is the pro-apoptotic protein, p53-upregulated modulator of apoptosis (PUMA). miR-BART5-5p has been shown to regulate PUMA. Either depletion of this v-miR or induction of PUMA expression is adequate to prompt apoptosis (Choy et al., 2008; Cai et al., 2009).

Other EBV miRs contributing to the inhibition of apoptosis are: miR-BART4-5p (BID), miR-BART8 (STAT1), and miR-BART13-3p (CAPRIN2) (Marquitz et al., 2011; Huang and Lin, 2014; Riley et al., 2012). All are involved in the reduction of pro-apoptotic protein levels such as, BID, STAT1 and CAPRIN2, respectively. mRNA-bound v-miRs were captured using advanced techiques such as photoactivable ribonucleoside enhanced crosslinking and immunoprecipitacion (PAR-CLIP) (Kang et al., 2015). Numerous studies have identified host mRNAs with anti-apoptotic functions targeted by EBV miRs including CASZ1 and DICE1 (miR-BART3-5p), OCT1 (miR-BART6-5p), SH2B3 and CREBBP (miR-BART16), and PAK2 and TP53INP1 (BART22) (Lei et al., 2013; Marquitz et al., 2011; Vereide et al., 2014; Chen et al., 2017; Kang et al., 2015; Harold et al., 2016).

Furthermore, several KSHV miRNAs (miR-K12-1, miR-K12-3, miR-K12-4) (Table 4) have been shown to inhibit apoptosis by targeting proteins that are involved in the initiation of apoptosis (Sorel and Dewals, 2016). Suffert et al. (2011), reported that cells stably expressing KSHV miRNA were protected from apoptosis. Using microarray profiling, cellular targets that were down-regulated with KSHV miRNA expression were identified. One of the targets was identified as Caspase 3 (Casp3), a critical factor for the control of apoptosis, and validated via luciferase assays, quantitative PCR, and western blotting. After identifiying the specific miRNAs involved, Suffert et al. (2011), confirmed that inhibition of these miRNAs resulted in an increased expression of endogenous Casp3, and an increase in apoptosis.

On the contrary, the transcriptional regulator of interleukin-6, c-EBPβ is targeted by miR-K12-11 and is linked to B-cell lymphoproliferative disorders. Delivery of miR-K12-11 antagomirs resulted in higher c-EBPβ transcript levels and disrupted a potential KSHV-associated lymphomagenesis by targeting c-EBPβ (Boss et al., 2011; Dong et al., 2019). Existing data further indicate that the tumor necrosis factor-like weak inducer of apoptosis receptor (TWEAKR), was the most robustly downregulated gene in the presence of KSHV miR-K12-10a (miR-K10a) in KS tumor-derived human ECs, which in turn attenuates induction of caspase activation (Abend et al., 2010).

4.4. V-miR Regulation of Cell Movement and Migration

V-miRs have been demonstrated to regulate the migration of a wide variety of cell types through direct modulation of extracellular matrix, cell to matrix adhesions, cytoskeleton rearrangement and cell signaling cascades (Hu et al., 2015; Li et al., 2016; Cai et al., 2015). As previously discussed, there is evidence that EBV and KSHV v-miRs may play a role in oncogenesis (Table 5). One proposed mechanism is described via EBV BART miRNAs. Alteration of EBV-miR-BART1-5p expression results in increased invasion and migration of NPC cells in vitro and has been demonstrated to cause tumor metastasis in vivo (Cai et al., 2015). The mechanism includes targeting PTEN and thereby activate PTEN-dependent pathways including PI3K-Akt, FAK-p130 (Cas) and Shc-MAPK/ERK1/2 signaling, driving the epithelial-mesenchymal transition (EMT). EMT is an essential biological process that polarizes epithelial cells to mesenchymal-like cells, which includes the rearrangement of the cytoskeleton and improved migratory and invasive capacity. It is considered the initial step in tumor metastasis (Cai et al., 2015; Thiery and Sleeman, 2006). Again, a characteristic event of EMT involves rearrangement of the actin cytoskeleton. It has been demonstrated that even a slight upregulation of BART1-5p may trigger actin cytoskeleton rearrangement, thereby enhancing migration, metastasis, and invasion of NPC (Fig. 3). Further, when examining the organization of actin and associated proteins, a pattern most similar to migrating mesenchymal cells was visualized (Cai et al., 2015; Mayer, 2001). This included dynamic structures of actin filaments and associated proteins, otherwise referred to as actin stress fibers (Cai et al., 2015).

Table 5.

List of validated vmiR targets involved in the regulation of migration, angiogenesis and cancer development

Migration, Angiogenesis and Cancer development
V-miR	Target	Functional impact	References
KSHV miR-K3	GRK2	Promotes endothelial cell migration as well as invasion by activating the CXCR2/AKT signaling pathway	Hu et al. (2015)
KSHV miR-K12-1	p21	Promotes oncogenesis by targeting p21, causing cell cycle progression.	Gottwein and Cullen (2010)
KSHV miR-K12-3; KSHV miR-K12-7	C-EBPβ	Promotes oncogenesis and angiogenesis by targeting C-EBPβ, causing activation of IL6 and IL10.	Qin et al. (2010); Qin et al. (2011)
KSHV miR-K12-6-3p	SH3BGR	Promotes cell migration and angiogenesis via directly targeting and downregulating SH3BGR	Li et al. (2016)
KSHV miR-K12-10	TGFBR2	Reduced TGF-β signaling promoting oncogenesis	Lei et al. (2012)
KSHV miR-K12-11	C-EBPβ, SMAD5	Promotes oncogenesis	Lei et al. (2012); Feederle et al. (2011); Qin et al. (2011)
KSHV miR-K12-6-5p; KSV miR-K12-1; KSV miR- K12-2; KSV miR- K12-11.	MEG3, ANRIL, Loc541472, CD27-AS1	Maintain latency and regulate oncogenesis	Maeda et al. (2009)
EBV miR-BHRF1-1,−2,−3	EBNA-LP	Involved in B cell lymphogenesis.	Sethuraman et al. (2017)
EBV miR-BART1-5p;	PTEN	Increased invasion and migration of NPC by targeting PTEN	Cai et al. (2015)
EBV miR-BART3-5p	DICE1	Silencing tumor suppression activity of DICE1	Wong et al. (2012)
EBV miR-BART7-3p; EBV miR-19-3p	APC	Increased cell proliferation through β-catenin signaling	Kanda et al. (2015); Motsch et al. (2012)
EBV miR-BART17-5p	LMP1	Promote cancer development	Lo et al. (2007)
EBV miR-BART19-3p	WIF1	Activation of wnt pathway	Wong et al. (2012)
EBV-miR-BART22	NDRG1	Cell transformation and proliferation	Kanda et al. (2015)

Fig. 3. Viral microRNA target cell movement and migration pathways.

EBV and KSHV v-miRs impair cell migration, actin polymerization and cytoskeleton homeostasis to cause oncogenesis. For instance, EBV-miR-BART1-5p expression results in the increased invasion and migration of nasopharyngeal carcinoma (NPC) cells by targeting PTEN and facilitate epithelial-mesenchymal transition (EMT). Sprout initiation and endothelial migration and invasion are augmented by KSHV-miR-K3 by suppressing GRK2 expression and increasing the levels of CXCR/AKT. KSHV-miR-K6-3p induces migration and angiogenesis by negative regulation of SH3BGR and upregulation of STAT3.

Kaposi’s sarcoma, caused by KSHV, is an angiogenic, vascular tumor of epithelial spindle cells. It has been demonstrated that KSHV miRNAs are involved in numerous cellular pathways such as cell migration and cytoskeleton arrangement that may contribute to the pathogenesis of KSHV-associated tumors, (Li et al., 2016). More specifically, kshv-miR-K3 promotes endothelial cell migration as well as invasion by activating the CXCR2/AKT signaling pathway. Kshv-miR-K3 directly targets G protein-coupled receptor (GPCR) kinase 2 (GRK2) to impair the signaling cascade. It has been proposed that this may play a role in the dissemination of KSHV-induced tumors (Fig. 4) (Hu et al., 2015). Li et al. (2016) demonstrated that KSHV-miR-K6-3p promoted cell migration and angiogenesis via direct targeting and downregulation of SH3 domain binding glutamate-rich protein (SH3BGR). SH3 domains are involved in protein-protein interactions and have been implicated in numerous processes including cell motility and cytoskeletal rearrangement (Mayer, 2001). Li et al. (2016) reported that SH3BGR is typically negatively regulated by STAT3. By downregulating SH3BGR, STAT3 was relieved and able to mediate the miR-K6-3p-induced cell migration and angiogenesis. miR-K6-3p and its the downstream pathway are proposed as promising therapeutic targets for the treatment of KSHV-associated malignancies (Fig. 3)

Fig. 4. KSHV encodes multiple v-miRs that promote angiogenesis and metastasis.

KSHV-encoded v-miRs are involved in the pathogenesis of numerous cancers such as Kaposi’s sarcoma, which is an angiogenic, vascular tumor of endothelial cells. miR-K12-6-3p is involved in endothelial cell migration and angiogenesis by inhibiting glutamate-rich protein (SH3BGR) expression and enhanced cell migration and angiogenesis by activating STAT3. KSHV-miR-K12-3 and KSHV-miR-K12-7 repress C-EBPβ, a transcriptional repressor of IL-6 and IL-10 causing cell growth of virus-infected cells and angiogenesis.

4.5. V-miR Regulation of Angiogenesis and Cancer Development

Angiogenesis is a complex cellular mechanism required for the formation of new blood vessels from the existing vasculature or from bone marrow-derived endothelial progenitors, allowing tumor growth and development at early stages of carcinogenesis (Weis and Cheresh, 2011). Lately, the impact of v-miRs during angiogenesis have been explored. These angiomiRs may regulate multiple molecules targeting angiogenesis, such as, cytokines, metalloproteinases and growth factors (Table 5) (Salinas-Vera et al., 2019).

EBV and KSHV are among seven known oncoviruses (Boshoff and Weiss, 2002; Maeda et al., 2009; Cesarman et al., 1995). EBV miRNAs play a key role in the pathogenesis of several EBV-associated cancers including NPC. In addition to NPC, high expression of EBV BART miRNAs have been detected in gastric carcinomas (Cai et al., 2015). V-miR analysis of NPC tissue versus non-tumor tissue revealed that 5–19% of miRNAs expressed in the tumor are EBV-encoded (Zhu et al., 2009). It is important to note that while no particular v-miR was consistently expressed in NPC lesions, few v-miRs were frequently detected at higher levels. For instance, miR-BART7-3P, miR-BART22, and miRNAs from the BART-cluster 1 were abundant in NPC tumors (Table 5) (Umbach et al., 2008; Zhu et al., 2009). As mentioned above, BART7-3p expression causes a growth advantage to EBV-associated NPC tumors by repression of DICE1. BART22 is highly express in Acquired Inmmunodeficiency Syndrome (AIDS)-related DLBCL (Diffuse Large B Cell Lymphoma) cells, NPC and Gastric Carcinoma (GC) (Xia et al., 2008; Imig et al., 2011). This v-miR inhibits the differentiation and promotes the metastasis of NPC cells by targeting NDRG1 (Valadi et al., 2007). EBV BHRF1 miRs (1, 2 and 3) cluster play an important role in B cell lymphogenesis. This cluster is highly expressed during stage III latency. Moreover, knockout of BHRF1 cluster miRNAs inhibited B cell transformation and exhibit attenuated growth of mutant cell lines (Table 5) (Feederle et al., 2011; Haar et al., 2015). Ectopic expression of these miRs however, failed to rescue the suppression of reduced cell growth in wild type cells (Poling et al., 2017). Instead of acting in trans by suppression of cellular targets, BHRF1 cluster miRs act in cis by preventing accumulation of EBV Epstein-Barr virus nuclear antigen (EBNA) leader protein (EBNA-LP) transcript. This v-miR cluster is located on EBNA-LP transcripts at the promoter (BHRF1 miR-1) and 3’UTR (BHRF1 miR-2 and −3). Excision of the BHRF1 miR stem-loop precursor from the EBNA-LP transcript is microprocessor (Drosha) complex-mediated and thus augments degradation of transcript by rendering it more susceptible to RNA degradation (Feederle et al., 2011; Haar et al., 2015). It has been proposed that alteration of miR-BART1-5p expression results in an increased invasion and migration of NPC cells in vitro and has been demonstrated to cause tumor metastasis in vivo by targeting PTEN (Cai et al., 2015).

In NK/T-cell lymphoma samples, miR-BART7-3p and miR-BART19-3p are highly expressed suggesting that inhibition of APC expression and increased cell proliferation through β-catenin signaling (Wong et al., 2012; Motsch et al., 2012). However, ebv-miR-BART19-3p targets APC and WIF1, both tumor-supressor genes, which are involved in cancer development by Wnt pathway activation (Wong et al., 2012). The immunogenic viral protein, Latent membrane protein 1 (LMP-1) is also reported to be targeted by BART1-5, −16, −17-5p and 5–5p. The inhibition of LMP-1 contributes to the development and progression of EBV-associated tumors (Lo et al., 2007; Verhoeven et al., 2016).

KSHV is involved in the pathogenesis of numerous cancers including Kaposi’s sarcoma (Cheung, 2004). A recent study investigated the role of a KSHV-encoded miRNA, miR-K12-6-3p, on endothelial cell migration and angiogenesis. It binds to and inhibit glutamate-rich protein (SH3BGR), and enhances cell migration and angiogenesis by activating the STAT3 pathway that is activated in many cancers, including Kaposi’s Sarcoma (Fig. 4) (Li et al., 2016). kshv-miR-K3 is reported to promote endothelial cell migration and invasion, which may contribute to the dissemination of tumors induced by KSHV (Hu et al., 2015). For oncogenic viruses, like KSHV, suppression of cell death and upregulation of cell cycle progression and cell growth pathways is critical for persistence and contributes to viral pathology. In this regard, sequence-dependent interaction of KSHV-miR-K12-1 with the 3′UTR of cyclin-dependent kinase inhibitor p21, a negative regulator of cell cycle arrest and apoptosis, and its suppression promotes oncogenesis (Gottwein and Cullen, 2010).

It is well known that KSHV v-miRs modulate cytokine secretion. In this context, both miR-K12-3 and miR-K12-7 repress C-EBPβ, a transcriptional repressor of IL-6 and IL-10 causing cell growth of KSHV-infected cells and angiogenesis (Fig. 4) (Qin et al., 2010; Qin et al., 2011). Direct interaction of miR-K12-11 with its cognate C-EBPβ mRNA may cause B-cell expansion and KSHV-associated lymphomagenesis (Boss et al., 2011). In addition, miR-K12-11 targets SMAD family member 5 (SMAD5) to inhibit the TGF-pathway thereby promoting cell survival and KSHV-associated oncogenesis (Gottwein and Cullen, 2010; Liu et al., 2012). These observations indicate that v-miRs achieve robust modulation of a specific pathway by concurrent suppression of multiple genes involved in the pathway. Other v-miRs can also work in concert to assure favorable functional outcomes for virus. For instance, miR-K12-10 targets TGFBR2 thus affecting the TGF-pathway and promoting oncogenesis in KSHV-infected cells (Gottwein and Cullen, 2010).

Although most of the studies focus on the v-miR dependent regulation of host protein coding transcripts, long noncoding RNAs (lncRNAs) are recently shown as another targets of v-miRs indicating a broader regulatory role of v-miRs in host-pathogen interaction (Sethuraman, 2017). KSHV-infected endothelial cells exhibit dysregulated expression of lncRNAs, many of which are predominantly nuclear retained. Interesintgly, KSHV encoded v-miRs and AGO2 (integral member of miRISC) were also present in the nucleus. miR-K12-6-3p and miR-K12-8-5p were predominantly nuclear retained while, miR-K12-11 and miR-K12-12 were partially nuclear restricted. Employing HITS-CLIP, 98 of the 126 nuclear retained lncRNAs were shown to contain putative v-miR binding sites. Among these four novel lncRNA viz., Loc541472, CD27-AS1, ANRIL and MEG3 were validated. Cotransfection of four KSHV miRNAs (miR-K12-1, K12-6-5p, K12-2 and K12-11) significantly downregualted ANRIL, an oncogenic lncRNA while, miR-K12-5, K12-6-5p and K12-8 cotransfection reduced expression of MEG3, a tumor suppressor. This was further validated by pull-down expeiments, where 24.5% of MEG3 and 43.7% of Loc541472 transcripts were associated with miR-K12-6-5p but not with other non-binding v-miRs. However, other lncRNAs lacking miRNA binding sites were shown to be regulated by viral proteins. These findings suggest that v-miRs work in concert with viral proteins to control multiple aspects involving dynamic host-pathogen and support a role of v-miRs in KSHV-mediated oncogenesis (Table 5) (Sethuraman et al., 2017).

5. Viral microRNAs mimicking host microRNAs

Viruses have evolved numerous ingenious mechanisms to overwhelm host. With limited genome size compared to the host, virus relies on multifaceted role of its encoded factors. Virus-host interaction is dynamic and a constant arms race in case of life-long infectious pathogens like Herpesviruses. One such mechanism involves evolution of viral molecules that display highly similar features (including shape and sequence) with the host molecules. Thus, molecular mimicry by pathogen encoded factors that are functionally similar to host effectors molecules can facilitate viral immune evasion (Tortorella et al., 2000; Alcami 2003). Large DNA viruses (including herpesviruses and poxviruses) are unique in mimicking host protein coding and nocoding RNAs that facilitate viral evasion of host antiviral defense by genetic acquisition of unique pro-viral host features (Tortorella et al., 2000; Alcami 2003). For instance, several members of HHV family encode for homologues of host cytokines, chemokines and and their receptors, adding another dimension to host-pathogen interaction and coevolution (Tortorella et al., 2000; Alcami 2003). For instance, EBV and HCMV encodes IL-10 homologues (vIL-10) which promote immunosuppression to facilitate viral persistence, (Kotenko et al., 2000; Imlack et al., 2002). Angiongenesis is a key clinical feature of KSHV which encoded for pro-angiogenic cytokine vIL6 (Jones et al., 1999).

In conjunction with protein-coding viral analogs, v-miRs provide yet another excellent example of how viruses can harness even a small stretch of nucleotide sequence to expand their functionality and manipulate host cellular processes. Two subsets of virus-encoded miRNAs have been established: (i) virus specific and (ii) host miRNA analogs. Multiple herpesviruses viz., KSHV, EBV, Marek’s Disease Virus 1 (MDV1), and Bovine Leukemia virus (BLV) encode for v-miRs that negatively regulate same mRNA target by complementary pairing as their counterpart host miRNAs (Rodney et al., 2012; Zhao et al., 2011). Multiple studies have shown that v-miRs can mimic host miRNAs by possessing identical seed sequence and share a common set of target transcripts with the host miRNAs. Mimicking a host miRNA allows a v-miR to enhance biological activities including regulation of latent/persistent infection, evasion of the innate and adaptive immune responses, promotion of cell viability, etc., (Zhao et al., 2011). Several of the KSHV miRNAs mimic cellular miRNAs (Gottwein et al., 2007; Skalsky et al., 2007). For instance, miR-K12-11 mimics miR-155 and promote the induction of KSHV-positive B-cell tumors in infected patients (Gottwein et al., 2007; Skalsky et al., 2007). miR-K12-6-5p is partially homologous to the seed sequence of miR-15a and miR-16 which exhibit tumor suppressor activity and induce apoptosis by silencing BCL2 (Skalsky et al.,2007; Cimmino et al., 2005). Furthermore, miR-214 and its viral analog miR-K6-5p share 6-mer seed homology and both promote tumorigenesis (Gottwein, 2012). Interestingly, miR-K3+1_5 share its target caspase 3, 7 and tumor necrosis factor receptor superfamily member 10B (TNFRSF10B) with miR-23 and inhibit the apoptosis in KSHV-infected B cells (Manzano et al., 2013). EBV also express miRNA mimics that suppress innate inflammatory pathways. As mentioned previously, ebv-miR-BART-15 exhibit seed sequence homology with a cellular miR-223 specifcally expressed in leukocytes and post-transcriptionally regulate one of the validated target, NLRP3 to suppress host immune responses (Haneklaus et al., 2012).

Kincaid et al., demonstrated that BLV-miR-B4 shared a partial seed sequence identity and shared two commons targets with the host miRNA, miR-29 (HMG-box transcription factor 1 (HBP1) (Han et al., 2010) and peroxidasin homolog (PXDN) (Santanam et al., 2010). Both genes are tumor suppressors which are downregulated by miR-29 targeting in B-cell tumors suggesting a possible mechanism contributing to BLV-induced tumorigenesis. Finally, Chicken miRNA gga-miR-15b mimics several MDV-1 miRNAs, including, MDV1-M2, MDV1-M3 and MDV1-M5 have been physically mapped flanking the MDV oncogene Meq (Burnside et al., 2006; Feitian et al., 2012). Overall, viral homologues, vmiRs, viral cytokine, chemokine and their receptors, confer robust immune evasion by preferential targeting of immune activation and antiviral pathways to facilitate viral replication and survival.

6. Viral microRNAs as diagnostic and prognostic markers

Detection of v-miRs, similar to endogenous miRNAs, have been associated with disease diagnosis and prognosis including various virus-associated cancers, oral inflammation, Alzheimer’s, multiple sclerosis, etc. (Naqvi et al., 2018; Naqvi et al., 2020). V-miRs, apart from providing clues onto their role in host-pathogen interactions, can yield information on the presence of pathogens and the status of the pathogen life cycle (Skalsky and Cullen, 2010). Recent studies also support utilization of extracelular RNAs as prognostic markers of therapeutic value, specifically in cancers. Circulating v-miRs also provide a valuable resource in disease pathogenesis and treatment. In this section, we will provide evidence supporting immense diagnostic and prognostic value of v-miRs (Condrat et al., 2020) (Fig. 4).

Expression profiles NPC biopsies identifed almost the entire reportoire of EBV encoded miRNAs indicating a critical role of v-miRs in carcinogenesis (Young and Dawson, 2014; Tsao et al., 2017). EBV v-miRs are differentially expressed in differentiated and undifferentiated NPC tissues suggesting their potential use as diagnostic and/or prognostic marker. For instance, higher expression of BART-7-3p in NPC cells renders them more susceptible to radiation treatment (Fig. 5) (Young and Dawson, 2014; Tsao et al., 2017; Zhang et al., 2015). Gao et al. (2017) described the underlying mechanism in which BART7-3p target GFPT1 transcript, a negative regulator of TGFB1. BART7-3p enhances TGFB1 levels, which in turn, augments responsiveness to radiation treatment. These findings suggest that BART7–3p can serve as marker of NPC progression and possible predictor of therapeutic outcomes. Futhermore, miR-BART8-3p could be another potential prognostic biomarker. Treatment with anticancer drugs (KU60019 or AZD6738) may augment radiosensitivity of NPC by suppressing the phosphorylation of ATM and ATR. Zhou et al. showed that higher levels of EBV encoded miR-BART8-3p can promote NPC radioresistance by directly inhibiting ATM and ATR (Fig. 5) (Zhou et al., 2019).

Fig. 5. Harnessing diagnostic and prognostic value of herpesvirus-encoded v-miRs as markers of disease.

This schematic provides a list of selected studies that demonstrate diagnostic/prognostic value of HHV-encoded miRNAs. Monitoring expression profiles of v-miRs can provide a glimpse of active viral infection in biological fluids or tissue biopsies. In case of oncoherpesviruses, sampling saliva or brush biopsy from tumor sites can be a valuable information of disease activity and its response to treatment. NPC: Nasopharyngeal cancer; NNKTL: Nasal natural killer/T-cell lymphoma; CNS: Central nervous system.

V-miR profiles were also examined in EBV-associated malignancy nasal natural killer/T-cell lymphoma (NNKTL). EBV v-miR profiling in patients and control sera identifed higher levels of miR-BART2-5p, miR-BART7-3p, miR-BART-13-3p and miR-BART1-5p (Komabayashi et al., 2017). Interestingly, patients treated with chemotherapy and radiation combination exhibit reduced serum profiles of the aforementioned v-miRs indicating their association in disease prognosis. Among these BART2-5p displayed the most significant changes. Similarly, higher levels of miR-BART1-5p, miR-BART4-5p and miR-BART20-5p were detected in patients with EBV positive gastric carcinoma compared to paired normal mucosal tissues (Fig. 5) (Kang et al., 2015). Using multivariate analysis including age and pathologic state, miR-BART20-5p levels were observed to correlate with worse recurrence-free survival indicating its potential use as a biomarker for disease progression and patient survival.

Expression levels of HSV-1 encoded miRNAs is associated with the development and diagnosis of prostate cancer (CaP), a leading cancer in men worldwide (Fig. 5). Yun et al. (2015) detected high levels of miR-H9-5p in the urine of patients with CaP. Compared with prostate serum antigen (PSA), a key biomarker for CaP, miR-H9-5p was a more reliable biomarker in distinguishing between cancer and noncancerous benign prostatic hyperplasia (BPH), specifically for indeterminate cases. Expression analysis (by RT-qPCR and in situ hybridization) of miR-H18 and miR-H9-5p showed higher levels of both HSV-1 v-miRs in CaP tissue and surrounding noncancerous tissue than in benign prostrate hyperplasia (BPH) tissue suggesting a possible association with tumorigenesis in the prostate (McNally et al., 2020). These v-miRs can be used as biomarker for tumor-containing prostate and can potentially be used to detect early tumorigenesis.

Increasing lines of evidence indicate a plausible role of members of HHV family in neurological disorders (Lin et al., 2002; Lamade and Strank, 2012). For instance, EBV genome and other viral encoded molecules have been consistently detected in different cohorts of subjects with neurological diseases (Lin et al., 2002; Hassani et al., 2018). Wang et al. (2017) evaluated EBV v-miRs in relapse-remitting multiple sclerosis patients and control subjects (n=30) and observed higher circulating levels of BHRF1-2-5p and BHRF1-3 in diseased subjects, which correlated with the clinical score of the disease progression. It can be noted that precursor of both of these v-miRs are encoded from the 3’UTR of viral transcript EBNA-LP. Using reporter assays, they demonstrate that BHRF1-2-5p post-transcriptionally regulate MALT1, an important regulator of NFκB activation, T and B cell proliferation, apoptosis (Ruland et al., 2003).

Studies from our lab have also shown increased prevalence of HHV v-miRs in oral health and disease (Naqvi et al., 2018; Ruland et al., 2003; Zhong et al., 2017). In pulpitis, a necrotic, inflammatory disease of teeth, we noticed higher expression of multiple HHV v-miRs viz., KSHV miR-K12-3, HSV-1 miR-H1, HCMV miR-US4 and miR-UL70 (Zhong et al., 2017). Expression of KSHV miR-K12-3, HSV-1 miR-H1 and HCMV miR-US4 was examined in another oral inflammatory disease, periodontitis. Interestingly, similar expression pattern of high expression and increased prevalence of these v-miRs was observed in periodontitis subjects compared to healthy controls (Fig. 5) (Naqvi et al., 2018; Naqvi et al., 2019). These findings, strongly support a likely association of v-miR levels and pathogenesis of inflammatory disease, in these cases, of oral mucosal origin. Involvement of multiple overlapping, yet mututally exclusive components may be required for the disease pathogenesis. Clinical findings from our lab and others indicate HHV derived v-miRs as key component in the disease development. As mentioned above, we recently showed that v-miR expressing cells exhibit altered profiles of endogenous miRNAs and mRNAs suggesting global impact on cellular functions. Interestingly, v-miR transfected cells exhibit attenuated phagocytosis of bacteria and reduced secretion of various cytokines strongly supporting immunomodulatory impact of v-miRs (Naqvi et al., 2018; Naqvi et al., 2018). From an oral disease perspective, this could significantly impact the pathogenesis of bacteria-mediated oral inflammatory diseases. V-miRs can thus be considered an important link between host-bacteria interaction. Overall, all these studies demonstrate a potential use of v-miRs in disease diagnosis and prognosis.

Limitations of v-miRs detection, however, should be mentioned. Indeed, the most commonly used and promising miRNA detection methods have benefits and limitions. Cloning method and Northern blot as validation methods were the first common methods to discover miRNAs, however, these methods are low sensitivity, low-throughput and high time-consuming (Lee et al., 1993, Grishok et al., 2001; Lau et al., 2001; Lee et al, 2001; Chen et al., 2009). In situ hybridization techique is recomendable only to detect high abundance miRNA species. Similar to cloning and Northern blotting, it encounters low-throughput, low sensitivity and specificity issues (Li et al., 2009; Nelson et al, 2008). Bead-based flow cytometry assays provide highly robust, sensitive, and reproducible detection of miRNAs. There are mulitple limitations of this technology that needs to be addressed including high input RNA, time-consuming, low-throughput, and complex data analysis (Jill Koshiol et al., 2010). Oligonucleotide microarray (microchip) and reverse transcription quantitative PCR (RT-qPCR) are two of the most commonly used high-throughput miRNA detection methods for known miRNAs but not without limitations (Creighton et al., 2005; Chen et al, 2009; Nelson et al., 2008). Microarray requires high total RNA amount (between 0.2–2 μg of RNA), exhibit low sensitivity and specificity, provide relative miRNA abundance (semi-quantitative) and the potential cross-hybridization of related miRNAs is high (Chen et al., 2009; Chen et al., 2009). Regarding RT-qPCR, the throughput, sensitivity, specificity is high but the main limitation is based on the primer complementarity over short sequences at the 3’-end (Chen et al., 2009; Chen et al., 2009). Many studies proposed a good practice to profile miRNAs by microarray and their validation with RT-qPCR, however there are no standard guidelines based on this practice (Croce et al., 2009). Next-generation, high resolution, deep sequencing technologies allows quantification of miRNAs as well as discovery of new miRNAs species (Creighton et al., 2009). They present high-throughput, high sensitivity, high specificity methods but are time-consuming, high cost, requires higher input RNA (1–2 μg), and specialized laboratory equipment (Creighton et al., 2009; Ansorge et al., 2009). In addition, the inherent bias in sequence-specific amplification, low copy number miRNA species may not provide accurate and reproducible results. In brief, the current and new technologies to discover and validate new miRNAs have benefits and limitations but still necessary to advance the research and further improvisation in improving sensitivity, specificity and cost will revolutionize their value in harnessing v-miR expression monitoring in diagnostics and prognostics.

7. Viral microRNAs in therapeutics

V-miRs regulate a huge repertoire of host mRNAs, which makes them a promising therapeutic target for many diseases such as virus-associated cancers (NPC, NNKTL, gastric cancer, prostate cancer) (Young and Dawson, 2014; Tsao et al., 2017; Zhang et al., 2015; Gao et al., 2017; Komabayashi et al., 2017; Kang et al., 2015; Yun et al., 2015; McNally et al., 2020), oral inflammatory diseases (pulpitis, gingivitis, periodontitis) (Naqvi et al., 2018; Naqvi et al., 2019; Naqvi and Slots 2021; Zhong et al., 2017; Zhong et al., 2012), immune disorders (Wang et al.,2017; Ruland et al., 2003), neurological disorders (Coscoy et al., 2007; Damania et al., 2007; Klein et al., 2007; Lin et al., 2002; Lamade and Strank, 2012; Hassani et al., 2018), etc. In the past decade, microRNA therapeutics is an emerging field in biopharmaceutical companies as next-generation of drugs. Indeed, mulitple clinical trials are testing several parameters such as, absorption, distribution, metabolism, excretion and delivery system of miRNAs (Hemida et al., 2010). Another class of small RNAs viz., short interfering RNA (siRNA) have shown promising antiviral function by supressing viral gene expression in the recent clinical trials (Hemida et al., 2010). For instance, the clinical trial with siRNA NucB1000 against HBV has shown promising results and can be utilized as antiviral therapy (Le, 2008). NucB1000 contains a plasmid DNA formulated with delivery system which can transcribe four siRNAs molecules that target four viral genes. RNAi of viral genes can inhibit viral replication and favor resolution of HBV infection (Le, 2008). On the other hand, a novel attractive antiviral therapy is based on the inhibition of specific viral proteases or polymerases by silencing the action of v-miRs gaining the control over the virus or even its elimination (Anderson et al., 2009; De Clercq & Neyts, 2009).

The well-known AMOs (Anti-miRNA oligonucleotides) is a type of anti-miRNA therapeutic strategy based on the silencing of miRNA (including v-miR) by complementary interaction. They are classified based on their chemical composition: first generation: 2’-O-methyl AMOs, second generation: LNA-modified oligonucleotides and third generation: phosphodiamidate morpholino oligomers (PMO), PNA, microRNA sponge and antagomirs (Esau et al., 2008; Krutzfeldt et al., 2005; Krutzfeldt et al., 2007; Mattes et al., 2007; Fabani et al., 2008; Weiler et al., 2006). All of them exhibit high functional efficacy in miRNA silencing and can serve as potential therapeutic tools. Sponge-based antagomirs have shown promising results (Mishra et al., 2020). Low toxicity, high specificity designing and targeted delivery of antagomirs can mitigate numerous problems including off-targets, and side-effects observed with small RNA delivery (He et al., 2008, Tahamtan et al., 2016). Cytomegalovirus-infected mouse (MCMV) treated with antagomirs against MCMV v-miRs augmented its immunogenicity upon new infections of MCMV (Mishra et al., 2020). miR-UL112-1, a v-miR expressed by cytomegalovirus, evades the immune system by stablishing latent viral infection. Antagomirs against miR-UL112-1 force the virus to get into the lytic phase and activates immune system to promote viral clearance (Moens, 2009). However, targeting of v-miRs (encoded by KSHV, EBV and other HHV) with seed-sequence homology with cellular miRNAs can be extremely tricky as it may perturb normal cellular functions (Moens, 2009). Ebv-miR-BART5 targets PUMA and inhibit apoptosis. The development of anti-miR-BART5 could increase the expression of PUMA, which in turn would allow an increase in the apoptosis (Choy et al., 2008). A recent study has demonstrated antitumor activity for CXCL-11 in EBV-positive cells tumors by targeting of miR-BHRF1-3 using AMOs against this v-miR, restoring antiviral immunity (Xia et al., 2008). Mice treated with gold nanoparticles containing anti-ebv-miR-BART7-3p showed reduced tumorigenicity of EBV-positive cells (Cai et al., 2015). Finally, Wang et al. (2019) reported the use of iRGD-tagged exosomes containing antagomiR targeting ebv-BART10-5p and cellular miR-18a to inhibit the angiogenesis in NPC in vitro and in vivo by targeting the tumor suppressor, Spry3 and its downstream targets. Both antagomirs exert a synergistic effect to inhibit angiogenesis and cell growth in EBV-associated cancer. In summary, while a large number of cellular miRNA have been examined for therapeutic targeting, only a few studies have assessed v-miRs. Developing viral treatment modalities targeting v-miRs will open new research avenues, advance novel clinical intervention to improve the quality of health care in medicine and dentistry.

Conclusion and perspective

Detection of one or more human herpesviral genomes in chronic inflammatory diseases including cancers, neurological disorders, periodontal disease, and periapical periodontitis have indicated their role in the etiopathogenesis. However, the underlying virulence factors and their functional characterization remain poorly studied. Recent studies have reported frequent detection of v-miRs in diseased human biopsies and their accumulation correlates with diagnosis and prognosis. For instance, a recent study by Fuentes-Mattei et al. (2017) highlights superior potential of v-miR as diagnostic marker in plasma collected from post-surgical/post-chemotherapy sepsis, chronic lymphocytic leukemia, and post-abdominal surgery. High sensitivity of v-miRs (EBV {miR-BART4 or miR-BHRF1-1} or KSHV {miRK12-10b, miR-K12-12, or miR-K12-4-3p}) detection over seropositive assays further supporting the diagnostic resolution of noncoding RNAs over serology methods. Identification of unique v-miR repertoire in specific tissues and improvising v-miR detection techniques is a challenge for virologists to advance the utility of these noncoding RNA from bench to chairside.

Dissecting the hitherto unknown role of HHV-encoded v-miRs will be critical in our understanding of their function in the pathogenesis of various viral diseases. Accumulation of HHV members, which exhibit tropism for immune cells, in inflammatory microenvironment is not surprising. However, utilizing molecular changes that can yield knowledge on active viral replication can provide clinically relevant information. Identification of disease-associated v-miRs required in active disease and delineating mechanisms that confer viral persistence and immune evasion demands further investigation. v-miRs, by virtue of their ability to move across bystander cells via exosomal pathway, can modulate biological processes such as proliferation, apoptosis or carcinogenesis in a wide range of host cells; however this require further in vivo and ex vivo analysis. Elucidation of unique subset of v-miRs in shaping host-pathogen interaction will be important in devising new treatment modalities. Using anti-v-miR strategies in conjunction with anti-herpetic drugs (like acyclovir) may unravel alternate clinical approaches to treat recurrent diseases.

Highlights.

1. Human herpesviruses (HHV) are ubiquitous DNA viruses that cause recurrent infections and cancers.

2. Most HHV members encode multiple microRNAs (miRs) that perform multifaceted functions.

3. Viral miRs (v-miRs) can target host and viral transcripts to shape host-pathogen interaction.

4. HHV v-miRs impair pathways including apoptosis and angiogenesis to promote viral persistence.

5. v-miR can serve as valuable diagnostics/prognostic to monitor disease and treatment response.