Human cytomegalovirus (HCMV)-encoded microRNAs: potential biomarkers and clinical applications

ABSTRACT

HCMV-encoded microRNAs (hcmv-miRNAs) are non-coding and non-immunogenic molecules that target numerous cellular genes and allow the virus to modulate the host’s signalling pathways, thus favouring viral survival and replication. Given their capacity to silence the human genes involved in various physiological processes, these hcmv-miRNAs have now emerged as a potential clinical biomarker in many human diseases. In this review, we summarize the evidence published on the diagnostic and prognostic value of hcmv-miRNAs in several human diseases and their clinical implications. Specifically, we discuss the role of hcmv-miRNAs in the development of cardiovascular diseases and cancer by silencing tumour suppressors. We also examine the current knowledge on the utility of some hcmv-miRNAs in predicting HCMV viraemia recurrence in transplant patients, as well as the interference of hcmv-miRNAs in the development of an appropriate immune response against other viral infections, which might have therapeutic implications.

Abbreviations: HCMV, human cytomegalovirus; hcmv-miRNA, HCMV-encoded microRNAs

Introduction

Human cytomegalovirus (HCMV) is a double-stranded linear DNA β-herpesvirus whose genome is approximately 230 kb in size. The worldwide CMV seroprevalence depends on socio-economic conditions and is higher in individuals from lower socio-economic groups. The estimated global mean seroprevalence for the general population is 83% and ranges from 90% in the Eastern Mediterranean region to 66% in Europe [1,2]. Although infection is usually asymptomatic, it can cause serious complications in immunocompromised patients such as solid organ or haematopoietic stem cell transplant patients [2–7].

After primary infection, HCMV develops several immune evasion mechanisms to prevent eradication of the virus before latency is established. These mechanisms operate by inhibiting innate immunity and preventing the development of adaptive immunity. This occurs through the expression of homologous anti-inflammatory cytokine proteins or by interfering with the antigen presentation, among others [8]. The high prevalence of HCMV worldwide and its ability to persist for life in a latent state inside human cells suggest that this virus has evolved to develop very well-designed survival strategies. One of these strategies is mediated by microRNAs (miRNAs), which are small non-coding RNA and non-immunogenic molecules of around 22 nucleotides that silence gene expression post-transcriptionally by targeting the 3ʹUTR of mRNAs [9–15]. These molecules exhibit great plasticity since a single miRNA can target many different transcripts, and a transcript can be regulated by many different miRNAs [11].

RNA silencing is part of a primitive immune system against viruses in plants and insects, but its role in viral infection in human cells had not been investigated. Since the initial discovery of viral miRNAs in 2004 [16,17], several viruses have been reported to express their own miRNAs. The miRNAs encoded by herpesviruses, such as EBV and HCMV, have been shown to induce RNA silencing as a method for the gene regulation of host and viral genes in a non-immunogenic manner [16–18].

The miRNAs encoded by HCMV and other herpesviruses have been reported to target numerous cellular and viral transcripts. Through their cellular targets, herpesvirus miRNAs promote immune evasion, cell survival and tumorigenesis, whereas the viral targets seem to be crucial for maintaining latency [15,18]. HCMV-encoded miRNAs (hcmv-miRNAs) allow the virus to evade host immune response and to regulate the establishment of lytic (productive) and latent (non-productive) infection. In fact, HCMV is the virus with the largest number of genes (miRNAs and non-miRNAs) dedicated to this purpose, since more than 50% of the genes encoded by HCMV are focused on immune evasion [4,19]. According to the miRNA database (www.mirbase.org), 26 HCMV miRNAs and their target molecules have been identified to date. Unlike other herpesviruses, whose genes are found in localized clusters in the genome, HCMV miRNA genes are scattered throughout the viral genome [5,14,20–22].

In recent years, the study of the human or viral miRNA expression profile has shown clinical utility as a biomarker in human pathologies, such as viral infections [23,24], or in oncology, where miRNAs have been reported to have diagnostic and prognostic value [25–27]. Given the role of hcmv-miRNAs in the gene regulation of cellular transcripts through gene silencing and the impact of HCMV infection on immunocompromised individuals, hcmv-miRNAs are also good candidates to be used as biomarkers in human diseases. For this reason, the analysis of the hcmv-miRNA expression profile and its potential clinical applications has gained increasing importance. In this review, we summarize recent evidence on the potential role of hcmv-miRNAs as diagnostic and prognostic biomarkers in several human diseases and their clinical implications.

Biogenesis of miRNAs

Hcmv-miRNA biogenesis occurs the same way as human miRNAs (h-miRNAs) in a process requiring multiple steps (Fig. 1) [28,29]. Biogenesis starts in the nucleus where the miRNA genes are transcribed by RNA polymerase II, resulting in a hairpin-like primary transcript called primary miRNA (pri-miRNA). The pri-miRNA is then processed by the Drosha-DGCR8 enzyme complex, which is responsible for excising the hairpin structure from the rest of the transcript. This step produces a new structure of about 70 nucleotides known as the precursor miRNA (pre-miRNA), which is transported from the nucleus to the cytoplasm where the rest of the steps occur.

Figure 1.

Biogenesis of hcmv-microRNAs. This is a multi-step process that takes place in the cell nucleus and cytoplasm. Enzymes such as RNA polymerase II, DGCR8 Drosha, Exportin-5, DICER and AGO are involved in the process. Created with BioRender.com

In the cytoplasm, the DICER enzyme recognizes the structure and cuts the upper loop of the pre-miRNA hairpin, resulting in a small structure of two almost complementary strands that constitute the mature miRNA duplex. One of the strands is degraded, while the other one constitutes the guide strand, which is loaded into the Argonaute protein (AGO) to form the miRNA-AGO complex. This complex drives the guide strand to an mRNA target in a complementary nucleotides-based way. The mRNA target site is usually in the 3ʹ UTR, whereas the complementary sequence on miRNA, called the seed sequence, is located at nucleotides 2–8. This sequence is considered the strongest target site. In addition to the seed, nucleotides 13–16, or the ‘supplemental region’, can also contribute to target recognition. The interactions of the miRNA-AGO complex with a target mRNA through the miRNA seed sequence result in translation repression or degradation. In contrast, extensive pairing to the 3ʹ end destabilizes the complex, thus promoting miRNA unloading and the target cleavage and decay [10,11,14,30].

Circulating miRNAs

In addition to gene regulation inside the cells (intracellular signalling), miRNAs can also be packaged in exosomes and circulate through biological fluids until they reach the target cells, where they can also be functional [31]. These extracellular vesicles contain miRNAs as well as mRNA and other non-coding RNA and transport miRNAs in a paracrine and endocrine manner. Exosomal miRNAs are free of AGO protein and are instead specifically recognized by individual proteins that identify the specific binding motif of miRNAs and selectively load them into exosomes [32].

Circulating miRNA can reach distant sites and modulate the correct functionality of tissue cells, thus contributing to the onset of disease. Initial studies showing the association between miRNAs and human disease were limited almost exclusively to cancer [33,34], while the association between miRNA expression and non-neoplastic diseases was identified later. It has now been demonstrated that the dysregulation of miRNA expression plays an important role in the progression of several conditions, such as cardiovascular diseases, neurodegenerative diseases, retinal disorder, viral infections and diabetes, among others. Therefore, miRNA expression profiles can be important diagnostic, prognostic and therapeutic tools for several diseases [35,36].

Nevertheless, viral miRNAs can also be packaged inside exosomes, since HCMV uses the cellular exosomes pathway to introduce its miRNAs in distant cells and modulate the host immune response to ensure viral persistence. The dissemination of hcmv-miRNAs through cellular exosomes may impair the function of different cells at tissue or even at systemic level, thus contributing to disease manifestations. Consequently, circulating hcmv-miRNAs have been reported to be potentially useful biomarkers for the diagnosis or prognosis of various diseases [11,37].

Clinical applications of HCMV-encoded miRNAs

The usefulness of hcmv-miRNAs as potential biomarkers in several human diseases has been analysed in the literature, as summarized in Fig. 2 and Table 1.

Figure 2.

Upregulation of hcmv-miRNAs (intracellular or circulating) in pathological conditions and their target genes. In cardiovascular diseases, a high level of hcmv-miR-UL112-3p is associated with atherosclerosis and related diseases. The target transcripts blocked by this miRNA are: (1) the IRF-1 gene, which is related to hypertension; (2) the NOS gene, which is related to atherosclerosis, and (3) an undetermined gene, which is related to diabetes. The hcmv-miR-US25-1-5p (4) has shown to be associated with atherosclerotic processes, whereas a high level of hcmv-miR-US33-5p is related to AAD (5). In addition, HCMV-encoded miRNAs are also involved in the development and prognosis of GBM. Specifically, hcmv-miR-UL112-3p (6) and hcmv-miR-UL70-3p (7) are associated with this type of cancer. Furthermore, there is evidence that hcmv-miR-UL112-3p (8) and hcmv-miR-US25-1-5p (9) are involved in the development of liver damage and neurological disorders in newborns suffering from congenital HCMV infection. In solid organ transplantation, C-MYC gene blockage mediated by hcmv-miR-UL22A-5p (10) is related to recurrent HCMV replication in these patients. Finally, there is evidence that IFNα therapy in chronic hepatitis B patients is less effective due to ERAP1 gene blockage mediated by hcmv-miR-US4-5p (11). Created with BioRender.com

Table 1.

Hcmv-miRNAs upregulated in human pathological conditions. The target genes of these miRNAs and their potential clinical utility are indicated

Hcmv-miRNAs	Target genes	Related disease	Potential clinical utility	References
hcmv-miR-UL112-3p	NOS	Atherosclerosis	Diagnostic biomarker	(41)
	IRF-1	Hypertension	Diagnostic biomarker	(28)
	undetermined	Diabetes/Glioblastoma multiforme	Diagnostic/ prognostic biomarker	(29)
	TUSC3	Glioblastoma multiforme	Prognostic biomarker	(33)
	undetermined	Congenital CMV infection	Diagnostic biomarker	(35,36)
hcmv-miR-US25-1-5p	BRCC3	Atherosclerosis	Diagnostic biomarker	(31)
	undetermined	Congenital CMV infection	Diagnostic biomarker	(35,36)
hcmv-miR-US33-5p	undetermined	Acute aortic dissection	Diagnostic biomarker	(32)
hcmv-miR-UL70-3p	undetermined	Glioblastoma multiforme	Prognostic biomarker	(34)
hcmv-miR-UL22A-5p	C-MYC	Solid organ transplant	CMV replication biomarker	(38)
hcmv-miR-US4-5p	ERAP1	Hepatitis B	Predictive biomarker of therapy efficacy	(40)

BRCC3, BRCA1/BRCA2-Containing Complex Subunit 3; C-MYC, V-Myc Avian Myelocytomatosis Viral Oncogene Homolog; ERAP1, Endoplasmic Reticulum Aminopeptidase 1; IRF-1, Interferon Regulatory Factor 1; ; TUSC3, Tumour Suppressor Candidate 3; NOS, Nitric Oxide Synthase

Cardiovascular diseases

Several studies have shown that chronic HCMV infection in vascular cells could be related to the development of cardiovascular diseases such as atherosclerosis. Atherosclerosis is characterized by the accumulation of abnormal vascular cells and apoptotic cellular debris. However, it is a complex disease and the mechanisms responsible for its development have not yet been fully discovered. The primary site of HCMV infections and reservoirs are vascular cells, which facilitate the spread and persistence of the virus in the individual. Furthermore, HCMV seems to be involved in the activation of pro-inflammatory signals mediated by IL1-beta or TNF alpha. Thus, it can promote higher cell proliferation, thickening of the vascular wall and atherosclerotic plaque formation [38].

Li et al. [39] analysed human and viral miRNA expression, including hcmv-miR-UL112, in hypertensive patients and healthy individuals. The objective of their study was to verify the role of these miRNAs in the development and progression of hypertension. To determine the differential miRNA expression in hypertensive patients, the authors profiled the plasma miRNA expression of 1700 miRNAs in 13 hypertensive patients and 5 healthy donors using microarray. They observed that hcmv-miR-UL112 was upregulated in hypertensive patients compared to healthy individuals (2.72-fold change; p = 0.002). Furthermore, they determined that this miRNA targets the mRNA of the IRF-1 gene, which is involved in the control of inflammation and cell proliferation. Therefore, the dysregulation of IRF-1, mediated by hcmv-miR-UL112, would support the proliferation and dysfunction of vascular cells. The authors concluded that this dysregulation could be associated with the development and progression of hypertension and consequent atherosclerosis (Fig. 2).

Some years later, Mohammad et al. [40] studied the prevalence and expression level of hcmv-miR-UL112-3p in 87 plasma or serum samples from healthy individuals and three groups of patients with different diseases: diabetes mellitus, rheumatoid arthritis and glioblastoma. The authors quantified the mean copy number of miR-UL112-3p per 10 ng of total RNA and observed that miR-UL112-3p levels were significantly higher in diabetes mellitus patients compared with healthy controls (mean copy number; 8 versus 1; p = 0.003) (Fig. 2). Therefore, given that the characteristic hyperglycaemia of diabetes mellitus is also one of the main risk factors for the development of atherosclerosis, this miRNA may be useful as a biomarker.

More recently, Shen et al. [41] examined the in vitro effect of hcmv-miR-UL112 on vascular endothelial cell growth and migration. Endothelial cells are the first site of HCMV infection during primary infection and a potential reservoir for this virus. The authors used a recombinant adenoviral vector to stably express hcmv-miR-UL112 in vascular endothelial cells. They demonstrated that the overexpression of hcmv-miR-UL112 significantly promoted the growth of vascular endothelial cells at 48, 72 and 96 h after infection (p < 0.05) as compared to the control cells. The molecular mechanism analysis demonstrated that the endothelial nitric oxide synthase gene was a target for this miRNA and that inhibition of this enzyme activates the MAPK signalling pathway in stress response, which stimulates cell proliferation and dysfunction (Fig. 2).

Also in relation to atherosclerosis, but focusing on a different hcmv-miRNA, Fan et al. [42] investigated whether hcmv-miR-US25-1 expression accelerated the development and progression of atherosclerosis, since this viral miRNA downregulates multiple cell cycle genes through mRNA 5ʹ UTRs. For this purpose, they analysed atherosclerosis patients with and without HCMV infection. The authors observed a significant upregulation of hcmv-miR-US25- 1 in HCMV infected versus non-infected subjects (2.72-fold change vs. 1.00; p = 0.0013) and in HCMV-infected atherosclerosis versus non-infected atherosclerosis patients (2.31-fold change vs. 1.00; p = 0.0036) subjects. In addition, in vitro cell line experiments showed that hcmv-miR-US25-1 transfection reduced the viability and aggravated the apoptosis induced after treatment with oxidized low-density protein (with 16.3% vs. 32.7% of apoptotic cells at 24 h, and 35.6% vs. 56.5% at 48 h). The authors speculated that this effect on apoptosis might be related to the fact that hcmv-miR-US25-1 downregulated BRCC3, a gene involved in the repair of DNA damage. Using western analysis and luciferase reporter constructions, they demonstrated that hcmv-mir-US25-1 suppressed the translation of mRNA BRCC3 by targeting the 5ʹ UTR. A significant BRCC3 reduction (60–80%) in protein level by miRNA transfection was also reported. Hence, hcmv-miR-US25-1 also appears to be involved in the development and aggravation of atherosclerosis (Fig. 2).

Acute aortic dissection (AAD) is another cardiovascular disease that involves the tearing of the inner layer of the aorta artery, the main human artery responsible for transporting blood from the heart to the rest of the body. AAD is one of the most catastrophic cardiovascular diseases and has a high morbidity and mortality rate, especially when it is not recognized and promptly treated [43]. Therefore, the discovery of new biomarkers that facilitate early diagnosis may be of clinical relevance. In the study carried out by Dong et al. [44], the authors aimed to determine whether some selected human or HCMV miRNAs could be good candidates as a diagnostic biomarker for AAD. To this end, they screened the expression of 1205 different h-miRNAs and 142 viral miRNAs in plasma samples from patients with AAD, from chest pain patients without AAD (myocardial infarction, aortic aneurysm and pulmonary embolism) and from healthy individuals. They observed a significant increase in the expression of hcmv-miR-US33-5p in the plasma of AAD patients compared to chest pain patients without AAD (21.97-fold change; p = 0.011) (Fig. 2). Using RT-PCR, the authors demonstrated in their study that hcmv-miR-US33-5p has a high diagnostic accuracy and a long time-window for the diagnosis of AAD. These findings show that the dysregulation of different hcmv-miRNAs is associated with the development and progression of cardiovascular diseases and that hcmv-miRNAs may be sensitive and specific biomarkers for these diseases.

Cancer

There is increasing evidence that supports the role of infectious agents in the development and progression of human cancer. In this regard, HCMV has been reported to play an oncomodulatory role in the development of glioblastoma multiforme (GBM), a type of aggressive cancer affecting the central nervous system [45]. GBM is considered the most lethal primary tumour of the central nervous system in adulthood since the average survival rate is 15–18 months after diagnosis, which is related to treatment resistance and recurrence in these patients [46].

Liang et al. [45], based on the influence of HCMV on the development and progression of GBM, published the results of a study on hcmv-miRNA expression in patients with GBM and compared the expression in cancer cells and adjacent healthy cells of the same patients. The authors found that hcmv-miR-UL112-3p expression was significantly up-regulated (3-fold change) in GBM tissues compared to adjacent normal tissue. In addition, high hcmv-miR-UL112-3p expression was related to poor prognostic features and lower overall survival rates. To investigate the molecular mechanism behind this correlation, the authors used in vitro cultures of GBM cells and observed that the overexpression of this miRNA promoted cell growth. They identified TUSC3, a potential tumour suppressor gene, as a new target of miR-UL112-3p, thus demonstrating that hcmv-miR-UL112-3p might act as a tumour regulator by directly targeting TUSC3 in GBM. Therefore, their data reveal that miR-UL112-3p may be a novel agent and may play a critical role in GBM progression (Fig. 2).

However, the association between hcmv-miR-UL112-3p and GBM has not been confirmed in other studies. In the previously mentioned study of Mohammad et al. [40] on the role of hcmv-miR-UL112-3p in patients with different diseases, serum samples from GBM patients did not show a statistically significant different expression of hcmv-miR-UL112-3p compared to healthy individuals.

Recently, in relation to the same type of cancer, Ulasov et al. [46] analysed the association between hcmv-miR-UL70-3p and the activation of glioma stemness. The authors determined the expression level of this miRNA in cancer tissue samples from GBM patients and in healthy individuals. Quantitative PCR analysis showed a more than 10-fold expression of hcmv-miR-UL70-3p in tumour tissue compared to samples from healthy individuals, suggesting that this miRNA can be a contributing factor for GBM. Furthermore, the authors observed that high miR-UL70-3p expression increases the migratory and invasive capacity of cancer cells. To determine the role of hcmv-miR-UL70-3p expression, they treated GBM cells with a miRNA inhibitor and found a significant suppression of cell invasion (12.07 ± 7.82) compared to GBM cells without an inhibitor (36.57 ± 12.72, p < 0.0001). In addition, inhibition experiments demonstrated that the hcmv-miR-UL70-3p inhibitor decreased SOX2 stem cell marker expression, thus suggesting that SOX2 was ain direct target of hcmv-miR-UL70-3p, and that this miRNA may exert its effect through the upregulation of SOX2. Therefore, the authors concluded that hcmv-miR-UL70-3p could promote tumour development and progression (Fig. 2).

Overall, although more studies are needed to demonstrate the role of hcmv-miR-UL112-3p and miR-UL70-3p in silencing tumour suppressor genes, the evidence reported in this review seems to indicate that they might be potential silencers of these genes and that their overexpression could be used as a biomarker for worse prognosis in GBM patients.

Congenital HCMV infection

Congenital HCMV infection occurs in 0.2–2% of births in developed countries. Although most infants suffering from congenital HCMV infection are asymptomatic, in some cases the infection can different organs such as the liver, lung or even the nervous system, which is associated with the appearance of neurological disorders such as intellectual disability and cerebral palsy [47,48].

In relation to liver damage, it has been reported that liver lesions are relatively common in infants under 6 months of age since they are highly susceptible to HCMV infection. For this reason, Zhang et al. [47] carried out a comparative study to assess the relationship between the expression of some hcmv-miRNAs in extracellular vesicles and the development of liver damage in newborns suffering from HCMV infection. They analysed the expression level in HCMV-infected infants compared to healthy infants. Total RNAs were extracted in extracellular vesicles to detect miR-US25-1 and miR-UL112-3p. The authors found that the levels of both miRNAs were significantly higher (4-fold increase) than those of non-infected infants and that the levels of miR-US25-1-5p were also significantly higher than those of miR-UL112-3p. Furthermore, high expression of both miRNAs was associated with some clinical biochemical indices of liver damage. In particular, the overexpression of hcmv-miR-UL112-3p was associated with a higher serum level of direct bilirubin (µmol/L), whereas the overexpression of hcmv-miR-US25-1-5p was significantly positively correlated (p < 0.05) with an increased level of serum level of γ-glutamyl transpeptidase (U/L), direct bilirubin (µmol/L) and total bile acid (µmol/L). Therefore, the authors suggest that high expression levels of these hcmv-miRNAs might be the cause of liver damage in HCMV-infected newborns (Fig. 2).

As concerns typical neurological disorders in newborns with congenital HCMV infection, Kawano et al. [48] analysed the association between hcmv-miRNAs and clinical features in patients with congenital HCMV infection. For this purpose, they studied the expression level of hcmv-miR-UL112-3p, hcmv-miR-US25-1-5p and hcmv-miR-US25-2-5p in plasma from infants under 6 months of age suffering from congenital HCMV infection with and without clinical features such as hearing disorders, intrauterine growth restriction, developmental retardation and abnormalities on brain imaging. Levels of miR-US25-1-5p and miR-US25-2-5p were higher in some newborns with abnormal compared to normal brain imaging, but no significant differences were observed (Fig. 2). Although the literature is scarce, miR-US25-1 and miR-UL112-3p have been shown to be related to liver damage in congenital HCMV-infected newborns. However, further studies are required to confirm the role of these miRNAs.

HCMV infection in transplant patients

HCMV infection remains a significant complication in immunocompromised patients such as transplant recipients who have undergone both solid organ and haematopoietic progenitor cell transplantation. In immunocompetent patients, the immune system is able to control HCMV replication, so these individuals are usually asymptomatic or mildly symptomatic [19]. However, transplant patients undergo long-term immunosuppressive treatment that can frequently lead to uncontrolled virus replication and occasionally severe disease [49]. In addition, in haematopoietic stem cell transplantation, HCMV infection is one of the main factors associated with mortality and serious infectious complications due to the immune reconstitution process [2]. For this reason, it would be beneficial to evaluate whether hcmv-miRNAs could be useful as predictive biomarkers of HCMV replication in transplant patients [50,51].

To the best of our best knowledge, the only existing publication on solid organ transplantation is that of Lisboa et al. [50]. In this study, the authors analysed the expression level of different hcmv-miRNAs in blood samples from HCMV-viraemic solid organ transplanted patients to determine if these miRNAs could be predictive of HCMV recurrence. Only detection of hcmv-miR-UL22A-5p at the beginning of antiviral therapy was independently predictive of HCMV recurrence (odds ratio 3.024, 95% CI 1.35–6.8, p = 0.007). They then investigated the effects of hcmv-miR-UL22A-5p at the protein level and found that transfection of hcmv-miR-UL22A-5p modified the expression pattern of several proteins involved in antigen processing and presentation indirectly via C-MYC targeting, a transcriptional regulator that modulates the host cellular immunity. Therefore, the authors suggest that hcmv-miR-UL22A-5p could be evaluated as a clinical biomarker (Fig. 2).

In haematopoietic stem cell transplantation, the determination of hcmv-miRNAs in both donors and recipients could also be useful in predicting HCMV replication after transplantation. However, the existing literature on this topic is also scarce. Talaya et al. [51] assessed the role of some hcmv-miRNAs as biomarkers for HCMV viraemia after transplantation. They analysed hcmv-miR-UL22A-5p, UL36-5p, UL33-5p, UL148D and UL112-3p expression in plasma samples of recent haematopoietic stem cell transplant patients. The authors did not find differential expression between transplant patients with or without subsequent HCMV viraemia. Therefore, they concluded that none of the miRNAs analysed could be considered potential biomarkers of HCMV viraemia. Despite the limited literature on the role of hcmv-miRNAs as a predictor of HCMV replication/recurrence in immunocompromised patients, hcmv-miR-UL22A-5p is a promising candidate in solid organ transplantation, although further research is needed to confirm these findings.

Response to treatment against viral infections

Occasionally, HCMV infection can modulate host immune response and influence infection by other viruses, such as the hepatitis B virus. Hepatitis B infection remains endemic in large geographic areas and represents a major global healthcare challenge as it causes acute and chronic liver diseases. Chronic hepatitis B (CHB) is a leading cause of mortality worldwide, with approximately 780,000 deaths annually [52]. Although IFNalpha (IFNα) is widely used for the treatment of acute or chronic HBV infection, due to the multiple contraindications and safety concerns of IFN-based regimens, the use of molecular biomarkers to predict the efficacy of IFNα treatment prior to treatment would be extremely useful.

In this regard, a recent study by Pan et al. [53] suggests that certain hcmv-miRNAs may interfere with the T-cell-mediated immune response required to achieve optimal efficacy of IFNα treatment in individuals with CHB. They analysed whether a specific profile of serum hcmv-miRNAs can be used as a fingerprint to predict the efficacy of IFNα treatment in CHB patients. After comparing the expression level of hcmv-miRNAs in patients who were responsive and not responsive to the treatment, the authors observed that the absolute level of hcmv-miR-UL148D and hcmv-miR-US4-1 was 3.0- and 5.2-fold greater, respectively, in the IFNα non-responsive group. However, when the authors attempted to establish a threshold for miRNA expression to discriminate between responsive and non-responsive patients, only hcmv-miR-US4-1 accurately predicted the efficacy of IFNα therapy. Specifically, serum levels of hcmv-miR-US4-1 correctly predicted 42 out of 50 CHB patients who were responsive to IFNα therapy and 33 out of 46 CHB patients who were non-responsive. The authors hypothesized that since hcmv-miR-US4-1 downregulates aminopeptidase ERAP1 expression, which is involved in antigen presentation to cytotoxic CD8 + T cells, the inhibition of HBV peptide presentation by hcmv-miR-US4-1 might contribute to the ineffective response of CHB patients to IFNα treatment. Hence, hcmv-miR-US4-1 could be considered a potential predictive biomarker of the efficacy of IFNα therapy (Fig. 2).

Future perspectives and conclusions

Although it is well known that HCMV has developed a multitude of mechanisms to persist for life inside the individual, some of these mechanisms, such as protein synthesis, only occur during the lytic cycle. However, the biogenesis of hcmv-miRNAs takes place during the lytic and latent cycle of the virus. For this reason, there is increasing interest in investigating the role that these miRNAs play in the development or progression of human diseases. Furthermore, miRNAs can travel through biological fluids to other target cells via the exosomal pathway, which gives us an idea of their potential as non-invasive biomarkers. Given that the existing literature on the pathophysiological role of hcmv-miRNAs and their potential utility as prognostic, diagnostic or therapeutic efficacy biomarkers is still scarce, this area is worth exploring.

The available studies reveal interesting associations between some hcmv-miRNAs and certain cardiovascular diseases, such as atherosclerosis; or some types of cancer, such as GBM, where certain hcmv-miRNAs can provide useful information about the diagnosis or prognosis of this disease. Nevertheless, despite the relevance of HCMV infection in transplant patients and the need for new biomarkers to prevent HCMV complications, the literature on the potential role of hcmv-miRNAs in predicting HCMV recurrence in transplant patients is very limited, especially in haematopoietic stem cell transplantation, where hcmv-miRNAs have not yet been found to be associated with HCMV infection. Last, but not least, the expression of certain hcmv-miRNAs is involved in reducing the efficacy of treatments for other viral diseases, which may have important therapeutic implications for this and other viral infections.

The literature reviewed in this manuscript highlights hcmv-miR-UL112-3p as one of the hcmv-miRNAs with the greatest potential for clinical and therapeutic applications since it can alter the expression of genes involved in different cellular signalling pathways. Given its association with the development and progression of GBM, hcmv-miR-UL112-3p is a potential therapeutic target. Since this miRNA directly targets and suppresses the expression of the TUSC3 tumour suppressor gene, a feasible strategy to block GBM progression would be to inhibit the suppressive effect of hcmv-miR-UL112-3p on TUSC3 [54]. There are two main miRNA-based therapeutic strategies: miRNA antagonists and miRNA mimics. A miRNA antagonist (antisense) is chemically modified to inhibit miRNA through complementary binding to the miRNA strand. This strategy could be applied to inhibit miRNAs with undesired functions. MiRNA mimics are synthetic double-stand RNA which mimic endogenous miRNAs. They are used to enhance levels of miRNAs which are crucial to control disease progression [55,56]. Antisense therapy has been used to reverse the inhibition of chemokine RANTES secretion by hcmv-miR-148D [57] or sequester the liver-specific miRNA-122 and downregulate hepatitis C virus replication [58]. More recently, these strategies have been used in other herpesviruses such as Kaposi’s sarcoma-associated herpesvirus, in which antisense drugs have been successfully tested to inhibit some KSHV-miRNAs, thus preventing the proliferation of lymphoma cells [59]. Therefore, targeting hcmv-miRNAs and viral miRNAs in general constitutes a promising therapeutic strategy that might change conventional treatments.

Funding Statement

This work was supported by the Consejería de Salud y Familias, Junta de Andalucia [B-0010-2017]; REIPI [RD16/0016/0008]; Consejería de Economía, Conocimiento, Empresas y Universidad, Junta de Andalucia [CV20/81659].

Disclosure of potential conflicts of interest

No potential conflict of interest was reported by the author(s).