MicroRNAs (miRNAs) are a novel class of small non-coding single-stranded RNAs that regulate gene expression at the posttranscriptional level by binding to target messenger RNAs (mRNAs) [1,2]. Although it is likely that not all of their functions have been fully identified, emerging evidence indicates that miRNAs play a major role in fundamental cellular processes, the regulation of the immune system, and in the onset and progression of many diseases [3,4,5]. Using in silico approaches it has been estimated that miRNAs regulate more than 60% of human protein-coding genes [6], and over 2,000 human mature miRNAs have been annotated (miRBase v19.0; http://www.mirbase.org/). They are transcribed in the nucleus by RNA polymerase II as primary miRNAs (pri-miRNA) that harbor the mature miRNA sequence within the stem of an imperfect ~80 nt hairpin RNA (reviewed in [7]). The pri-miRNA is processed by the microprocessor, consisting of nuclear RNAse III enzyme Drosha and the double stranded RNA-binding protein partner DiGeorge syndrome Critical Region 8 (DGCR8), into precursor miRNA (pre-miRNA) that is subsequently transported to the cytoplasm. The pre-miRNA is cleaved by the cytoplasmic enzyme Dicer into an imperfect 22 nucleotide RNA duplex characterized by two nucleotide 3’ overhangs at each end. Generally, the miRNA strand that exhibits weaker 5’ base pairing is preferentially loaded onto RNA-Induced Silencing Complex (RISC) that guides the recognition of partial matches, generally within the 3’ untranslated region (UTR) of mRNAs. The binding of miRNA to its cognate sequence on the mRNA leads to translational repression or enhanced mRNA degradation (Figure 1). Two independent recent studies have determined the kinetics of translational repression and mRNA decay and have found that miRNAs seem to first block translation of their mRNA target and, subsequently, to mediate its degradation [8,9], although whether this is a general mechanism remains to be demonstrated.

Interestingly, recent data support the notion that miRNAs are key players in virus-host interactions and viral pathogenesis [7,10,11,12,13,14,15,16]. The role of miRNAs in the complex regulatory network that controls both viral and host gene expression in the infected cell is starting to be elucidated for some pathogenic viruses. DNA viruses can encode their own miRNAs, and more that 225 viral miRNAs have been identified, although the function of only a few miRNAs has been demonstrated [17,18]. In contrast, the existence of viral miRNAs in RNA viruses is controversial. At least theoretically, the lack of access to nuclear miRNA processing machinery, and the destabilizing effects of miRNA processing on RNA genomes are major barriers that RNA viruses would need to overcome. Remarkably, and despite those barriers, retroviruses, a flavivirus, and influenza virus have been engineered to express biologically active miRNAs or miRNA-like oligonucleotides when a pre-miRNA sequence is incorporated into the viral genome [19,20,21]. These data suggest that viruses with RNA genomes can express miRNAs through Drosha-independent mechanisms. In support of this hypothesis, Hussain, et al., [22] have identified a miRNA-like small RNA in the 3’UTR of West Nile virus, which is produced during viral infection in mosquito cells and, remarkably, leads to an accumulation of GATA4 mRNA that facilitates virus replication. In addition, viral infections trigger changes in the cellular microRNAome that can modulate the expression of host proteins to the benefit of the virus. For example, Hepatitis C virus (HCV) infection enhances miR-130a expression, which in turn inhibits endogenous Interferon-induced transmembrane protein 1 (IFITM1) expression in a hepatoma cell line [23]. Furthermore, cellular miRNAs can target and repress the expression of viral mRNAs [24,25,26]. Although there are some examples on how cellular miRNAs can stimulate virus replication through indirect or unknown mechanisms [27,28], at least one cellular miRNA (miR-122) facilitates viral infection (HCV) through direct target of the 5’UTR of the viral genome [29,30].

Hepatitis C virus (HCV) infection is the leading cause of chronic hepatitis, liver cirrhosis and hepatocellular carcinoma affecting 180 million people worldwide [31]. Currently, there are no protective vaccines against HCV. Although acute HCV infection resolves spontaneously in some patients [32], persistent infection with chronic liver disease develops in more than 70% of patients, of whom approximately 20% will develop cirrhosis [33]. The present standard of care, a combination of pegylated interferon (Peg-IFN)-α and ribavirin, is suboptimal and sustained virological response is achieved only in about 50% of patients (depending on the viral genotype) and the treatment is associated with several side-effects, some of which can be severe [34]. After more than two decades in which no new antivirals against HCV had been developed, two NS3/4A protease inhibitors, Telaprevir and Boceprevir, were approved by the FDA earlier this year. The drugs have improved response rates, however because they need to be administered with the standard treatment to achieve sustained viral clearance, the therapy is associated with a higher risk of adverse events [35,36]. The search for an interferon-free regimen and the discovery of the remarkable role of miR-122 in the HCV viral cycle has paved the way for a novel class of drugs called antagomirs, which silence endogenous microRNAs, to be investigated as a therapeutic option against chronic HCV infections [37,38]. The specific inhibitor of miR-122 (Miravirsen, a β-D-oxy-Locked Nucleic Acid (LNA)-modified phosphorothioate anti-sense oligonucleotide; Santaris Pharma A/S) is the first miRNA-targeting treatment to enter into human clinical trials, and it is currently in phase 2 for the treatment of HCV infection [38].

Activation of the innate immune response through Toll-Like Receptors (TLRs) has a profound impact on the cellular microRNAome. Several miRNAs have been shown to be up-regulated in response to TLR ligands, and many directly target components of the TLR signaling system, and downstream effectors such as interferons (IFNs), cytokines and chemokines (reviewed in [39,40]). Human Immunodeficiency Virus (HIV-1) infection of macrophages plays a key role in viral pathogenesis and progression to AIDS. Interestingly, our group has provided evidence that miR-155, a key regulator of inflammatory and immune responses, exerts an anti-HIV-1 effect by targeting several HIV-1 dependency factors involved in post-entry and pre-integration events, leading to severely diminished HIV-1 infection of macrophages [41]. Our studies provide evidence of novel microRNA-155 targets and may serve as the basis for innovative approaches to reduce cellular susceptibility to HIV-1 infection.

Due to their similar routes of transmission (sexual and blood-borne), co-infection of HIV-1 and HCV is relatively common. Of the 35 million people infected with HIV-1 worldwide, around 20% (seven million) had chronic hepatitis C [42]. This population is mainly represented by individuals with a past history of intravenous drug use, although frequent outbreaks of HCV among homosexual men have been reported in the last decade [43]. The effects of HIV-1 on the pathogenesis of HCV infection include increased viral loads, a higher rate of viral persistence, a faster rate of fibrosis progression, and higher rates of hepatic decompensation [44,45,46]. The reciprocal effects of HCV on HIV-1 disease progression are less clear. Based on our current understanding, the major contribution of HCV to mortality of HIV-1 co-infected individuals is attributable to accelerated liver disease and not to increased AIDS-related complications [47,48].

In this review, we focus on the dynamic interactions between HCV and the host miRNA pathway and its effect on disease pathogenesis. In addition, based on data from the literature on HCV and HIV-1 mono-infection, we have identified some miRNAs of interest in the setting of HCV-HIV-1 co-infection. We discuss their potential role in co-infection with HCV and HIV-1 viruses.

The HIV-1 life cycle is fundamentally different from that of HCV. HIV-1 is a retrovirus that enters susceptible host cells such as CD4+ T cells and macrophages containing two copies of positive sense, single-stranded RNA (which encodes for nine genes) and enzymes needed for its productive infection such as reverse transcriptase, protease and integrase. The viral RNA undergoes reverse transcription to form double stranded viral DNA, which is transported to the nucleus for integration into the host genome [114]. On the other hand, HCV replicates via an RNA-dependent RNA polymerase and does not have a DNA intermediate [49]. Despite their well-characterized differences at the molecular biology level, the pathogenesis of HCV and HIV-1 co-infection remains poorly understood.

The development of HCV infection is dramatically influenced during co-infection with HIV-1, resulting in augmented viral replication and accelerated progression to chronic hepatitis [115]. About 30% of HIV infected individuals are estimated to be co-infected with HCV [116]. With the development of the current regimens of anti-retroviral therapy (cART), life expectancy of HIV-1 infected individuals has extended significantly. Cirrhosis and end-stage liver disease has become one of the predominant causes of morbidity and mortality in HIV-1/HCV co-infected patients on cART, accounting for up to 50% of all deaths [117,118]. In addition, it has been characterized that patients with HIV-1/HCV co-infection have higher HCV RNA abundance as compared to HCV mono-infected patients [119,120], insinuating potential molecular mechanisms that may be altered in the co-infected patients.

The mechanisms underlying the mutually influential roles of HIV-1 and HCV in co-infected patients are still incompletely understood. Some studies have reported that HCV may negatively affect proliferation of T cells by up-regulating apoptotic pathways [121,122]. On the other hand, HIV-1 infection of CD4+ T cells has a profound effect in impairing adaptive immune responses towards HCV infected cells contributing to poor natural clearance of the virus [123]. A recent study has suggested that the chronic immune activation characteristic of HIV-1 infection and progression to AIDS [124,125] is a distinguishing feature of the host response to HCV/HIV-1 co-infection [126].

Previous studies have demonstrated that HCV mono- and HCV/HIV-1 co-infection induce unique immunologic gene expression profiles in PBMCs [127]. While HIV-1 has evolved to successfully infect cells of the monocyte/macrophages lineage [128], productive infection of HCV in these cells is controversial [129]. However, preliminary evidence of potential HCV replication in monocytes and macrophages has been reported but it requires substantial exploration [130,131]. Despite numerous reports in addition to the ones mentioned above that have studied HIV-1/HCV co-infection, very few have characterized specific alterations at a molecular level that could play a crucial role in HIV-1/HCV co-infected patients.

The role of miRNAs in HIV-1 infection is a vastly expanding field [132,133,134]. The importance of microRNA modulation in HIV-1 replication has been reported, albeit only in the context of HIV-1 mono-infection. In a seminal paper in 2007, Huang et al., found that the 3' ends of HIV-1 messenger RNAs are targeted by a cluster of cellular miRNAs including miR-28, miR-125b, miR-150, miR-223 and miR-382, which are enriched in resting CD4+ T cells as compared to activated CD4+ T cells [135]. Their data demonstrates that cellular miRNAs are pivotal in HIV-1 latency and suggests that manipulation of cellular miRNAs could be a novel approach for purging the HIV-1 reservoir. In addition, a potential role for miR-28, miR-150, miR-223 and miR-382 as “anti-HIV-1 miRNAs” has been recently reported in monocytes [136]. The authors suggested that the existence of these anti-HIV miRNAs contributed to the low susceptibility of monocytes to HIV-1, and differentiation of monocytes into macrophages reduced these miRNAs facilitating infection and active replication [136]. However, their role as anti-HIV-1 miRNAs in monocytes and their modulation during monocyte-to-macrophage differentiation remains highly controversial issues [137]. Likewise, miR-198 and miR-27b in monocytes and CD4+ T cells, respectively, have been shown to inhibit HIV-1 replication by repression of Cyclin T1 expression, an important cellular factor required for Tat-mediated transactivation of HIV-1 LTR-directed gene expression [138,139]. In addition to the cellular miRNAs, HIV-1’s own TAR miRNA is encoded in the viral LTR and manipulating this miRNA through drugs has been shown to reduce viral transcription (19). Overall, these reports emphasize the key role of miRNAs in suppressing HIV-1 infections.

Given the plethora of publications characterizing the importance of various microRNAs in HIV-1 and HCV infection independently, it is surprising that no reports thus far have characterized the alterations in microRNA expression profiles upon HIV-1/HCV co-infection in vitro or in patients. Based on data in the literature, we have identified several miRNAs that could be of relevance for the pathogenesis of HCV/HIV-1 co-infection.

The role of miRNAs in regulating not only normal cellular processes but also their contribution to viral pathogenesis and the replication of viruses is already remarkable, despite the fact that we may have uncovered only a fraction of the likely very large number of functional interactions of microRNAs with implications in viral pathogenesis. HCV in particular appears to be profoundly influenced by the host intracellular miRNA environment for its replicative cycle. The ability of the virus to hijack the cellular miRNA machinery for its survival serves as yet another example of how viruses co-evolve with the host. HCV infection is associated with intracellular miRNA changes that are induced as a combined consequence of the host response and perhaps direct effects of the virus. These miRNA alterations have been proposed to serve as useful biomarkers for diagnosis, prognosis and response to therapy during HCV infection. Furthermore, over the past few years, the concept of extracellular, circulating miRNAs has gained momentum for its potential to serve as non-invasive molecular markers for various diseased states [73,151,152]. Circulating miRNAs shown to exist in two forms—as exosomes/microvesicles encapsulated and as Ago2-associated miRNAs that are highly stable and resistant to nuclease degradation [153,154,155]. One of the mechanisms for release of miRNAs into the circulation is from cells undergoing necrosis and apoptosis. In regard to HCV infection, serum miR-122 is found to be elevated in chronically HCV infected patients likely due to hepatic tissue damage and it correlates with levels of serum alanine aminotransferase and aspartate aminotransferase, and with the histologic activity index score in the liver [91]. In contrast, other studies have found no correlation of miR-122 expression of liver biopsies with viral load from subjects with chronic hepatitis C undergoing IFN therapy; and markedly decreased pretreatment miR-122 levels in subjects who had no virological response during later IFN therapy [99]. Therefore, the potential use of miR-122 as a biomarker in chronic hepatitis C and HCC needs to be further defined and remains as a possibility that requires further investigation. Apart from miR-122, HCV patients also exhibit increased serum levels of miR-34a and -16 wherein both miR-122 and -34a are reflective of the liver fibrotic stage and hepatic inflammation [92]. In-depth knowledge of miRNA modulation in HCV associated disease will aid in truly exploiting the potential of miRNAs to serve as biomarkers with a real diagnostic or prognostic value.

Apart from the biomarker purpose, identification of miRNA alterations that make the cellular environment more conducive to HCV replication could reveal targets for antagomir-based therapy against HCV infection. Indeed, mounting knowledge on the role of cellular miR-122 in maintaining HCV abundance has ultimately led to the development of the first antagomir based therapeutic intervention against HCV infection. The advancement of Miravirsen to phase 2a clinical trials [38] represents not only a milestone for the field of HCV research but also for the antimiR-based technology as a promising therapeutic platform. However, caution needs to be maintained, as it remains unknown if long-term suppression of an endogenous miRNA will adversely affect the homeostatic balance maintained by the body.

A current challenge is to get a better molecular understanding of HCV/HIV-1 co-infection. This knowledge is essential to clearly comprehend the differential molecular mechanisms that may contribute to pathogenesis in infected patients. The expression of miRNAs is highly specific to the cell type or tissues being studied. A difficulty towards the understanding of the role of the miRNAome in HCV/HIV-1 co-infection is to identify cell types with biological significance for both viruses to perform mechanistic studies in cell culture. It is important to point out that most of the studies in the literature characterizing microRNA changes upon HCV infection are in hepatoma cell lines or liver tissue from HCV-infected patients. However, the liver is neither targeted by HIV-1 to establish a productive infection nor has a major role in the development of AIDS. In addition, the ability of HCV to productively infect human cells other than hepatocytes is controversial. In spite of these major differences in tropism, both viruses influence the complex pathogenesis in HCV/HIV-1 co-infected individuals, albeit to varying extents. A recent report characterized microRNA changes in PBMCs from HIV-1 infected patients [156]. These authors noted that 33% of global microRNAs that were up-regulated by HCV in hepatoma cell lines, as reported by another group [88], were also up-regulated in PBMCs from HIV-1 infected patients in their study. These data suggests that host cells might respond to pathogens using similar miRNAs as part of the interactions between the pathogen and the host. Conversely, there was no overlap in the miRNAs down-regulated by each virus, which indicates that viruses might target certain miRNAs as part of a general virus-host interaction to aid replication and survival.

Emerging evidence demonstrates that secreted miRNAs are key regulators of the tumor microenvironment in the liver, and neurodegenaration in the central nervous system [157,158]. The recent discovery of miRNAs as paracrine agonists of TLR7 and TLR8 [157] could represent new avenues for the treatment of chronic viral infections, such as HCV and HIV-1. Furthermore, although the role of exosomes in cell-to-cell communication is well known (reviewed in [159]), several groups have recently demonstrated that secreted miRNAs enclosed in exosomes have an important role in cell-to-cell communication (reviewed in [160]). In particular, the functional transfer of miRNAs between cells of the same or distinct type has been reported [161,162,163]. Indeed, there are significant recent advances on the intercellular transfer of miRNAs by exosomes in several clinically relevant scenarios, such as cancer [163,164,165,166], the regulation of the immune system [167,168], and liver [169,170], central nervous system [171], and infectious diseases [172]. To add to this intriguing interplay between exosome-mediated cell-to-cell communication and horizontal modulation of gene expression by miRNAs, some viruses including HIV-1 and HCV, utilize exosomes for intercellular communication [173,174,175,176], and viruses that express miRNAs exploit the endosomal-exosomal pathway for intercellular cross-talk and immune evasion [177]. Thus, considering the different tropism of HIV-1 and HCV viruses, it is possible that circulating miRNAs in exosomes secreted by non-hepatocyte HIV-1-infected cells were taken by HCV-infected hepatocytes as a part of the cell-to-cell communication process, and affect the expression of some cellular factors in the HCV-infected hepatocyte which may ultimately impact disease outcome in HCV/HIV-1 co-infected patients.

Overall, the modulation of the expression of miRNAs in the human population represents a difficult task in the years to come. Could it be possible in the future to successfully fight against viral infections using “antagomir” or “mimics” as therapeutic approaches with none or minimal side effects? The challenge is on us.