Pediatric Pulmonology
Review
Respiratory Syncytial Virus Infection of Airway Cells: Role of microRNAs Giovanni A. Rossi,
1 MD, *
Michela Silvestri,
PhD,
1
and Andrew A. Colin,
MD
2
Summary. MicroRNAs (miRNAs) are small single-stranded RNA molecules involved in the regulation of gene expression at the post-transcriptional level. In the airways, miRNAs are implicated in the modulation of antiviral defense, through modulation of both innate and adaptive immune response in inflammatory and immune effector cells but also in parenchymal cells. The first target of respiratory viruses are airway epithelial cells. Following infection, an altered expression of distinct miRNAs occurs in airway cells aimed at inhibiting viral replication and preserving the airway epithelial barrier, while at the same time viruses induce or repress the expression of other miRNAs that favor viral replication. Understanding the changes in miRNA expression profile, identification of miRNAs target genes and their contribution to the pathogenesis of the disease may help the intricate mechanisms of virus-host interaction. Further understanding of these molecular mechanisms could lead to development of new antiviral treatments in common, high impact, respiratory disorders for which specific treatments are not available. Respiratory syncytial virus (RSV) airway infection is a common example of virus modifying miRNAs expression to favor immune evasion, and constitutes the salient feature of this ß 2015 Wiley Periodicals, Inc. review. Pediatr Pulmonol. Key words: RNA expression; airway epithelial cells; innate immune response. Funding source: Ricerca Corrente, Italian Ministry of Health, Rome, Italy.
INTRODUCTION
RSV, a negative-strand non-segmented RNA pneumovirus of the family Paramyxoviridae, is a common human pathogen that causes cold-like symptoms in most healthy adults and children.1 It takes, however, a whole new dimension in infants for both its immediate and long-term effects. RSV is more likely to affect the lower respiratory tract (LRT), and is the most common respiratory virus isolated from the majority of infants hospitalized for bronchiolitis during the RSV season.1–3 The primary infection usually causes a severe illness, while subsequent infections tend to induce bronchial obstructive episodes that become progressively milder over later childhood.1–3 The dynamics of the RSVhost interface in response to infection is complex and not completely understood. RSV is a “parasite” that must interface with host cellular machinery to achieve an optimal balance between viral and cellular gene expression and co-opt host genes to favor viral replication.4–6 There is convincing evidence that degree of virus replication correlates with disease severity.7,8 In the earliest phases of the infection, activation of defense ß 2015 Wiley Periodicals, Inc.
mechanisms and regulation of viral replication is governed by an array of mechanisms4,5,9 that involve gene expression that are subsequently further modulated by other factors. Of the latter microRNA (miRNA)
1 Pulmonary and Allergy Disease Paediatric Unit and Cystic Fibrosis Center, Istituto Giannina Gaslini, Genoa, Italy. 2 Division of Pediatric Pulmonology, Miller School of Medicine, University of Miami, Miami, Florida.
Conflict of interest: none.
Correspondence to: Giovanni A. Rossi, MD, Pediatric Pulmonology and Allergy Unit and Cystic Fibrosis Center, Istituto Giannina Gaslini, Via Gerolamo Gaslini 5, 16147 Genoa, Italy. E-mail: [email protected] Received 18 December 2014; Revised 2 March 2015; Accepted 5 March 2015. DOI 10.1002/ppul.23193 Published online in Wiley Online Library (wileyonlinelibrary.com).
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species have recently emerged as gene expression regulators that play a modulatory role in virus infection by modifying host responses in inflammatory and immune cells as well as airway epithelial cells.10 RSV Replication in Airway Epithelial Cells
RSV possess a 15 kb non-segmented, negative-sense, single-stranded RNA (ssRNA) genome containing 10 genes.1,4,5 Eleven proteins are translated from these 10 genes: three transmembrane surface glycoproteins (the attachment G protein, the fusion F protein, and the small hydrophobic SH protein), three proteins associate with genomic RNA to form the nucleocapsid (the N protein, the P phosphoprotein and the large L polymerase subunit), two nonstructural proteins (the NS1 and NS2 proteins), two transcription and RNA replication factors (M2-1 and M2-2), and one unglycosylated matrix M protein.4,5 RSV attachment to airway epithelial cells occurs via the surface G protein that binds cell-surface glycosaminoglycans, while host cell membrane fusion is mediated by the surface F protein.4,5,9 The viral envelope is then incorporated into the cell membrane, the nucleocapsid is released into the cytoplasm where the L protein initiates viral transcription and subsequent replication. Transcription involves a sequential stop-start mechanism that produces subgenomic mRNAs: the N, P, and L proteins constitute the replicase, whereas the transcriptase also requires the M2-1 and M2-2 proteins.4,5,9 The NS1 and NS2 proteins are not essential for viral replication in vitro but interfere with cell defense mechanisms by downregulating antiviral response and their ablation significantly attenuates RSV growth.4,5,9 RSV mRNA can be detected in airway epithelial cells within 4 hr postinfection, accumulates until about 15 hr after infection and then remains at constant levels allowing RNA replication, protein synthesis and hence viral assembly.4,5,9 The virions then mature in clusters at the apical surface of the airway epithelial cells in a filamentous form and extend from the plasma membrane: budding appears to be the reverse of penetration and occurs in vitro on the apical cell surface.4,5,9 Infection of the neighboring cells may also take place through membrane fusion with syncytia formation through the F protein.9 Host Immune Response to RSV Infection
All viral infections are characterized by rapid induction of immune mediators through the activation of the pattern recognition receptors (PRRs) that, as their name denotes, use a wide variety of pathogen-associated molecular patterns (PAMPs) to identify impending risks. In airway epithelial cells, the PRRs involved in the induction of the innate response include the toll-like receptor (TLR) 3 and 4, the cytoplasmic retinoic acid inducible gene (RIG)-I like receptor dsRNA helicase (RLH) enzyme and the Pediatric Pulmonology
melanoma differentiation-associated protein (MDA) 5, that is also part of the RIG-I-like receptor (RLR) family.4,5,9,11 Recognition of RSV proteins and replication products by PRRs results in activation of transcription factors, such as the nuclear factor kappalight-chain-enhancer of activated B cells (NF-kB), the activator protein 1 (AP-1), the c-Jun-N-terminal kinase (JNK), the interferon regulatory factor (IRF)-1, -3 and -7 and the Janus kinase (JAK)/signal transducers and activators of transcription (STAT) signaling pathway.4,5,9,11 The sum of activation of these transcription factors leads to downstream signaling to create a broadly effective antiviral state. RSV Strategies to Escape Host Immune Response
RSV utilizes a number of strategies to escape host cell surveillance through mechanisms leading to neutralization of pro-apoptotic signals and to inhibition of antiviral activities.4,5,9 Rapid induction of apoptosis (programmed cell demise, that otherwise can be thought of as cell suicide) of a virus-infected cell is a vital aspect of innate protection since it aborts viral replication and spread of infection and allows the infected cell to be phagocytosed. An infected cell failing to undergo apoptosis will replicate virus, produce proinflammatory mediators and undergo cell death by lysis thus releasing a large numbers of progeny viruses that infect the neighboring cells.4,5 RSV infection stimulates the expression of nerve growth factor (NGF) and its tropomyosin-related kinase A (TrkA) high affinity receptor and markedly down-regulates the low affinity p75 neurotrophin receptor (p75NTR).12 This pattern of neurotrophin expression boosts viral replication by delaying the infected cell apoptosis via activation of the phosphoinositidide 3-kinase pathway.12 In addition, RSV may antagonize the interferon (IFN)-mediated antiviral responses in epithelial cells by inducing the suppressor to cytokines signaling (SOCS) gene expression.13 SOCS proteins inhibit the transcription of type I IFN-a and-b mRNAs and utilize a feedback loop to inhibit effective cytokine responses.11,13 IFN-a and-b production by the infected host epithelial cells is also suppressed by the NS1 and NS2 proteins; these block IRF-3 activation, decrease the expression of STAT2 and down-regulate the RIG-I-mediated type I IFN promoter activation.4,5,11,14 A breakthrough in biology has been the discovery of small noncoding RNAs (sncRNAs), such as transfer RNAs (tRNAs) and microRNAs (miRNAs), that appear to play regulatory functions in RSV infection. A recent report showed up-regulation of a tRNA-RNA fragment (tRF-5-GluCTC) by RSV infection promoting RSV replication.15 More information, however, is emerging on the key role of miRNAs, in RSV strategies of escaping host cell surveillance.10,16
Modulation of RSV Infection by MicroRNAs
miRNAs and Viral Infections
miRNAs are short (20–23-nucleotide), endogenous, ssRNA molecules that regulate gene expression.10,17 Most miRNA genes are transcribed by RNA polymerase II into primary miRNA transcripts that are processed in the nucleus by a complex containing the RNase III endonuclease Drosha (Fig. 1). The resulting precursor miRNAs are transported to the cytoplasm where the mature miRNAs are excised by a complex containing the endonuclease Dicer. Mature miRNAs are incorporated into the RNA-induced silencing complex, which binds to the three prime untranslated region (30 -UTR) of target mRNAs, repressing their translation and/or inducing their degradation (Fig. 1).10,16 The human genome may encode over 1,000 miRNAs which appear to target about 60% of the human genes, and it hence stands to reason that posttranscriptional regulation of gene expression by miRNAs is critical for a wide range of cellular functions.17 Even
3
though the biological significance of miRNAs is understood only in a limited number of instances, specific miRNAs have been implicated in various biological events, including cell proliferation and differentiation, apoptosis, morphogenesis, development, and oncogenesis.17–19 More recently, miRNAs have been shown to affect numerous processes pertaining to viral infections, such as modulation of the innate and adaptive immune responses, of cell cycle progression, and of apoptosis induction.20 Virus infection alters host gene transcription and transcript translation not only through antagonism to host protein function or induction of RNA stress granules, but also by altering patterns of host gene expression through processes that involve miRNAs.20 miRNAs, RSV Replication and Host Immune Response
RSV infection is influenced by a variety of miRNAs, which are all encoded by host cells. Biosynthesis of RSVencoded miRNAs is virtually absent, since the life cycle of this virus is exclusively cytoplasmatic and hence implies lack of access to the nuclear endonuclease Drosha, the obligatory enzyme for generation of primary miRNA transcripts.16 As reported below, RSV-induced miRNA expression occurs through different mechanisms and modulates a variety of host cell functions, some advantageous and others adverse for the virus. Induction of miRNA Expression
Fig. 1. Biogenesis and mechanism of action of miRNAs. miRNAs are synthesized in the nucleus by RNA polymerase II into primary miRNA transcripts. The primary miRNAs are then cleaved by a complex containing the RNase III endonuclease Drosha and then transported to the cytoplasm by the exportin-5RAN-GTP complex. Here the mature miRNAs are excised to a short RNA duplex by the endonuclease Dicer. One strand of the duplex is incorporated into the RNA-induced silencing complex (RISC), which binds to the three prime untranslated region (30 UTR) of target mRNAs, repressing their translation and/or inducing their degradation. The second strand can be loaded into another RISC complex or can be degraded.16,17
During RSV infection, both viral proteins and mediators released by the host cells were found to be involved in RSV-induced miRNA expression.21 In a study using normal human bronchial epithelial cell line cultures, RSV inoculation caused a significant decrease in the expression of miR-221 and a robust induction of two miRNA; let-7i and miR-30b. The study also showed that the intensity of the response depended on virus concentration in the inoculum and active viral replication (Fig. 2).22 However, the increase in let-7i induction was observed 24 and 48 hr post virus inoculation, while detectable miR-30b was found only 48 hr post inoculation, pointing to different pathways. The hypothesis of differential pathways was further supported by the observation that let-7i, but not miR-30b levels, were enhanced by the presence IFN-b in a concentrationdependent manner, whereas miR-30b induction required mechanisms involving the NF-kB pathway.22,23 Indeed, both NF-kB activation and IFN-b production are induced in airway epithelial cells following RSV infection.4,5 The roles of let-7i and miR-30b have not been directly studied in RSV infection, but in biliary and in hepatoma cell lines both miRNA contribute to strengthening of epithelial immune responses against viral infections and inhibition of viral replication.24,25 Another important finding in that study was that up-regulation of let-7i and miR-30b was Pediatric Pulmonology
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miRNAs were transfected into non-RSV-infected cells, to screen their role in the modulation of neurotrophin expression. Maximal down-regulation of both NGF and TrkA, the receptor that increases antiapoptotic factors, was detected in cells over-expressing miR-221.27,28 In contrast p75NTR, the receptor that promotes apoptosis, was not affected by the over-expression of miR-221.27,28 As expected, cells transfected with miR-221 were more prone to apoptosis and, after infection with RSV, showed significantly lower NGF protein expression and a sharp reduction in the proportion of infected cells. Thus, by silencing miR-221, RSV up-regulates the NGF-TrkA axis that favors viral replication by inhibiting the apoptosis of infected cells (Fig. 3). The G Protein and the Complexity of miRNA Functions
Fig. 2. Regulation of miRNA expression. RSV infection induces let-7i and miR-30b expression that is dependent on the viral inoculum dose and on active RSV replication. Let-7i appears to be enhanced by the presence IFN-b, while induction of miR-30b requires NF-kB activation. Expression of let-7i and miR-30b, miRNA that favor inhibition of viral replication, is inhibited by NS1 or NS2 proteins.
The attachment G protein also regulates host cell defensive functions through a variety of mechanisms, including the modulation of miRNAs encoded by host cells.4,5,29 The high glycosylation of G protein helps evade effective recognition by the immune system, and its high variability allows easy escape from neutralizing antibodies.4,5,29 Furthermore, during viral replication, a soluble form of G protein is released and binds RSVspecific antibodies and hence reducing the concen-
further enhanced when the cells were infected by RSV strains lacking NS1 or NS2 genes. NS1 and NS2 proteins may antagonize the up-regulation of let-7i and of miR30b expression by inhibiting the production of type I IFNs4,5 and of other cytokines involved in miRNA transcription.22 Thus, RSV appears to counteract the miRNA-related response of the host cells through NS1 and NS2 viral proteins that, via different mechanisms, reduce the induction of miRNA involved in the inhibition of viral infection (Fig. 2). Interestingly, NS1 and NS2 proteins may also favor RSV replication through NF-kBdependent, IFN-independent pathways, by suppressing premature host cell apoptosis.26 Neurotrophin Activity, miRNA, and Cell Apoptosis
Host cell apoptosis during RSV infections may also be controlled by miRNAs that regulate neurotrophin activity. A study of RSV infection of human bronchial epithelial cell primary cultures showed that among the miRNAs significantly affected by the infection, six (miR-27a, miR221, miR-339-5p, miR-453, miR-574, and miR-744) were highly complementary to NGF and/or the NGF receptors TrkA and p75NTR.27 With the exception of miR744, all the other miRNAs appeared to be significantly down-regulated. To screen their role in the modulation of neurotrophin expression, precursors for each of these Pediatric Pulmonology
Fig. 3. Modulation of neurotrophin expression and RSV replication. RSV infection of bronchial epithelial cells is associated with inhibition of miRNA-221 expression. By silencing miR-221, RSV prevents the miR-221-mediated down-regulation of NGF and TrkA receptor expression and promotes viral replication by inhibiting bronchial epithelial cells apoptosis. p75NTR, the receptor that promotes apoptosis, is not affected by changes in miR-221 expression.27
Modulation of RSV Infection by MicroRNAs 4,5,29
trations available for RSV neutralization. A study using recombinant RSV (6340WT) or recombinant RSV lacking the G gene (RSVDG), revealed that in human alveolar epithelial cells both replicated to similar levels but showed a differential pattern of induction of miRNA expression.30 As compared with 6340WT infected cells, those infected by RSVDG showed lower let-7f levels, higher levels of miR-337 and miR-24 and similar levels of miR-520a and miR-26b. The most interesting among these miRNA appeared to be let-7f because, after infection with 6340WT, the induction of the “let-7 family” was consistent among replicates, and let-7f showed the highest up-regulation among various let-7 members.30 By increasing the transcriptional activity of let-7f, the RSV G protein significantly down-regulated three “cell proliferation and survival” genes (CCND1, DYRK2, and ELF4), one “chemokine” gene (CCL7/ MCP3) and the SOCS 3 gene (Fig. 4). The degree of inhibition induced by let-7f appeared to be elevated (approximately >40%) for CCND1, CCL7/ MCP3, ELF4, and SOCS3 genes and lower for DYRK2 gene.30 CCND1 promotes G1-to-S phase transition of the cell cycle, whilst DYRK2 and ELF4 promote apoptosis.31,32 In addition, ELF4 up-regulates
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IFN expression in a feed-forward loop and directly activates the T-cell antigen receptor signaling in cytotoxic CD8þ T-cells to induce the arrest of their cell cycle and increase their survival.34 Consistently, ELF4 deficiency resulted in enhanced viral susceptibility.33,34 CCL7/MCP3 is a chemokine, induced by cytokines or exogenous agents (bacteria, viruses) that attracts and activates several types of leukocytes involved in anti-viral defense, including monocytes, lymphocytes, granulocytes, NK cells, and dendritic cells.35 Thus, the let-7f-mediated inhibition of CCND1, DYRK2, ELF4, and CCL7/MCP3 translation can be one of the mechanisms employed by RSV to favor delayed viral clearance (Fig. 4). In contrast, by enhancing the transcriptional activity of let-7f, the RSV G protein supports the host response by decreasing the expression of SOCS3, a regulator of cytokine signaling expression that negatively regulate type I IFNs36 (Fig. 4). These observations substantiate the multifaceted functions of miRNAs in regulating the balance between virus replication and the antiviral host responses. However, disproportion between the “pro” and “con” actions of the increased transcriptional activity of let-7f appears to shift the balance toward a dominant effect of slowing viral clearance. CONCLUSIONS
Fig. 4. The “multifaceted role” of the G protein. By increasing let7f levels in airway cells, the G protein down-regulates genes that favor host cell response to infection. These include the CCND1 gene that promotes host cell cycle progression, the DYRK2 and ELF4 genes, that favor host cell apoptosis and the CCL7/MCP3 chemokine gene, that attracts and activates several types of leukocytes involved in anti-viral defense. Inhibition of these genes translation favors RSV replication delaying viral clearance. By contrast, the G protein induces down-regulation the SOCS3 gene that negatively regulates type I IFNs, thus favoring the host response.
As part of their evolutionary strategies both viruses and host cells are capable to manipulate the miRNAome to regulate the overall innate response, in a virus- and cell-typespecific fashion. Via activation or inhibition of pathways of the classical regulators, such as NF-kB and STAT, and the production of cytokines, such as type I IFNs, RSV up- or down-regulates a variety of miRNA expression. This enhances evasion of immune recognition prolongs host cell survival and establishes viral latency. Consequently, miRNAs may play an important role in modulating the severity and duration of RSV infection and, putatively, its long-lasting sequelae. In the various studies described, different experimental systems were used (including different type of airway epithelial cells and of RSV strains) to evaluate diverse functional effects that involve diverse miRNA. Therefore with the data currently available it is difficult to state with certainty whether the patterns of miRNAs-induced response to RSV infection are truly consistent and uniform. With these limitations in mind, a significant RSV-induced decrease in expression of miR-221, the miRNA endowed of anti-neurotrophic, pro-apoptotic activity, was reported in both the Othumpangat12 and in the Thornburg21 studies. Future research should demonstrate whether the exogenous administration of synthetic miRNAs could antagonize viral replication and hence provide a novel strategy for the therapy of this common infection. Pediatric Pulmonology
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REFERENCES 1. Wright M, Piedimonte G. Respiratory syncytial virus prevention and therapy: Past, present, and future. Pediatr Pulmonol 2011;46:324–347. 2. Hall CB, Weinberg GA, Iwane MK, Blumkin AK, Edwards KM, Staat MA, Auinger P, Griffin MR, Poehling KA, Erdman D, Grijalva CG, Zhu Y, Szilagyi P. The burden of respiratory syncytial virus infection in young children. N Engl J Med 2009;360:588– 598. 3. Midulla F, Scagnolari C, Bonci E, Pierangeli A, Antonelli G, De Angelis D, Berardi R, Moretti C. Respiratory syncytial virus, human bocavirus and rhinovirus bronchiolitis in infants. Arch Dis Child 2010;95:35–41. 4. Openshaw PJ, Tregoning JS. Immune responses and disease enhancement during respiratory syncytial virus infection. Clin Microbiol Rev 2005;18:541–555. 5. Rossi GA, Colin AA. Infantile RSV and HRV infections: respective role in inception and persistence of wheezing. Eur Resp J 2015; [Epub ahead of print]. 6. Guo-Parke H, Canning P, Douglas I, Villenave R, Heaney LG, Coyle PV, Lyons JD, Shields MD, Power UF. Relative respiratory syncytial virus cytopathogenesis in upper and lower respiratory tract epithelium. Am J Respir Crit Care Med 2013;188:842–851. 7. El Saleeby CM, Bush AJ, Harrison LM, Aitken JA, Devincenzo JP. Respiratory syncytial virus load, viral dynamics, and disease severity in previously healthy naturally infected children. J Infect Dis 2011;204:996–1002. 8. Scagnolari C, Midulla F, Selvaggi C, Monteleone K, Bonci E, Papoff P, Cangiano G, Di Marco P, Moretti C, Pierangeli A, Antonelli G. Evaluation of viral load in infants hospitalized with bronchiolitis caused by respiratory syncytial virus. Med Microbiol Immunol 2012;201:311–317. 9. Oshansky CM, Zhang W, Moore E, Tripp RA. The host response and molecular pathogenesis associated with respiratory syncytial virus infection. Future Microbiol 2009;4:279–297. 10. Głobinska A, Pawełczyk M, Kowalski ML. MicroRNAs and the immune response to respiratory virus infections. Expert Rev Clin Immunol 2014;10:963–971. 11. Barik S. Respiratory syncytial virus mechanisms to interfere with type 1 interferons. Curr Top Microbiol Immunol 2013;372:173– 191. 12. Othumpangat S, Gibson LF, Samsell L, Piedimonte G. NGF is an essential survival factor for bronchial epithelial cells during respiratory syncytial virus infection. PLoS One 2009;4:e6444. 13. Hashimoto K, Ishibashi K, Ishioka K, Zhao D, Sato M, Ohara S, Abe Y, Kawasaki Y, Sato Y, Yokota S, Fujii N, Peebles RS, Jr, Hosoya M, Suzutani T. RSV replication is attenuated by counteracting expression of the suppressor of cytokine signaling (SOCS) molecules. Virology 2009;391:162–170. 14. Ling Z, Tran KC, Teng MN. Human respiratory syncytial virus nonstructural protein NS2 antagonizes the activation of beta interferon transcription by interacting with RIG-I. J Virol 2009;83:3734–3742. 15. Wang Q, Lee I, Ren J, Ajay SS, Lee YS, Bao X. Identification and Functional characterization of tRNA-derived RNA Fragments (tRFs) in respiratory syncytial virus infection. Molecular Ther 2013;21:368–379. 16. Bartel DP. MicroRNAs: genomics, biogenesis, mechanism, and function. Cell 2004;116:281–297. 17. Bushati N, Cohen SM. MicroRNA functions. Annu Rev Cell Dev Biol 2007;23:175–205. 18. Fabian MR, Sonenberg N, Filipowicz W. Regulation of mRNA translation and stability by microRNAs. Ann Rev Biochem 2010;79:351–379.
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19. Friedman RC, Farh KK, Burge CB, Bartel DP. Most mammalian mRNAs are conserved targets of microRNAs. Genome Res 2009;19:92–105. 20. Grundhoff A, Sullivan CS. Virus-encoded microRNAs. Virology 2011;411:325–343. 21. Winter J, Jung S, Keller S, Gregory RI, Diederichs S. Many roads to maturity: microRNA biogenesis pathways and their regulation. Nat Cell Biol 2009;11:228–234. 22. Thornburg NJ, Hayward SL. Crowe JE Jr. Respiratory syncytial virus regulates human microRNAs by using mechanisms involving beta interferon and NF-kB. MBio 2012;3:e00220–e00212. 23. Zhou R, Hu G, Gong AY, Chen XM. Binding of NF-kappaB p65 subunit to the promoter elements is involved in LPS-induced transactivation of miRNA genes in human biliary epithelial cells. Nucleic Acids Res 2010;38:3222–3232. 24. Chen XM, Splinter PL, O’Hara SP, LaRusso NF. A cellular microRNA, let-7i, regulates Toll-like receptor 4 expression and contributes to cholangiocyte immune responses against Cryptosporidium parvum infection. J Biol Chem 2007;282:28929–28938. 25. Zhang X, Daucher M, Armistead D, Russell R, Kottilil S. MicroRNA expression profiling in HCV-infected human hepatoma cells identifies potential anti-viral targets induced by interferon-a. PLoS ONE 2013;8:e55733. 26. Bitko V, Shulyayeva O, Mazumder B, Musiyenko A, Ramaswamy M, Look DC, Barik S. Nonstructural proteins of respiratory syncytial virus suppress premature apoptosis by an NF-kappaBdependent, interferon-independent mechanism and facilitate virus growth. J Virol 2007;81:1786–1795. 27. Othumpangat S, Walton C, Piedimonte G. MicroRNA-221 modulates RSV replication in human bronchial epithelium by targeting NGF expression. PLoS ONE 2012;7:e30030. 28. Freund-Michel V, Frossard N. The nerve growth factor and its receptors in airway inflammatory diseases. Pharmacol Ther 2008;117:52–76. 29. Tripp RA, Moore D, Jones L, Sullender W, Winter J, Anderson LJ. Respiratory syncytial virus G and/or SH protein alters Th1 cytokines, natural killer cells, and neutrophils responding to pulmonary infection in BALB/c mice. J Virol 1999;73:7099– 7107. 30. Bakre A, Mitchell P, Coleman JK, Jones LP, Saavedra G, Teng M, Tompkins SM, Tripp RA. Respiratory syncytial virus modifies microRNAs regulating host genes that affect virus replication. J Gen Virol 2012;93:2346–2356. 31. Taira N, Nihira K, Yamaguchi T, Miki Y, Yoshida K. DYRK2 is targeted to the nucleus and controls p53 via Ser46 phosphorylation in the apoptotic response to DNA damage. Mol Cell 2007;25:725– 738. 32. Sashida G, Bae N, Di Giandomenico S, Asai T, Gurvich N, Bazzoli E, Liu Y, Huang G, Zhao X, Menendez S, Nimer SD. The MEF/ ELF4 transcription factor fine tunes the DNA damage response. Cancer Res 2011;71:4857–4865. 33. You F, Wang P, Yang L, Yang G, Zhao YO, Qian F, Walker W, Sutton R, Montgomery R, Lin R, Iwasaki A, Fikrig E. ELF4 is critical for induction of type I interferon and the host antiviral response. Nat Immunol 2013;14:1237–1246. 34. Yamada T, Park CS, Mamonkin M, Lacorazza HD. Transcription factor ELF4 controls the proliferation and homing of CD8þ T cells via the Kr€uppel-like factors KLF4 and KLF2. Nat Immunol 2009;10:618–626. 35. Menten P, Wuyts A, Van Damme J. 2001. Monocyte chemotactic protein-3. Eur Cytokine Netw 2001;12:554–560. 36. Moore EC, Barber J, Tripp RA. Respiratory syncytial virus (RSV) attachment and nonstructural proteins modify the type I interferon response associated with suppressor of cytokine signaling (SOCS) proteins and IFN-stimulated gene-15 (ISG15). Virol J 2008;5:116.
Review
Respiratory Syncytial Virus Infection of Airway Cells: Role of microRNAs Giovanni A. Rossi,
1 MD, *
Michela Silvestri,
PhD,
1
and Andrew A. Colin,
MD
2
Summary. MicroRNAs (miRNAs) are small single-stranded RNA molecules involved in the regulation of gene expression at the post-transcriptional level. In the airways, miRNAs are implicated in the modulation of antiviral defense, through modulation of both innate and adaptive immune response in inflammatory and immune effector cells but also in parenchymal cells. The first target of respiratory viruses are airway epithelial cells. Following infection, an altered expression of distinct miRNAs occurs in airway cells aimed at inhibiting viral replication and preserving the airway epithelial barrier, while at the same time viruses induce or repress the expression of other miRNAs that favor viral replication. Understanding the changes in miRNA expression profile, identification of miRNAs target genes and their contribution to the pathogenesis of the disease may help the intricate mechanisms of virus-host interaction. Further understanding of these molecular mechanisms could lead to development of new antiviral treatments in common, high impact, respiratory disorders for which specific treatments are not available. Respiratory syncytial virus (RSV) airway infection is a common example of virus modifying miRNAs expression to favor immune evasion, and constitutes the salient feature of this ß 2015 Wiley Periodicals, Inc. review. Pediatr Pulmonol. Key words: RNA expression; airway epithelial cells; innate immune response. Funding source: Ricerca Corrente, Italian Ministry of Health, Rome, Italy.
INTRODUCTION
RSV, a negative-strand non-segmented RNA pneumovirus of the family Paramyxoviridae, is a common human pathogen that causes cold-like symptoms in most healthy adults and children.1 It takes, however, a whole new dimension in infants for both its immediate and long-term effects. RSV is more likely to affect the lower respiratory tract (LRT), and is the most common respiratory virus isolated from the majority of infants hospitalized for bronchiolitis during the RSV season.1–3 The primary infection usually causes a severe illness, while subsequent infections tend to induce bronchial obstructive episodes that become progressively milder over later childhood.1–3 The dynamics of the RSVhost interface in response to infection is complex and not completely understood. RSV is a “parasite” that must interface with host cellular machinery to achieve an optimal balance between viral and cellular gene expression and co-opt host genes to favor viral replication.4–6 There is convincing evidence that degree of virus replication correlates with disease severity.7,8 In the earliest phases of the infection, activation of defense ß 2015 Wiley Periodicals, Inc.
mechanisms and regulation of viral replication is governed by an array of mechanisms4,5,9 that involve gene expression that are subsequently further modulated by other factors. Of the latter microRNA (miRNA)
1 Pulmonary and Allergy Disease Paediatric Unit and Cystic Fibrosis Center, Istituto Giannina Gaslini, Genoa, Italy. 2 Division of Pediatric Pulmonology, Miller School of Medicine, University of Miami, Miami, Florida.
Conflict of interest: none.
Correspondence to: Giovanni A. Rossi, MD, Pediatric Pulmonology and Allergy Unit and Cystic Fibrosis Center, Istituto Giannina Gaslini, Via Gerolamo Gaslini 5, 16147 Genoa, Italy. E-mail: [email protected] Received 18 December 2014; Revised 2 March 2015; Accepted 5 March 2015. DOI 10.1002/ppul.23193 Published online in Wiley Online Library (wileyonlinelibrary.com).
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species have recently emerged as gene expression regulators that play a modulatory role in virus infection by modifying host responses in inflammatory and immune cells as well as airway epithelial cells.10 RSV Replication in Airway Epithelial Cells
RSV possess a 15 kb non-segmented, negative-sense, single-stranded RNA (ssRNA) genome containing 10 genes.1,4,5 Eleven proteins are translated from these 10 genes: three transmembrane surface glycoproteins (the attachment G protein, the fusion F protein, and the small hydrophobic SH protein), three proteins associate with genomic RNA to form the nucleocapsid (the N protein, the P phosphoprotein and the large L polymerase subunit), two nonstructural proteins (the NS1 and NS2 proteins), two transcription and RNA replication factors (M2-1 and M2-2), and one unglycosylated matrix M protein.4,5 RSV attachment to airway epithelial cells occurs via the surface G protein that binds cell-surface glycosaminoglycans, while host cell membrane fusion is mediated by the surface F protein.4,5,9 The viral envelope is then incorporated into the cell membrane, the nucleocapsid is released into the cytoplasm where the L protein initiates viral transcription and subsequent replication. Transcription involves a sequential stop-start mechanism that produces subgenomic mRNAs: the N, P, and L proteins constitute the replicase, whereas the transcriptase also requires the M2-1 and M2-2 proteins.4,5,9 The NS1 and NS2 proteins are not essential for viral replication in vitro but interfere with cell defense mechanisms by downregulating antiviral response and their ablation significantly attenuates RSV growth.4,5,9 RSV mRNA can be detected in airway epithelial cells within 4 hr postinfection, accumulates until about 15 hr after infection and then remains at constant levels allowing RNA replication, protein synthesis and hence viral assembly.4,5,9 The virions then mature in clusters at the apical surface of the airway epithelial cells in a filamentous form and extend from the plasma membrane: budding appears to be the reverse of penetration and occurs in vitro on the apical cell surface.4,5,9 Infection of the neighboring cells may also take place through membrane fusion with syncytia formation through the F protein.9 Host Immune Response to RSV Infection
All viral infections are characterized by rapid induction of immune mediators through the activation of the pattern recognition receptors (PRRs) that, as their name denotes, use a wide variety of pathogen-associated molecular patterns (PAMPs) to identify impending risks. In airway epithelial cells, the PRRs involved in the induction of the innate response include the toll-like receptor (TLR) 3 and 4, the cytoplasmic retinoic acid inducible gene (RIG)-I like receptor dsRNA helicase (RLH) enzyme and the Pediatric Pulmonology
melanoma differentiation-associated protein (MDA) 5, that is also part of the RIG-I-like receptor (RLR) family.4,5,9,11 Recognition of RSV proteins and replication products by PRRs results in activation of transcription factors, such as the nuclear factor kappalight-chain-enhancer of activated B cells (NF-kB), the activator protein 1 (AP-1), the c-Jun-N-terminal kinase (JNK), the interferon regulatory factor (IRF)-1, -3 and -7 and the Janus kinase (JAK)/signal transducers and activators of transcription (STAT) signaling pathway.4,5,9,11 The sum of activation of these transcription factors leads to downstream signaling to create a broadly effective antiviral state. RSV Strategies to Escape Host Immune Response
RSV utilizes a number of strategies to escape host cell surveillance through mechanisms leading to neutralization of pro-apoptotic signals and to inhibition of antiviral activities.4,5,9 Rapid induction of apoptosis (programmed cell demise, that otherwise can be thought of as cell suicide) of a virus-infected cell is a vital aspect of innate protection since it aborts viral replication and spread of infection and allows the infected cell to be phagocytosed. An infected cell failing to undergo apoptosis will replicate virus, produce proinflammatory mediators and undergo cell death by lysis thus releasing a large numbers of progeny viruses that infect the neighboring cells.4,5 RSV infection stimulates the expression of nerve growth factor (NGF) and its tropomyosin-related kinase A (TrkA) high affinity receptor and markedly down-regulates the low affinity p75 neurotrophin receptor (p75NTR).12 This pattern of neurotrophin expression boosts viral replication by delaying the infected cell apoptosis via activation of the phosphoinositidide 3-kinase pathway.12 In addition, RSV may antagonize the interferon (IFN)-mediated antiviral responses in epithelial cells by inducing the suppressor to cytokines signaling (SOCS) gene expression.13 SOCS proteins inhibit the transcription of type I IFN-a and-b mRNAs and utilize a feedback loop to inhibit effective cytokine responses.11,13 IFN-a and-b production by the infected host epithelial cells is also suppressed by the NS1 and NS2 proteins; these block IRF-3 activation, decrease the expression of STAT2 and down-regulate the RIG-I-mediated type I IFN promoter activation.4,5,11,14 A breakthrough in biology has been the discovery of small noncoding RNAs (sncRNAs), such as transfer RNAs (tRNAs) and microRNAs (miRNAs), that appear to play regulatory functions in RSV infection. A recent report showed up-regulation of a tRNA-RNA fragment (tRF-5-GluCTC) by RSV infection promoting RSV replication.15 More information, however, is emerging on the key role of miRNAs, in RSV strategies of escaping host cell surveillance.10,16
Modulation of RSV Infection by MicroRNAs
miRNAs and Viral Infections
miRNAs are short (20–23-nucleotide), endogenous, ssRNA molecules that regulate gene expression.10,17 Most miRNA genes are transcribed by RNA polymerase II into primary miRNA transcripts that are processed in the nucleus by a complex containing the RNase III endonuclease Drosha (Fig. 1). The resulting precursor miRNAs are transported to the cytoplasm where the mature miRNAs are excised by a complex containing the endonuclease Dicer. Mature miRNAs are incorporated into the RNA-induced silencing complex, which binds to the three prime untranslated region (30 -UTR) of target mRNAs, repressing their translation and/or inducing their degradation (Fig. 1).10,16 The human genome may encode over 1,000 miRNAs which appear to target about 60% of the human genes, and it hence stands to reason that posttranscriptional regulation of gene expression by miRNAs is critical for a wide range of cellular functions.17 Even
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though the biological significance of miRNAs is understood only in a limited number of instances, specific miRNAs have been implicated in various biological events, including cell proliferation and differentiation, apoptosis, morphogenesis, development, and oncogenesis.17–19 More recently, miRNAs have been shown to affect numerous processes pertaining to viral infections, such as modulation of the innate and adaptive immune responses, of cell cycle progression, and of apoptosis induction.20 Virus infection alters host gene transcription and transcript translation not only through antagonism to host protein function or induction of RNA stress granules, but also by altering patterns of host gene expression through processes that involve miRNAs.20 miRNAs, RSV Replication and Host Immune Response
RSV infection is influenced by a variety of miRNAs, which are all encoded by host cells. Biosynthesis of RSVencoded miRNAs is virtually absent, since the life cycle of this virus is exclusively cytoplasmatic and hence implies lack of access to the nuclear endonuclease Drosha, the obligatory enzyme for generation of primary miRNA transcripts.16 As reported below, RSV-induced miRNA expression occurs through different mechanisms and modulates a variety of host cell functions, some advantageous and others adverse for the virus. Induction of miRNA Expression
Fig. 1. Biogenesis and mechanism of action of miRNAs. miRNAs are synthesized in the nucleus by RNA polymerase II into primary miRNA transcripts. The primary miRNAs are then cleaved by a complex containing the RNase III endonuclease Drosha and then transported to the cytoplasm by the exportin-5RAN-GTP complex. Here the mature miRNAs are excised to a short RNA duplex by the endonuclease Dicer. One strand of the duplex is incorporated into the RNA-induced silencing complex (RISC), which binds to the three prime untranslated region (30 UTR) of target mRNAs, repressing their translation and/or inducing their degradation. The second strand can be loaded into another RISC complex or can be degraded.16,17
During RSV infection, both viral proteins and mediators released by the host cells were found to be involved in RSV-induced miRNA expression.21 In a study using normal human bronchial epithelial cell line cultures, RSV inoculation caused a significant decrease in the expression of miR-221 and a robust induction of two miRNA; let-7i and miR-30b. The study also showed that the intensity of the response depended on virus concentration in the inoculum and active viral replication (Fig. 2).22 However, the increase in let-7i induction was observed 24 and 48 hr post virus inoculation, while detectable miR-30b was found only 48 hr post inoculation, pointing to different pathways. The hypothesis of differential pathways was further supported by the observation that let-7i, but not miR-30b levels, were enhanced by the presence IFN-b in a concentrationdependent manner, whereas miR-30b induction required mechanisms involving the NF-kB pathway.22,23 Indeed, both NF-kB activation and IFN-b production are induced in airway epithelial cells following RSV infection.4,5 The roles of let-7i and miR-30b have not been directly studied in RSV infection, but in biliary and in hepatoma cell lines both miRNA contribute to strengthening of epithelial immune responses against viral infections and inhibition of viral replication.24,25 Another important finding in that study was that up-regulation of let-7i and miR-30b was Pediatric Pulmonology
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miRNAs were transfected into non-RSV-infected cells, to screen their role in the modulation of neurotrophin expression. Maximal down-regulation of both NGF and TrkA, the receptor that increases antiapoptotic factors, was detected in cells over-expressing miR-221.27,28 In contrast p75NTR, the receptor that promotes apoptosis, was not affected by the over-expression of miR-221.27,28 As expected, cells transfected with miR-221 were more prone to apoptosis and, after infection with RSV, showed significantly lower NGF protein expression and a sharp reduction in the proportion of infected cells. Thus, by silencing miR-221, RSV up-regulates the NGF-TrkA axis that favors viral replication by inhibiting the apoptosis of infected cells (Fig. 3). The G Protein and the Complexity of miRNA Functions
Fig. 2. Regulation of miRNA expression. RSV infection induces let-7i and miR-30b expression that is dependent on the viral inoculum dose and on active RSV replication. Let-7i appears to be enhanced by the presence IFN-b, while induction of miR-30b requires NF-kB activation. Expression of let-7i and miR-30b, miRNA that favor inhibition of viral replication, is inhibited by NS1 or NS2 proteins.
The attachment G protein also regulates host cell defensive functions through a variety of mechanisms, including the modulation of miRNAs encoded by host cells.4,5,29 The high glycosylation of G protein helps evade effective recognition by the immune system, and its high variability allows easy escape from neutralizing antibodies.4,5,29 Furthermore, during viral replication, a soluble form of G protein is released and binds RSVspecific antibodies and hence reducing the concen-
further enhanced when the cells were infected by RSV strains lacking NS1 or NS2 genes. NS1 and NS2 proteins may antagonize the up-regulation of let-7i and of miR30b expression by inhibiting the production of type I IFNs4,5 and of other cytokines involved in miRNA transcription.22 Thus, RSV appears to counteract the miRNA-related response of the host cells through NS1 and NS2 viral proteins that, via different mechanisms, reduce the induction of miRNA involved in the inhibition of viral infection (Fig. 2). Interestingly, NS1 and NS2 proteins may also favor RSV replication through NF-kBdependent, IFN-independent pathways, by suppressing premature host cell apoptosis.26 Neurotrophin Activity, miRNA, and Cell Apoptosis
Host cell apoptosis during RSV infections may also be controlled by miRNAs that regulate neurotrophin activity. A study of RSV infection of human bronchial epithelial cell primary cultures showed that among the miRNAs significantly affected by the infection, six (miR-27a, miR221, miR-339-5p, miR-453, miR-574, and miR-744) were highly complementary to NGF and/or the NGF receptors TrkA and p75NTR.27 With the exception of miR744, all the other miRNAs appeared to be significantly down-regulated. To screen their role in the modulation of neurotrophin expression, precursors for each of these Pediatric Pulmonology
Fig. 3. Modulation of neurotrophin expression and RSV replication. RSV infection of bronchial epithelial cells is associated with inhibition of miRNA-221 expression. By silencing miR-221, RSV prevents the miR-221-mediated down-regulation of NGF and TrkA receptor expression and promotes viral replication by inhibiting bronchial epithelial cells apoptosis. p75NTR, the receptor that promotes apoptosis, is not affected by changes in miR-221 expression.27
Modulation of RSV Infection by MicroRNAs 4,5,29
trations available for RSV neutralization. A study using recombinant RSV (6340WT) or recombinant RSV lacking the G gene (RSVDG), revealed that in human alveolar epithelial cells both replicated to similar levels but showed a differential pattern of induction of miRNA expression.30 As compared with 6340WT infected cells, those infected by RSVDG showed lower let-7f levels, higher levels of miR-337 and miR-24 and similar levels of miR-520a and miR-26b. The most interesting among these miRNA appeared to be let-7f because, after infection with 6340WT, the induction of the “let-7 family” was consistent among replicates, and let-7f showed the highest up-regulation among various let-7 members.30 By increasing the transcriptional activity of let-7f, the RSV G protein significantly down-regulated three “cell proliferation and survival” genes (CCND1, DYRK2, and ELF4), one “chemokine” gene (CCL7/ MCP3) and the SOCS 3 gene (Fig. 4). The degree of inhibition induced by let-7f appeared to be elevated (approximately >40%) for CCND1, CCL7/ MCP3, ELF4, and SOCS3 genes and lower for DYRK2 gene.30 CCND1 promotes G1-to-S phase transition of the cell cycle, whilst DYRK2 and ELF4 promote apoptosis.31,32 In addition, ELF4 up-regulates
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IFN expression in a feed-forward loop and directly activates the T-cell antigen receptor signaling in cytotoxic CD8þ T-cells to induce the arrest of their cell cycle and increase their survival.34 Consistently, ELF4 deficiency resulted in enhanced viral susceptibility.33,34 CCL7/MCP3 is a chemokine, induced by cytokines or exogenous agents (bacteria, viruses) that attracts and activates several types of leukocytes involved in anti-viral defense, including monocytes, lymphocytes, granulocytes, NK cells, and dendritic cells.35 Thus, the let-7f-mediated inhibition of CCND1, DYRK2, ELF4, and CCL7/MCP3 translation can be one of the mechanisms employed by RSV to favor delayed viral clearance (Fig. 4). In contrast, by enhancing the transcriptional activity of let-7f, the RSV G protein supports the host response by decreasing the expression of SOCS3, a regulator of cytokine signaling expression that negatively regulate type I IFNs36 (Fig. 4). These observations substantiate the multifaceted functions of miRNAs in regulating the balance between virus replication and the antiviral host responses. However, disproportion between the “pro” and “con” actions of the increased transcriptional activity of let-7f appears to shift the balance toward a dominant effect of slowing viral clearance. CONCLUSIONS
Fig. 4. The “multifaceted role” of the G protein. By increasing let7f levels in airway cells, the G protein down-regulates genes that favor host cell response to infection. These include the CCND1 gene that promotes host cell cycle progression, the DYRK2 and ELF4 genes, that favor host cell apoptosis and the CCL7/MCP3 chemokine gene, that attracts and activates several types of leukocytes involved in anti-viral defense. Inhibition of these genes translation favors RSV replication delaying viral clearance. By contrast, the G protein induces down-regulation the SOCS3 gene that negatively regulates type I IFNs, thus favoring the host response.
As part of their evolutionary strategies both viruses and host cells are capable to manipulate the miRNAome to regulate the overall innate response, in a virus- and cell-typespecific fashion. Via activation or inhibition of pathways of the classical regulators, such as NF-kB and STAT, and the production of cytokines, such as type I IFNs, RSV up- or down-regulates a variety of miRNA expression. This enhances evasion of immune recognition prolongs host cell survival and establishes viral latency. Consequently, miRNAs may play an important role in modulating the severity and duration of RSV infection and, putatively, its long-lasting sequelae. In the various studies described, different experimental systems were used (including different type of airway epithelial cells and of RSV strains) to evaluate diverse functional effects that involve diverse miRNA. Therefore with the data currently available it is difficult to state with certainty whether the patterns of miRNAs-induced response to RSV infection are truly consistent and uniform. With these limitations in mind, a significant RSV-induced decrease in expression of miR-221, the miRNA endowed of anti-neurotrophic, pro-apoptotic activity, was reported in both the Othumpangat12 and in the Thornburg21 studies. Future research should demonstrate whether the exogenous administration of synthetic miRNAs could antagonize viral replication and hence provide a novel strategy for the therapy of this common infection. Pediatric Pulmonology
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