Host MicroRNA Regulation of Human Cytomegalovirus Immediate Early Protein Translation Promotes Viral Latency
TL;DR: Members of a family of small RNAs, termed microRNAs, encoded by human myeloid progenitor cells are capable of repressing a key viral protein, thus enabling the virus to ensure a quiet/latent state of this pathogen.
Abstract: Reactivation of human cytomegalovirus (HCMV) is a significant cause of disease and death in immunocompromised patients, underscoring the need to understand how latency is controlled. Here we demonstrate that HCMV has evolved to utilize cellular microRNAs (miRNAs) in cells that promote latency to regulate expression of a viral protein critical for viral reactivation. Our data reveal that hsa-miR-200 miRNA family members target the UL122 (immediate early protein 2) 3′ untranslated region, resulting in repression of this viral protein. Utilizing recombinant viruses that mutate the miRNA-binding site compared to the sequence of the wild-type virus results in lytic rather than latent infections in ex vivo infections of primary CD34+ cells. Cells permissive for lytic replication demonstrate low levels of these miRNAs. We propose that cellular miRNA regulation of HCMV is critical for maintenance of viral latency.
IMPORTANCE Human cytomegalovirus (HCMV) is a herpesvirus that infects a majority of the population. Once acquired, individuals harbor the virus for life, where the virus remains, for the most part, in a quiet or latent state. Under weakened immune conditions, the virus can reactivate, which can cause severe disease and often death. We have found that members of a family of small RNAs, termed microRNAs, encoded by human myeloid progenitor cells are capable of repressing a key viral protein, thus enabling the virus to ensure a quiet/latent state. As these progenitor cells mature further down the myeloid lineage toward cells that support active viral replication, the levels of these microRNAs decrease. Together, our data suggest that host cell microRNA regulation of HCMV is important for the quiet/latent state of this pathogen.
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TL;DR: It is shown that following viral infection or stimulation of cells with an inactivated virus, deletion of the m6A ‘writer’ METTL3 or ‘reader’ YTHDF2 led to an increase in the induction of interferon-stimulated genes, and propagation of different viruses was suppressed in an interferON-signaling-dependent manner.
Abstract: N6-methyladenosine (m6A) is the most common mRNA modification. Recent studies have revealed that depletion of m6A machinery leads to alterations in the propagation of diverse viruses. These effects were proposed to be mediated through dysregulated methylation of viral RNA. Here we show that following viral infection or stimulation of cells with an inactivated virus, deletion of the m6A 'writer' METTL3 or 'reader' YTHDF2 led to an increase in the induction of interferon-stimulated genes. Consequently, propagation of different viruses was suppressed in an interferon-signaling-dependent manner. Significantly, the mRNA of IFNB, the gene encoding the main cytokine that drives the type I interferon response, was m6A modified and was stabilized following repression of METTL3 or YTHDF2. Furthermore, we show that m6A-mediated regulation of interferon genes was conserved in mice. Together, our findings uncover the role m6A serves as a negative regulator of interferon response by dictating the fast turnover of interferon mRNAs and consequently facilitating viral propagation.
271 citations
TL;DR: New studies that suggest that distinct sites of cellular latency could exist in the human host are focused on, which argues for multiple latent phenotypes that could impact differently on the biology of this virus in vivo.
Abstract: Human cytomegalovirus (HCMV) infection remains a major cause of morbidity in patient populations. In certain clinical settings, it is the reactivation of the pre-existing latent infection in the host that poses the health risk. The prevailing view of HCMV latency was that the virus was essentially quiescent in myeloid progenitor cells and that terminal differentiation resulted in the initiation of the lytic lifecycle and reactivation of infectious virus. However, our understanding of HCMV latency and reactivation at the molecular level has been greatly enhanced through recent advancements in systems biology approaches to perform global analyses of both experimental and natural latency. These approaches, in concert with more classical reductionist experimentation, are furnishing researchers with new concepts in cytomegalovirus latency and suggest that latent infection is far more active than first thought. In this review, we will focus on new studies that suggest that distinct sites of cellular latency could exist in the human host, which, when coupled with recent observations that report different transcriptional programmes within cells of the myeloid lineage, argues for multiple latent phenotypes that could impact differently on the biology of this virus in vivo. Finally, we will also consider how the biology of the host cell where the latent infection persists further contributes to the concept of a spectrum of latent phenotypes in multiple cell types that can be exploited by the virus.
140 citations
Cites background from "Host MicroRNA Regulation of Human C..."
...HCMV latency [49] has postulated that an abundance of a cellular miRNA—mir200—targets the UL122 transcript for degradation, thereby preventing IE86 protein expression also [50]....
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TL;DR: This systematic analysis of the massive transcriptome RNA sequencing atlas generated by the Genotype-Tissue Expression project reveals that HCMV persistence in vivo is prevalent in diverse tissues and suggests that latency is governed mainly by quantitative changes, with a limited number of qualitative changes, in viral gene expression.
Abstract: Primary infection with human cytomegalovirus (HCMV) results in a lifelong infection due to its ability to establish latent infection, with one characterized viral reservoir being hematopoietic cells. Although reactivation from latency causes serious disease in immunocompromised individuals, our molecular understanding of latency is limited. Here, we delineate viral gene expression during natural HCMV persistent infection by analyzing the massive transcriptome RNA sequencing (RNA-seq) atlas generated by the Genotype-Tissue Expression (GTEx) project. This systematic analysis reveals that HCMV persistence in vivo is prevalent in diverse tissues. Notably, we find only viral transcripts that resemble gene expression during various stages of lytic infection with no evidence of any highly restricted latency-associated viral gene expression program. To further define the transcriptional landscape during HCMV latent infection, we also used single-cell RNA-seq and a tractable experimental latency model. In contrast to some current views on latency, we also find no evidence for any highly restricted latency-associated viral gene expression program. Instead, we reveal that latency-associated gene expression largely mirrors a late lytic viral program, albeit at much lower levels of expression. Overall, our work has the potential to revolutionize our understanding of HCMV persistence and suggests that latency is governed mainly by quantitative changes, with a limited number of qualitative changes, in viral gene expression.IMPORTANCE Human cytomegalovirus is a prevalent pathogen, infecting most of the population worldwide and establishing lifelong latency in its hosts. Although reactivation from latency causes significant morbidity and mortality in immunocompromised hosts, our molecular understanding of the latent state remains limited. Here, we examine the viral gene expression during natural and experimental latent HCMV infection on a transcriptome-wide level. In contrast to the classical views on herpesvirus latency, we find no evidence for a restricted latency-associated viral gene expression program. Instead, we reveal that latency gene expression largely resembles a late lytic viral profile, albeit at much lower levels of expression. Taken together, our data transform the current view of HCMV persistence and suggest that latency is mainly governed by quantitative rather than qualitative changes in viral gene expression.
131 citations
TL;DR: This review article describes current techniques that can be used to identify miRNAs involved in the modulation of viral infection and to characterize their targets and mode of action, and presents different reported examples of miRNA-mediated regulation of viruses, which can have a positive outcome either for the host or for the virus.
Abstract: Every living organism has to constantly face threats from the environment and deal with a large number of pathogens against which it has to defend itself to survive. Among those, viruses represent a large class of obligatory intracellular parasites, which rely on their host machinery to multiply and propagate. As a result, viruses and their hosts have engaged in an ever-evolving arms race to be able to maintain their existence. The role played by micro (mi)RNAs in this ongoing battle has been extensively studied in the past fifteen years and will be the subject of this review article. We will mainly focus on cellular miRNAs and their implication during viral infection in mammals. Thus, we will describe current techniques that can be used to identify miRNAs involved in the modulation of viral infection and to characterize their targets and mode of action. We will also present different reported examples of miRNA-mediated regulation of viruses, which can have a positive outcome either for the host or for the virus. In addition, the mode of action is also of a dual nature, depending on the target of the miRNA. Indeed, the regulatory small RNA can either directly guide an Argonaute protein on a viral transcript, or target a cellular mRNA involved in the host antiviral response. We will then see whether and how viruses respond to miRNA-mediated targeting. Finally, we will discuss how our knowledge of viral targeting by miRNA can be exploited for developing new antiviral therapeutic approaches.
130 citations
TL;DR: It is demonstrated that the replication of a wide range of pathogenic viruses is not enhanced in human cells engineered to be unable to produce mi RNAs, indicating that viruses have evolved to be resistant to inhibition by miRNAs.
Abstract: The issue of whether viruses are subject to restriction by endogenous microRNAs (miRNAs) and/or by virus-induced small interfering RNAs (siRNAs) in infected human somatic cells has been controversial. Here, we address this question in two ways. First, using deep sequencing, we demonstrate that infection of human cells by the RNA virus dengue virus (DENV) or West Nile virus (WNV) does not result in the production of any virus-derived siRNAs or viral miRNAs. Second, to more globally assess the potential of small regulatory RNAs to inhibit virus replication, we used gene editing to derive human cell lines that lack a functional Dicer enzyme and that therefore are unable to produce miRNAs or siRNAs. Infection of these cells with a wide range of viruses, including DENV, WNV, yellow fever virus, Sindbis virus, Venezuelan equine encephalitis virus, measles virus, influenza A virus, reovirus, vesicular stomatitis virus, human immunodeficiency virus type 1, or herpes simplex virus 1 (HSV-1), failed to reveal any enhancement in the replication of any of these viruses, although HSV-1, which encodes at least eight Dicer-dependent viral miRNAs, did replicate somewhat more slowly in the absence of Dicer. We conclude that most, and perhaps all, human viruses have evolved to be resistant to inhibition by endogenous human miRNAs during productive replication and that dependence on a cellular miRNA, as seen with hepatitis C virus, is rare. How viruses have evolved to avoid inhibition by endogenous cellular miRNAs, which are generally highly conserved during metazoan evolution, remains to be determined. IMPORTANCE Eukaryotic cells express a wide range of small regulatory RNAs, including miRNAs, that have the potential to inhibit the expression of mRNAs that show sequence complementarity. Indeed, previous work has suggested that endogenous miRNAs have the potential to inhibit viral gene expression and replication. Here, we demonstrate that the replication of a wide range of pathogenic viruses is not enhanced in human cells engineered to be unable to produce miRNAs, indicating that viruses have evolved to be resistant to inhibition by miRNAs. This result is important, as it implies that manipulation of miRNA levels is not likely to prove useful in inhibiting virus replication. It also focuses attention on the question of how viruses have evolved to resist inhibition by miRNAs and whether virus mutants that have lost this resistance might prove useful, for example, in the development of attenuated virus vaccines.
126 citations
References
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TL;DR: A novel microRNA quantification method has been developed using stem–loop RT followed by TaqMan PCR analysis, which enables fast, accurate and sensitive miRNA expression profiling and can identify and monitor potential biomarkers specific to tissues or diseases.
Abstract: A novel microRNA (miRNA) quantification method has been developed using stem–loop RT followed by TaqMan PCR analysis. Stem–loop RT primers are better than conventional ones in terms of RT efficiency and specificity. TaqMan miRNA assays are specific for mature miRNAs and discriminate among related miRNAs that differ by as little as one nucleotide. Furthermore, they are not affected by genomic DNA contamination. Precise quantification is achieved routinely with as little as 25 pg of total RNA for most miRNAs. In fact, the high sensitivity, specificity and precision of this method allows for direct analysis of a single cell without nucleic acid purification. Like standard TaqMan gene expression assays, TaqMan miRNA assays exhibit a dynamic range of seven orders of magnitude. Quantification of five miRNAs in seven mouse tissues showed variation from less than 10 to more than 30 000 copies per cell. This method enables fast, accurate and sensitive miRNA expression profiling and can identify and monitor potential biomarkers specific to tissues or diseases. Stem–loop RT–PCR can be used for the quantification of other small RNA molecules such as short interfering RNAs (siRNAs). Furthermore, the concept of stem–loop RT primer design could be applied in small RNA cloning and multiplex assays for better specificity and efficiency.
4,599 citations
"Host MicroRNA Regulation of Human C..." refers methods in this paper
...Expression of the hsa-miR-200 cluster was then detected utilizing modified TaqMan-based stem-loop reverse transcription (RT)quantitative PCR (qPCR) (36)....
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TL;DR: A program is presented, RNA-hybrid, that predicts multiple potential binding sites of miRNAs in large target RNAs and applied this method to the prediction of Drosophila miRNA targets in 3'UTRs and coding sequence.
Abstract: MicroRNAs (miRNAs) are short RNAs that post-transcriptionally regulate the expression of target genes by binding to the target mRNAs. Although a large number of animal miRNAs has been defined, only a few targets are known. In contrast to plant miRNAs, which usually bind nearly perfectly to their targets, animal miRNAs bind less tightly, with a few nucleotides being unbound, thus producing more complex secondary structures of miRNA/target duplexes. Here, we present a program, RNA-hybrid, that predicts multiple potential binding sites of miRNAs in large target RNAs. In general, the program finds the energetically most favorable hybridization sites of a small RNA in a large RNA. Intramolecular hybridizations, that is, base pairings between target nucleotides or between miRNA nucleotides are not allowed. For large targets, the time complexity of the algorithm is linear in the target length, allowing many long targets to be searched in a short time. Statistical significance of predicted targets is assessed with an extreme value statistics of length normalized minimum free energies, a Poisson approximation of multiple binding sites, and the calculation of effective numbers of orthologous targets in comparative studies of multiple organisms. We applied our method to the prediction of Drosophila miRNA targets in 3′UTRs and coding sequence. RNAhybrid, with its accompanying programs RNAcalibrate and RNAeffective, is available for download and as a Web tool on the Bielefeld Bioinformatics Server (http://bibiserv.techfak.uni-bielefeld.de/rnahybrid/).
2,236 citations
"Host MicroRNA Regulation of Human C..." refers methods in this paper
...Minimum free energies and binding hybridization of candidate miRNAs were calculated by use of the BiBiServe RNA hybrid algorithm (27)....
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TL;DR: Loss of expression of the miR-200 family members may play a critical role in the repression of E-cadherin by ZEB1 and ZEB2 during EMT, thereby enhancing migration and invasion during cancer progression.
1,577 citations
"Host MicroRNA Regulation of Human C..." refers background in this paper
...The miRNA 200 (miR-200)-regulated positive control contained the 3=UTR sequence of SIP1/ZEB2, which is regulated by the miR-200 family members (28), and the retroviral constructs which expressed the miR-200 family members were a kind gift of Yibin Kang (Princeton University)....
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...The positive control contains the 3=UTR of ZEB2, a published target of the hsa-miR-200 family members (28)....
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TL;DR: To identify other miRNA genes in pathogenic viruses, a new miRNA gene prediction method with small-RNA cloning from several virus-infected cell types was combined and predicted miRNAs in several large DNA viruses.
Abstract: Epstein-Barr virus (EBV or HHV4), a member of the human herpesvirus (HHV) family, has recently been shown to encode microRNAs (miRNAs). In contrast to most eukaryotic miRNAs, these viral miRNAs do not have close homologs in other viral genomes or in the genome of the human host. To identify other miRNA genes in pathogenic viruses, we combined a new miRNA gene prediction method with small-RNA cloning from several virus-infected cell types. We cloned ten miRNAs in the Kaposi sarcoma-associated virus (KSHV or HHV8), nine miRNAs in the mouse gammaherpesvirus 68 (MHV68) and nine miRNAs in the human cytomegalovirus (HCMV or HHV5). These miRNA genes are expressed individually or in clusters from either polymerase (pol) II or pol III promoters, and share no substantial sequence homology with one another or with the known human miRNAs. Generally, we predicted miRNAs in several large DNA viruses, and we could neither predict nor experimentally identify miRNAs in the genomes of small RNA viruses or retroviruses.
1,208 citations
TL;DR: Recombineering facilitates many kinds of genomic experiment that have otherwise been difficult to carry out, and should enhance functional genomic studies by providing better mouse models and a more refined genetic analysis of the mouse genome.
Abstract: Highly efficient phage-based Escherichia coli homologous recombination systems have recently been developed that enable genomic DNA in bacterial artificial chromosomes to be modified and subcloned, without the need for restriction enzymes or DNA ligases. This new form of chromosome engineering, termed recombinogenic engineering or recombineering, is efficient and greatly decreases the time it takes to create transgenic mouse models by traditional means. Recombineering also facilitates many kinds of genomic experiment that have otherwise been difficult to carry out, and should enhance functional genomic studies by providing better mouse models and a more refined genetic analysis of the mouse genome.
794 citations
"Host MicroRNA Regulation of Human C..." refers methods in this paper
...protocols (30) or to generate the TB40/EgfpIE2cis mutant using I-sce...
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