宿主-病毒在miRNA水平上的相互作用

微RNA(microRNA,miRNA)是近来发现的重要基因调节子,在许多生物学过程包括抗病毒防御中发挥着重要作用.越来越多的证据表明一些病毒或者编码它们自己的miRNAs或者颠覆细胞miRNAs.由此,宿主和病毒编码miRNAs及其靶标形成了宿主和病毒间新一调节层面的相互作用.深入理解宿主-病毒间miRNAs介导的相互作用,不仅有利于阐明病毒致病的分子基础,而且有利于制定更好的治疗策略.     

Evolutionarily conserved function of a viral microRNA

MicroRNAs (miRNAs) are potent RNA regulators of gene expression. Some viruses encode miRNAs, most of unknown function. The majority of viral miRNAs are not conserved, and whether any have conserved functions remains unclear. Here, we report that two human polyomaviruses associated with serious disease in immunocompromised individuals, JC virus and BK virus, encode miRNAs with the same function as that of the monkey polyomavirus simian virus 40 miRNAs. These miRNAs are expressed late during infection to autoregulate early gene expression. We show that the miRNAs generated from both arms of the pre-miRNA hairpin are active at directing the cleavage of the early mRNAs. This finding suggests that despite multiple differences in the miRNA seed regions, the primary target (the early mRNAs) and function (the downregulation of early gene expression) are evolutionarily conserved among the primate polyomavirus-encoded miRNAs. Furthermore, we show that these miRNAs are expressed in individuals diagnosed with polyomavirus-associated disease, suggesting their potential as targets for therapeutic intervention.     

The oncogenic role of Epstein–Barr virus‐encoded microRNAs in Epstein–Barr virus‐associated gastric carcinoma

Epstein–Barr virus (EBV) infection is detected in various epithelial malignancies, such as nasopharyngeal carcinoma (NPC) and gastric cancer (GC). EBV comprises some unique molecular features and encodes viral genes and microRNAs (miRNAs) by its own DNA sequence. EBV genes are required to maintain latency and contribute to oncogenic property. miRNAs encoded by EBV have been shown to contribute to initiation and progression of EBV‐related malignancies. By a number of genomic profiling studies, some EBV miRNAs were confirmed to be highly expressed in EBV‐associated gastric cancer (EBVaGC) samples and cell lines. The majority host targets of the EBV miRNAs are important for promoting cell growth and inhibiting apoptosis, facilitating cell survival and immune evasion. However, the integrated molecular mechanisms related to EBV miRNAs remain to be investigated. In this review, we summarized the crucial role of EBV miRNAs in epithelial malignancies, especially in EBVaGC. Collectively, EBV miRNAs play a significant role in the viral and host gene regulation network. Understanding the comprehensive potential targets and relevant functions of EBV miRNAs in gastric carcinogenesis might provide better clinical translation.     

Micro RNA and viral infections in mammals

RNA silencing plays an important role in development through the action of micro (mi) RNAs that fine tune the expression of a large portion of the genome. But, in plants and insects, it is also a very important player in innate immune responses, especially in antiviral defense. It is now well established that the RNA silencing machinery targets plant as well as insect viruses. While the genetic basis underlying this defense mechanism in these organisms starts being elucidated, much less is known about the possible antiviral role of RNA silencing in mammals. In order to identify siRNAs coming from viruses in infected human cells, small RNAs from cells infected with RNA viruses, such as hepatitis C virus, yellow fever virus or HIV-1, were cloned and sequenced, but no virus-specific siRNAs could be detected. On the contrary, viral small RNAs were found in cells infected by the DNA virus Epstein-Barr. A closer look at these revealed that they were not siRNAs, but rather resembled miRNAs. This finding indicated that, rather than being targeted by RNA silencing, human DNA viruses seem to have evolved their own miRNAs to modulate the expression of host genes. This primary observation has been extended to other members of the herpesvirus family as well as other DNA viruses such as the polyomavirus SV40. Viral miRNAs have the potential to act both in cis to regulate expression of viral genes, or in trans on host genes. There are good indications for the cis mode of action, but the identification of cellular targets of these small viral regulators is only in its infancy.     

Computational prediction of intronic microRNA targets using host gene expression reveals novel regulatory mechanisms

Approximately half of known human miRNAs are located in the introns of protein coding genes. Some of these intronic miRNAs are only expressed when their host gene is and, as such, their steady state expression levels are highly correlated with those of the host gene's mRNA. Recently host gene expression levels have been used to predict the targets of intronic miRNAs by identifying other mRNAs that they have consistent negative correlation with. This is a potentially powerful approach because it allows a large number of expression profiling studies to be used but needs refinement because mRNAs can be targeted by multiple miRNAs and not all intronic miRNAs are co-expressed with their host genes.Here we introduce InMiR, a new computational method that uses a linear-Gaussian model to predict the targets of intronic miRNAs based on the expression profiles of their host genes across a large number of datasets. Our method recovers nearly twice as many true positives at the same fixed false positive rate as a comparable method that only considers correlations. Through an analysis of 140 Affymetrix datasets from Gene Expression Omnibus, we build a network of 19,926 interactions among 57 intronic miRNAs and 3,864 targets. InMiR can also predict which host genes have expression profiles that are good surrogates for those of their intronic miRNAs. Host genes that InMiR predicts are bad surrogates contain significantly more miRNA target sites in their 3' UTRs and are significantly more likely to have predicted Pol II and Pol III promoters in their introns.We provide a dataset of 1,935 predicted mRNA targets for 22 intronic miRNAs. These prediction are supported both by sequence features and expression. By combining our results with previous reports, we distinguish three classes of intronic miRNAs: Those that are tightly regulated with their host gene; those that are likely to be expressed from the same promoter but whose host gene is highly regulated by miRNAs; and those likely to have independent promoters.     

Virus-encoded microRNAs: novel regulators of gene expression

MicroRNAs (miRNAs) are a class of small RNAs that have recently been recognized as major regulators of gene expression. They influence diverse cellular processes ranging from cellular differentiation, proliferation, apoptosis and metabolism to cancer. Bioinformatic approaches and direct cloning methods have identified >3500 miRNAs, including orthologues from various species. Experiments to identify the targets and potential functions of miRNAs in various species are continuing but the recent discovery of virus-encoded miRNAs indicates that viruses also use this fundamental mode of gene regulation. Virus-encoded miRNAs seem to evolve rapidly and regulate both the viral life cycle and the interaction between viruses and their hosts.     

Expression of Ovine Herpesvirus -2 Encoded MicroRNAs in an Immortalised Bovine – Cell Line

Ovine herpesvirus-2 (OvHV-2) infects most sheep, where it establishes an asymptomatic, latent infection. Infection of susceptible hosts e.g. cattle and deer results in malignant catarrhal fever, a fatal lymphoproliferative disease characterised by uncontrolled lymphocyte proliferation and non MHC restricted cytotoxicity. The same cell populations are infected in both cattle and sheep but only in cattle does virus infection cause dysregulation of cell function leading to disease. The mechanism by which OvHV-2 induces this uncontrolled proliferation is unknown. A number of herpesviruses have been shown to encode microRNAs (miRNAs) that have roles in control of both viral and cellular gene expression. We hypothesised that OvHV-2 encodes miRNAs and that these play a role in pathogenesis. Analysis of massively parallel sequencing data from an OvHV-2 persistently-infected bovine lymphoid cell line (BJ1035) identified forty-five possible virus-encoded miRNAs. We previously confirmed the expression of eight OvHV-2 miRNAs by northern hybridization. In this study we used RT-PCR to confirm the expression of an additional twenty-seven OvHV-2-encoded miRNAs. All thirty-five OvHV-2 miRNAs are expressed from the same virus genome strand and the majority (30) are encoded in an approximately 9 kb region that contains no predicted virus open reading frames. Future identification of the cellular and virus targets of these miRNAs will inform our understanding of MCF pathogenesis.     

Critical role of the virus-encoded microRNA-155 ortholog in the induction of Marek's disease lymphomas

Notwithstanding the well-characterised roles of a number of oncogenes in neoplastic transformation, microRNAs (miRNAs) are increasingly implicated in several human cancers. Discovery of miRNAs in several oncogenic herpesviruses such as KSHV has further highlighted the potential of virus-encoded miRNAs to contribute to their oncogenic capabilities. Nevertheless, despite the identification of several possible cancer-related genes as their targets, the direct in vivo role of virus-encoded miRNAs in neoplastic diseases such as those induced by KSHV is difficult to demonstrate in the absence of suitable models. However, excellent natural disease models of rapid-onset Marek's disease (MD) lymphomas in chickens allow examination of the oncogenic potential of virus-encoded miRNAs. Using viruses modified by reverse genetics of the infectious BAC clone of the oncogenic RB-1B strain of MDV, we show that the deletion of the six-miRNA cluster 1 from the viral genome abolished the oncogenicity of the virus. This loss of oncogenicity appeared to be primarily due to the single miRNA within the cluster, miR-M4, the ortholog of cellular miR-155, since its deletion or a 2-nucleotide mutation within its seed region was sufficient to inhibit the induction of lymphomas. The definitive role of this miR-155 ortholog in oncogenicity was further confirmed by the rescue of oncogenic phenotype by revertant viruses that expressed either the miR-M4 or the cellular homolog gga-miR-155. This is the first demonstration of the direct in vivo role of a virus-encoded miRNA in inducing tumors in a natural infection model. Furthermore, the use of viruses deleted in miRNAs as effective vaccines against virulent MDV challenge, enables the prospects of generating genetically defined attenuated vaccines.     

Identification of host encoded microRNAs interacting with novel swine-origin influenza A (H1N1) virus and swine influenza virus

The discovery of microRNAs (miRNAs) is a remarkable breakthrough in the field of life science, and they are important actors which regulate gene expression in diverse cellular processes. Recently, several reports indicated that miRNAs can also target viruses and regulate virus replication. Here we discovered 36 pig-encoded miRNAs and 22 human-encoded miRNAs which have putative targets in swine influenza virus (SIV) and Swine-Origin 2009 A/H1N1 influenza virus (S-OIV) genes respectively. Interestingly, the putative interactions of ssc-miR-124a, ssc-miR-136 and ssc-miR-145 with their SIV target genes had been found to be maintained almost throughout all of the virus evolution. Enrichment analysis of previously reported miRNA gene expression profiles revealed that three miRNAs are expressed at higher levels in human lung or trachea tissue. The hsa-miR-145 and hsa-miR-92a putatively target the HA gene and hsa-miR-150 putatively targets the PB2 gene. Analysis results based on the location distribution from which virus was isolated and sequence conservation imply that some putative miRNA-mediated host-virus interactions may characterize the location-specificity.     

BmNPV-miR-415 up-regulates the expression of TOR2 via Bmo-miR-5738

MicroRNAs (miRNAs) have emerged as key players in host–pathogen interaction and many virus-encoded miRNAs have been identified (computationally and/or experimentally) in a variety of organisms. A novel Bombyx mori nucleopolyhedrosis virus (BmNPV)-encoded miRNA miR-415 was previously identified through high-throughput sequencing. In this study, a BmNPV-miR-415 expression vector was constructed and transfected into BmN cells. The differentially expressed protein target of rapamycin isoform 2 (TOR2) was observed through two-dimensional gel electrophoresis and mass spectrometry. Results showed that TOR2 is not directly a target gene of BmNPV-miR-415, but its expression is up-regulated by BmNPV-miR-415 via Bmo-miR-5738, which could be induced by BmNPV.     

Human cytomegalovirus expresses novel microRNAs during productive viral infection

MicroRNAs (miRNAs) are a large class of approximately 22-nucleotide non-coding RNAs that facilitate mRNA cleavage and translation repression through the RNA interference pathway. Until recently, miRNAs have been exclusively found in eukaryotic organisms. A non-immunogenic molecule requiring minimal genomic investment, these RNAs may offer an efficient means for viruses to modulate both their own and the host's gene expression during a productive viral infection. In this study we report that human cytomegalovirus (HCMV) expresses miRNAs during its productive lytic infection of four clinically relevant human cell types: fibroblast, endothelial, epithelial and astrocyte cells. The sequences of the miRNAs, expressed from the UL23 and US24 loci of the viral genome, were conserved among all HCMV strains examined and in chimpanzee cytomegalovirus. Furthermore, their expression was detected from both a laboratory-adapted strain and a clinical isolate of HCMV. The conservation of these miRNAs and their expression in different cell types suggests that they represent an evolutionarily primitive feature in the viral genome, and that virus-encoded miRNAs may be more common than previously believed.     

Host range and cell cycle activation properties of polyomavirus large T-antigen mutants defective in pRB binding.

We have examined the growth properties of polyomavirus large T-antigen mutants that are unable to bind pRB, the product of the retinoblastoma tumor suppressor gene. These mutants grow poorly on primary mouse cells yet grow well on NIH 3T3 and other established mouse cell lines. Preinfection of primary baby mouse kidney (BMK) epithelial cells with wild-type simian virus 40 renders these cells permissive to growth of pRB-binding polyomavirus mutants. Conversely, NIH 3T3 cells transfected by and expressing wild-type human pRB become nonpermissive. Primary fibroblasts from mouse embryos that carry a homozygous knockout of the RB gene are permissive, while those from normal littermates are nonpermissive. The host range of polyomavirus pRB-binding mutants is thus determined by expression or lack of expression of functional pRB by the host. These results demonstrate the importance of pRB binding by large T antigen for productive viral infection in primary cells. Failure of pRB-binding mutants to grow well in BMK cells correlates with their failure to induce progression from G0 or G1 through the S phase of the cell cycle. Time course studies show delayed synthesis and lower levels of accumulation of large T antigen, viral DNA, and VP1 in mutant compared with wild-type virus-infected BMK cells. These results support a model in which productive infection by polyomavirus in normal mouse cells is tightly coupled to the induction and progression of the cell cycle.     

病毒miRNA与免疫逃逸

微小RNA（microRNA,miRNA）是一种非编码的小分子RNA,长度一般在22 nt左右,通过与mRNA 3＇UTR的特异性结合介导转录后调控过程。现已鉴定出的miRNA涵盖了从植物到人类的多个物种,并参与了调节生长、免疫、凋亡等多种生命活动。最近发现,DNA病毒感染宿主时也能编码产生miRNA,并在病毒免疫逃逸中扮演着重要角色。病毒感染是一个复杂的过程,病毒需要逃脱免疫系统才能对宿主产生持续性感染,而病毒miRNA能调控宿主和自身基因表达,帮助病毒感染宿主,且因其本身没有免疫原性,而成为病毒逃避免疫应答的重要工具,但其中的分子机制尚不十分清楚。该文就病毒miRNA如何调控病毒自身与宿主基因进行免疫逃逸的近期研究作一综述。     

Detection and evolutionary analysis of soybean miRNAs responsive to soybean mosaic virus

MicroRNAs (miRNA) are a class of non-coding RNAs that have important gene regulatory roles in various organisms. However, the miRNAs involved in soybean’s response to soybean mosaic virus (SMV) are unknown. To identify novel miRNAs and biotic-stress regulated small RNAs that are involved in soybean’s response to SMV, two small RNA libraries were constructed from mock-inoculated and SMV-infected soybean leaves and sequenced. This led to the discovery of 179 miRNAs, representing 52 families, among which five miRNAs belonging to three families were novel miRNAs in soybean. A large proportion (71.5 %) of miRNAs arose from segmental duplication, similar to the process that drives the evolution of protein-coding genes. In addition, we predicted 346 potential targets of these identified miRNAs, and verified 12 targets by modified 5′-RACE analysis. Finally, three miRNAs (miR160, miR393 and miR1510) that are involved in plant resistance were observed to respond to SMV infection. The interaction between miRNAs and resistance-related genes provides a novel mechanism for pathogens to evade host recognition.     

bmnpv-miR-3 facilitates BmNPV infection by modulating the expression of viral P6.9 and other late genes in Bombyx mori

During the last decade, microRNAs (miRNAs) have emerged as fine tuners of gene expression in various biological processes including host–pathogen interactions. Apart from the role of host encoded miRNAs in host–virus interactions, recent studies have also indicated the key role of virus-encoded miRNAs in the regulation of host defense responses. In the present study, we show that bmnpv-miR-3, a Bombyx mori nucleopolyhedrovirus (BmNPV) encoded miRNA, regulates the expression of DNA binding protein (P6.9) and other late genes, vital for the late stage of viral infection in the host, Bombyx mori. We have performed both cell culture and in vivo experiments to establish the role of bmnpv-miR-3 in the infection cycle of BmNPV. Our findings showed that bmnpv-miR-3 expresses during early stage of infection, and negatively regulates the expression of P6.9. There was an upregulation in P6.9 expression upon blocking of bmnpv-miR-3 by Locked Nucleic Acid (LNA), whereas overexpression of bmnpv-miR-3 resulted in a decreased expression of P6.9. Besides, a remarkable enhancement and reduction in the viral loads were observed upon blocking and overexpression of bmnpv-miR-3, respectively. Furthermore, we have also assessed the host immune response using one of the Lepidoptera-specific antimicrobial proteins, Gloverin-1 upon blocking and overexpression of bmnpv-miR-3, which correlated viral load with the host immune response. All these results together; clearly imply that bmnpv-miR-3-mediated controlled regulation of BmNPV late genes in the early stage of infection helps BmNPV to escape the early immune response from the host.