Host microRNAs exhibit differential propensity to interact with SARS-CoV-2 and variants of concern.

A significant number of SARS-CoV-2-infected individuals naturally overcome viral infection, suggesting the existence of a potent endogenous antiviral mechanism. As an innate defense mechanism, microRNA (miRNA) pathways in mammals have evolved to restrict viruses, besides regulating endogenous mRNAs. In this study, we systematically examined the complete repertoire of human miRNAs for potential binding sites on SARS-CoV-2 Wuhan-Hu-1, Beta, Delta, and Omicron. Human miRNA and viral genome interaction were analyzed using RNAhybrid 2.2 with stringent parameters to identify highly bonafide miRNA targets. Using publicly available data, we filtered for miRNAs expressed in lung epithelial cells/tissue and oral keratinocytes, concentrating on the miRNAs that target SARS-CoV-2 S protein mRNAs. Our results show a significant loss of human miRNA and SARS-CoV-2 interactions in Omicron (130 miRNAs) compared to Wuhan-Hu-1 (271 miRNAs), Beta (279 miRNAs), and Delta (275 miRNAs). In particular, hsa-miR-3150b-3p and hsa-miR-4784 show binding affinity for S protein of Wuhan strain but not Beta, Delta, and Omicron. Loss of miRNA binding sites on N protein was also observed for Omicron. Through Ingenuity Pathway Analysis (IPA), we examined the experimentally validated and highly predicted functional role of these miRNAs. We found that hsa-miR-3150b-3p and hsa-miR-4784 have several experimentally validated or highly predicted target genes in the Toll-like receptor, IL-17, Th1, Th2, interferon, and coronavirus pathogenesis pathways. Focusing on the coronavirus pathogenesis pathway, we found that hsa-miR-3150b-3p and hsa-miR-4784 are highly predicted to target MAPK13. Exploring miRNAs to manipulate viral genome/gene expression can provide a promising strategy with successful outcomes by targeting specific VOCs.

Overexpression of an insect virus encoded silencing suppressor does not enhance plants' susceptibility to its natural virus.

RNA silencing plays a key role in shielding plant and animal hosts against viral invasion and infection. Viruses encode RNA silencing suppressors (RSS) to block small RNA guided silencing of viral transcripts. The B2 protein encoded by Flock House virus (FHV) is a well-characterized RSS that facilitates infection in insects. It has been shown to act as a functional RSS in plants. FHVB2 over-expressing tobacco plants were used to study the effect of RSS on plant susceptibility to Tobacco mosaic virus (TMV), its natural pathogen. The major symptoms observed in TMV-infected transgenic plants were greenish mosaic, puckering and distortion of leaves, but the infected transgenic leaves were able to resist chlorophyll loss. The infected leaves of transgenic plants showed no significant difference in accumulation of virus when compared with that of the wild type plants. FHVB2 plants showed higher levels of H2O2 and the ROS scavenging enzymes, APX and SOD. This suggests that interference of FHVB2 with RNA silencing machinery may activate alternative defense pathways in the plants so that they are not overly sensitive to TMV infection. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s13337-020-00644-5.

Secondary siRNAs in Plants: Biosynthesis, Various Functions, and Applications in Virology.

The major components of RNA silencing include both transitive and systemic small RNAs, which are technically called secondary sRNAs. Double-stranded RNAs trigger systemic silencing pathways to negatively regulate gene expression. The secondary siRNAs generated as a result of transitive silencing also play a substantial role in gene silencing especially in antiviral defense. In this review, we first describe the discovery and pathways of transitivity with emphasis on RNA-dependent RNA polymerases followed by description on the short range and systemic spread of silencing. We also provide an in-depth view on the various size classes of secondary siRNAs and their different roles in RNA silencing including their categorization based on their biogenesis. The other regulatory roles of secondary siRNAs in transgene silencing, virus-induced gene silencing, transitivity, and trans-species transfer have also been detailed. The possible implications and applications of systemic silencing and the different gene silencing tools developed are also described. The details on mobility and roles of secondary siRNAs derived from viral genome in plant defense against the respective viruses are presented. This entails the description of other compatible plant-virus interactions and the corresponding small RNAs that determine recovery from disease symptoms, exclusion of viruses from shoot meristems, and natural resistance. The last section presents an overview on the usefulness of RNA silencing for management of viral infections in crop plants.

Identification and comparative analysis of microRNAs from tomato varieties showing contrasting response to ToLCV infections.

Increasing incidence of viral infections in crop plants adversely affects their growth and yield. Tomato (Solanum lycopersicum) is considered to be a favorite host for viruses with over 50 species of begomoviruses naturally infecting this crop. Tomato leaf curl virus (ToLCV) is among the most widespread and devastating begomoviruses affecting tomato production. microRNAs (miRs) have been established as key regulators of gene expression and plant development. The miR pathways are disturbed during infection by viruses. Thus, comprehension of regulatory miR networks is crucial in understanding the effect of viral pathogenicity. To identify key miRs involved in ToLCV infection, a high throughput approach involving next generation sequencing was employed. Healthy and infected leaf tissues of two tomato varieties, differing in their susceptibility to ToLCV infection were analyzed. NGS data analysis followed by computational predictions, led to identification of 91 known miRs, 15 novel homologs and 53 novel miRs covering two different varieties of tomato, susceptible (Pusa Ruby) and tolerant (LA1777) to ToLCV infection. The cleaved targets of these miRs were identified using online available degradome libraries from leaf, flower and fruit of tomato and showed their involvement in various biological pathways through KEGG Orthology. With detailed comparative profiling of expression pattern of these miRs, we could associate the specific miRs with the resistant and infected genotypes. This study depicted that in depth analysis of miR expression patterns and their functions will help in identification of molecules that can be used for manipulation of gene expression to increase crop production and developing resistance against diseases.

s8ORF2 protein of infectious salmon anaemia virus is a RNA-silencing suppressor and interacts with Salmon salar Mov10 (SsMov10) of the host RNAi machinery.

The infectious salmon anaemia virus (ISAV) is a piscine virus, a member of Orthomyxoviridae family. It encodes at least 10 proteins from eight negative-strand RNA segments. Since ISAV belongs to the same virus family as Influenza A virus, with similarities in protein functions, they may hence be characterised by analogy. Like NS1 protein of Influenza A virus, s8ORF2 of ISAV is implicated in interferon antagonism and RNA-binding functions. In this study, we investigated the role of s8ORF2 in RNAi suppression in a well-established Agrobacterium transient suppression assay in stably silenced transgenic Nicotiana xanthi. In addition, s8ORF2 was identified as a novel interactor with SsMov10, a key molecule responsible for RISC assembly and maturation in the RNAi pathway. This study thus sheds light on a novel route undertaken by viral proteins in promoting viral growth, using the host RNAi machinery.

An RNAi-based high-throughput screening assay to identify small molecule inhibitors of hepatitis B virus replication.

Persistent or chronic infection with the hepatitis B virus (HBV) represents one of the most common viral diseases in humans. The hepatitis B virus deploys the hepatitis B virus X protein (HBx) as a suppressor of host defenses consisting of RNAi-based silencing of viral genes. Because of its critical role in countering host defenses, HBx represents an attractive target for antiviral drugs. Here, we developed and optimized a loss-of-function screening procedure, which identified a potential pharmacophore that abrogated HBx RNAi suppression activity. In a survey of 14,400 compounds in the Maybridge Screening Collection, we prioritized candidate compounds via high-throughput screening based on reversal of green fluorescent protein (GFP)-reported, RNAi-mediated silencing in a HepG2/GFP-shRNA RNAi sensor line. The screening yielded a pharmacologically active compound, N-(2,4-difluorophenyl)-N'-[3-(1H-imidazol-1-yl) propyl] thiourea (IR415), which blocked HBx-mediated RNAi suppression indicated by the GFP reporter assay. We also found that IR415 reversed the inhibitory effect of HBx protein on activity of the Dicer endoribonuclease. We further confirmed the results of the primary screen in IR415-treated, HBV-infected HepG2 cells, which exhibited a marked depletion of HBV core protein synthesis and down-regulation of pre-genomic HBV RNA. Using a molecular interaction analysis system, we confirmed that IR415 selectively targets HBx in a concentration-dependent manner. The screening assay presented here allows rapid and improved detection of small-molecule inhibitors of HBx and related viral proteins. The assay may therefore potentiate the development of next-generation RNAi pathway-based therapeutics and promises to accelerate our search for novel and effective drugs in antiviral research.

RNAi Screening in Spodoptera frugiperda.

RNA interference is a potent and precise reverse genetic approach to carryout large-scale functional genomic studies in a given organism. During the past decade, RNAi has also emerged as an important investigative tool to understand the process of viral pathogenesis. Our laboratory has successfully generated transgenic reporter and RNAi sensor line of Spodoptera frugiperda (Sf21) cells and developed a reversal of silencing assay via siRNA or shRNA guided screening to investigate RNAi factors or viral pathogenic factors with extraordinary fidelity. Here we describe empirical approaches and conceptual understanding to execute successful RNAi screening in Spodoptera frugiperda 21-cell line.

Role of human GRP75 in miRNA mediated regulation of dengue virus replication.

In recent times, RNAi has emerged as an important defence system that regulates replication of pathogens in host cells. Many RNAi related host factors especially the host miRNAs play important roles in all intrinsic cellular functions, including viral infection. We have been working on identification of mammalian host factors involved in Dengue virus infection. In the present study, we identified Glucose Regulated Protein 75kDa (GRP75), as a host factor that is associated with dicer complex, in particular with HADHA (trifunctional enzyme subunit alpha, mitochondrial), an auxiliary component of dicer complex. Knockdown of GRP75 by respective siRNAs in Huh-7 cells resulted in the accumulation of dengue viral genomic RNA suggesting a role of GRP75 in regulating dengue virus replication in human cell lines. To elucidate the mode of action of GRP75, we over expressed the protein in Huh-7 cells and analysed the host miRNAs processing. The results revealed that, GRP75 is involved in processing of host miRNA, hsa-mir-126, that down regulates dengue virus replication. These findings suggest a regulatory role of human miRNA pathway especially GRP75 protein and hsa-mir-126 in dengue virus replication. These results thus provide insights into the role of miRNAs and RNAi machinery in dengue life cycle.

miRMOD: a tool for identification and analysis of 5' and 3' miRNA modifications in Next Generation Sequencing small RNA data.

In the past decade, the microRNAs (miRNAs) have emerged to be important regulators of gene expression across various species. Several studies have confirmed different types of post-transcriptional modifications at terminal ends of miRNAs. The reports indicate that miRNA modifications are conserved and functionally significant as it may affect miRNA stability and ability to bind mRNA targets, hence affecting target gene repression. Next Generation Sequencing (NGS) of the small RNA (sRNA) provides an efficient and reliable method to explore miRNA modifications. The need for dedicated software, especially for users with little knowledge of computers, to determine and analyze miRNA modifications in sRNA NGS data, motivated us to develop miRMOD. miRMOD is a user-friendly, Microsoft Windows and Graphical User Interface (GUI) based tool for identification and analysis of 5' and 3' miRNA modifications (non-templated nucleotide additions and trimming) in sRNA NGS data. In addition to identification of miRNA modifications, the tool also predicts and compares the targets of query and modified miRNAs. In order to compare binding affinities for the same target, miRMOD utilizes minimum free energies of the miRNA:target and modified-miRNA:target interactions. Comparisons of the binding energies may guide experimental exploration of miRNA post-transcriptional modifications. The tool is available as a stand-alone package to overcome large data transfer problems commonly faced in web-based high-throughput (HT) sequencing data analysis tools. miRMOD package is freely available at http://bioinfo.icgeb.res.in/miRMOD.

Mungbean yellow mosaic Indian virus encoded AC2 protein suppresses RNA silencing by inhibiting Arabidopsis RDR6 and AGO1 activities.

RNA silencing refers to a conserved RNA-directed gene regulatory mechanism in a wide range of eukaryotes. It plays an important role in many processes including growth, development, genome stability, and antiviral defense in the plants. Geminivirus encoded AC2 is identified as an RNA silencing suppressor protein, however, the mechanism of action has not been characterized. In this paper, we elucidate another mechanism of AC2-mediated suppression activity of Mungbean Yellow Mosaic India Virus (MYMIV). The AC2 protein, unlike many other suppressors, does not bind to siRNA or dsRNA species and its suppression activity is mediated through interaction with key components of the RNA silencing pathway, viz., RDR6 and AGO1. AC2 interaction inhibits the RDR6 activity, an essential component of siRNA and tasi-RNA biogenesis and AGO1, the major slicing factor of RISC. Thus the study identifies dual sites of MYMIV-AC2 interference and probably accounts for its strong RNA silencing suppression activity.

Association of HADHA with human RNA silencing machinery.

In RNA silencing, small RNAs produced by dicer mediate target repression guided by RNA induced silencing complex (RISC). To effectively mediate this response, one or more proteins are employed at each stage. In the present study, we investigated HADHA, a new component in the human RNA silencing machinery. Immunoprecipitation analysis revealed that, HADHA associates with dicer complex and immunohistochemical studies confirmed its co localization with Dicer in the cytoplasm. Further, over expression of HADHA resulted in higher abundance levels of mature miRNA against a reduction in respective precursor levels and vice versa in HADHA knocked down cells. These findings suggest an auxiliary role for HADHA in miRNA biogenesis and help in better understanding of molecular mechanisms underlying RNAi pathway in mammals.

De novo transcriptome assembly and analysis of Sf21 cells using illumina paired end sequencing.

Spodoptera is an important polyphagous agricultural insect pest in the tropical world. The genomic details are limited to understand the pest biology at molecular level. In the present study, we sequenced and assembled the transcriptome from Sf21 cells into a non redundant set of 24,038 contigs of ~ 47.38 Mb in size. A total of 26,390 unigenes were identified from the assembled transcripts and their annotation revealed the prevalent protein domains in Sf21 cells. The present study would provide a resource for gene discovery and development of functional molecular markers to understand the biology of S. frugiperda.

Dengue NS3, an RNAi suppressor, modulates the human miRNA pathways through its interacting partner.

RNAi acts as a host immune response against non-self molecules, including viruses. Viruses evolved to neutralize this response by expressing suppressor proteins. In the present study, we investigated dengue virus non structural protein 3 (dvNS3), for its RNAi-suppressor activity in human cell lines. Dengue virus (DV) NS3 reverts the GFP expression in GFP-silenced cell lines. Pull-down assays of dvNS3 revealed that it interacts with the host factor human heat shock cognate 70 (hHSC70). Down-regulation of hHSC70 resulted in accumulation of dengue viral genomic RNA. Also, the interaction of dvNS3 with hHSC70 perturbs the formation of RISC (RNA-induced silencing complex)-loading complex (RLC), by displacing TRBP (TAR RNA-binding protein) and possibly impairing the downstream activity of miRNAs. Interestingly, some of these miRNAs have earlier been reported to be down-regulated upon DV infection in Huh7 cells. Further studies on the miRNA-mRNA relationship along with mRNA profiling of samples overexpressing dvNS3 revealed up-regulation of TAZ (tafazzin) and SYNGR1 (synaptogyrin 1), known dengue viral host factors (DVHFs). Importantly, overexpression of dvNS3 in human embryonic kidney (HEK) 293T cells resulted in modulation of both mature and precursor miRNAs in human cell lines. Subsequent analysis suggested that dvNS3 induced stage-specific down-regulation of miRNAs. Taken together, these results suggest that dvNS3 affects biogenesis and function of host miRNAs to regulate DVHFs for favouring DV replication.

Identification and characteristics of microRNAs from army worm, Spodoptera frugiperda cell line Sf21.

microRNAs play important regulatory role in all intrinsic cellular functions. Amongst lepidopteran insects, miRNAs from only Bombyx mori have been studied extensively with a little focus on Spodoptera sp. In the present study, we identified a total of 226 miRNAs from Spodoptera frugiperda cell line Sf21. Of the total, 116 miRNAs were well conserved within other insects, like B. mori, Drosophila melanogaster and Tribolium castenum while the remaining 110 miRNAs were identified as novel based on comparative analysis with the insect miRNA data set. Landscape distribution analysis based on Sf21 genome assembly revealed clustering of few novel miRNAs. A total of 5 miRNA clusters were identified and the largest one encodes 5 miRNA genes. In addition, 12 miRNAs were validated using northern blot analysis and putative functional role assignment for 6 Sf miRNAs was investigated by examining their relative abundance at different developmental stages of Spodoptera litura and body parts of 6th instar larvae. Further, we identified a total of 809 potential target genes with GO terms for selected miRNAs, involved in different metabolic and signalling pathways of the insect. The newly identified miRNAs greatly enrich the repertoire of insect miRNAs and analysis of expression profiles reveal their involvement at various steps of biochemical pathways of the army worm.

Genome wide screening of RNAi factors of Sf21 cells reveal several novel pathway associated proteins.

BACKGROUND: RNA interference (RNAi) leads to sequence specific knock-down of gene expression and has emerged as an important tool to analyse gene functions, pathway analysis and gene therapy. Although RNAi is a conserved cellular process involving common elements and factors, species-specific differences have been observed among different eukaryotes. Identification of components for RNAi pathway is pursued intensively and successful genome-wide screens have been performed for components of RNAi pathways in various organisms. Functional comparative genomics analysis offers evolutionary insight that forms basis of discoveries of novel RNAi-factors within related organisms. Keeping in view the academic and commercial utility of insect derived cell-line from Spodoptera frugiperda, we pursued the identification and functional analysis of components of RNAi-machinery of Sf21 cell-line using genome-wide application. RESULTS: The genome and transcriptome of Sf21 was assembled and annotated. In silico application of comparative genome analysis among insects allowed us to identify several RNAi factors in Sf21 line. The candidate RNAi factors from assembled genome were validated by knockdown analysis of candidate factors using the siRNA screens on the Sf21-gfp reporter cell-line. Forty two (42) potential factors were identified using the cell based assay. These include core RNAi elements including Dicer-2, Argonaute-1, Drosha, Aubergine and auxiliary modules like chromatin factors, RNA helicases, RNA processing module, signalling allied proteins and others. Phylogenetic analyses and domain architecture revealed that Spodoptera frugiperda homologs retained identity with Lepidoptera (Bombyx mori) or Coleoptera (Tribolium castaneum) sustaining an evolutionary conserved scaffold in post-transcriptional gene silencing paradigm within insects. CONCLUSION: The database of RNAi-factors generated by whole genome association survey offers comprehensive outlook about conservation as well as specific differences of the proteins of RNAi machinery. Understanding the interior involved in different phases of gene silencing also offers impending tool for RNAi-based applications.

A draft genome assembly of the army worm, Spodoptera frugiperda.

Spodoptera is an agriculturally important pest insect and studies in understanding its biology have been limited by the unavailability of its genome. In the present study, the genomic DNA was sequenced and assembled into 37,243 scaffolds of size, 358 Mb with N50 of 53.7 kb. Based on degree of identity, we could anchor 305 Mb of the genome onto all the 28 chromosomes of Bombyx mori. Repeat elements were identified, which accounts for 20.28% of the total genome. Further, we predicted 11,595 genes, with an average intron length of 726 bp. The genes were annotated and domain analysis revealed that Sf genes share a significant homology and expression pattern with B. mori, despite differences in KOG gene categories and representation of certain protein families. The present study on Sf genome would help in the characterization of cellular pathways to understand its biology and comparative evolutionary studies among lepidopteran family members to help annotate their genomes.

Role of RNA interference (RNAi) in dengue virus replication and identification of NS4B as an RNAi suppressor.

RNA interference (RNAi) is an important antiviral defense response in plants and invertebrates; however, evidences for its contribution to mammalian antiviral defense are few. In the present study, we demonstrate the anti-dengue virus role of RNAi in mammalian cells. Dengue virus infection of Huh 7 cells decreased the mRNA levels of host RNAi factors, namely, Dicer, Drosha, Ago1, and Ago2, and in corollary, silencing of these genes in virus-infected cells enhanced dengue virus replication. In addition, we observed downregulation of many known human microRNAs (miRNAs) in response to viral infection. Using reversion-of-silencing assays, we further showed that NS4B of all four dengue virus serotypes is a potent RNAi suppressor. We generated a series of deletion mutants and demonstrated that NS4B mediates RNAi suppression via its middle and C-terminal domains, namely, transmembrane domain 3 (TMD3) and TMD5. Importantly, the NS4B N-terminal region, including the signal sequence 2K, which has been implicated in interferon (IFN)-antagonistic properties, was not involved in mediating RNAi suppressor activity. Site-directed mutagenesis of conserved residues revealed that a Phe-to-Ala (F112A) mutation in the TMD3 region resulted in a significant reduction of the RNAi suppression activity. The green fluorescent protein (GFP)-small interfering RNA (siRNA) biogenesis of the GFP-silenced line was considerably reduced by wild-type NS4B, while the F112A mutant abrogated this reduction. These results were further confirmed by in vitro dicer assays. Together, our results suggest the involvement of miRNA/RNAi pathways in dengue virus establishment and that dengue virus NS4B protein plays an important role in the modulation of the host RNAi/miRNA pathway to favor dengue virus replication.

Identification of tissue-preferential expression patterns of rice miRNAs.

It is imperative to understand the mechanisms of growth and development in higher plants for improving plant adaptation during different developmental stages. Plant microRNAs (miRs) play crucial regulatory roles in various developmental processes. As many as 15 miR families having multiple members are known to regulate plant development, yet the spatio-temporal expression patterns of individual members are not fully characterized. It is likely that different members of miR families can make specific contributions to the spatio-temporal control of targets. To understand the functional complexity of miRs and the amount of degeneracy existing in miR-mediated regulation of differentiated but developing tissues, we have identified the Osa-miR-sequences that are expressed in specific tissues. We adopted the approach of comparative miR profiling using next-generation sequencing technology followed by experimental validation. It was observed that 59 Osa-miR-sequences show tissue-preferential expression in local basmati rice variety; while 126 miRs belonging to 81 families are differentially regulated in these tissues. The 21 nt miRs were predominant in all tissues, but the 24 nt miRs were the most abundantly expressed. This indicates that target cleavage and chromatin state regulation are involved in organ development. This study also identified the expression patterns of individual members of Osa-miR families that were common and divergent between the indica and japonica rice varieties. The expression patterns of the predicted targets were also analyzed. The possible implications of the miR distribution patterns with respect to the regulation of their respective targets are discussed.

Micro-regulators of auxin action.

microRNAs (miRs) are 21- to 24-nucleotide-long RNA molecules that are mainly involved in regulating the gene expression at the post-transcriptional levels. They are present in a variety of organisms from algae to plants and play an important role in gene regulation. The identification of several diverging and converging functions of miRs indicates that they play versatile roles in regulating plant development including differentiation, organ development, phase change, signalling, disease resistance and response to environmental stresses. This article provides a concise update on the plant miR functions and their targets in the auxin pathway with focus on the interactions between miRs and auxin signalling to intricately regulate the plant responses.