Computational Prediction of RNA-RNA Interactions between Small RNA Tracks from Betacoronavirus Nonstructural Protein 3 and Neurotrophin Genes during Infection of an Epithelial Lung Cancer Cell Line: Potential Role of Novel Small Regulatory RNA.

Whether RNA-RNA interactions of cytoplasmic RNA viruses, such as Betacoronavirus, might end in the biogenesis of putative virus-derived small RNAs as miRNA-like molecules has been controversial. Even more, whether RNA-RNA interactions of wild animal viruses may act as virus-derived small RNAs is unknown. Here, we address these issues in four ways. First, we use conserved RNA structures undergoing negative selection in the genomes of SARS-CoV, MERS-CoV, and SARS-CoV-2 circulating in different bat species, intermediate animals, and human hosts. Second, a systematic literature review was conducted to identify Betacoronavirus-targeting hsa-miRNAs involved in lung cell infection. Third, we employed sophisticated long-range RNA-RNA interactions to refine the seed sequence homology of hsa-miRNAs with conserved RNA structures. Fourth, we used high-throughput RNA sequencing of a Betacoronavirus-infected epithelial lung cancer cell line (Calu-3) to validate the results. We proposed nine potential virus-derived small RNAs: two vsRNAs in SARS-CoV (Bats: SB-vsRNA-ORF1a-3p; SB-vsRNA-S-5p), one vsRNA in MERS-CoV (Bats: MB-vsRNA-ORF1b-3p), and six vsRNAs in SARS-CoV-2 (Bats: S2B-vsRNA-ORF1a-5p; intermediate animals: S2I-vsRNA-ORF1a-5p; and humans: S2H-vsRNA-ORF1a-5p, S2H-vsRNA-ORF1a-3p, S2H-vsRNA-ORF1b-3p, S2H-vsRNA-ORF3a-3p), mainly encoded by nonstructural protein 3. Notably, Betacoronavirus-derived small RNAs targeted 74 differentially expressed genes in infected human cells, of which 55 upregulate the molecular mechanisms underlying acute respiratory distress syndrome (ARDS), and the 19 downregulated genes might be implicated in neurotrophin signaling impairment. These results reveal a novel small RNA-based regulatory mechanism involved in neuropathogenesis that must be further studied to validate its therapeutic use.

High throughput sRNA sequencing revealed gene regulatory role mediated by pathogen-derived small RNAs during Sri Lankan Cassava Mosaic Virus infection in Cassava.

Small RNA (sRNA) mediated gene regulation during Sri Lankan Cassava Mosaic Virus (SLCMV) infection was studied from the Indian Cassava Cultivar H226. Our study generated high throughput sRNA dataset of 23.64 million reads from the control and SLCMV infected H226 leaf libraries. mes-miR9386 was detected as the most prominent miRNA expressed in control and infected leaf. Among the differentially expressed miRNAs, mes-miR156, mes- miR395 and mes-miR535a/b showed significant down regulation in the infected leaf. Genome-wide analysis of the three small RNA profiles revealed critical role of virus-derived small RNAs (vsRNAs) from the infected leaf tissues of H226. The vsRNAs were mapped to the bipartite SLCMV genome and high expression of siRNAs generated from the virus genomic region encoding AV1/AV2 genes in the infected leaf pointed towards the susceptibility of H226 cultivars to SLCMV. Furthermore, the sRNA reads mapped to the antisense strand of the SLCMV ORFs was higher than the sense strand. These vsRNAs were potential to target key host genes involved in virus interaction such as aldehyde dehydrogenase, ADP-ribosylation factor1 and ARF1-like GTP-binding proteins. The sRNAome-assisted analysis also revealed the origin of virus-encoded miRNAs from the SLCMV genome in the infected leaf. These virus-derived miRNAs were predicted to have hair-pin like secondary structures, and have different isoforms. Moreover, our study revealed that the pathogen sRNAs play a critical role in the infection process in H226 plants. Supplementary Information: The online version contains supplementary material available at 10.1007/s13205-023-03494-2.

SARS-CoV-2 RNAs are processed into 22-nt vsRNAs in Vero cells.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused the global pandemic, resulting in great fatalities around the world. Although the antiviral roles of RNA interference (RNAi) have been well studied in plants, nematodes and insects, the antiviral roles of RNAi in mammalians are still debating as RNAi effect is suspected to be suppressed by interferon (IFN) signaling pathways in most cell types. To determine the role of RNAi in mammalian resistance to SARS-CoV-2, we studied the profiling of host small RNAs and SARS-CoV-2 virus-derived small RNAs (vsRNAs) in the early infection stages of Vero cells, an IFN-deficient cell line. We found that host microRNAs (miRNAs) were dysregulated upon SARS-CoV-2 infection, resulting in downregulation of microRNAs playing antiviral functions and upregulation of microRNAs facilitating viral proliferations. Moreover, vsRNA peaked at 22 nt at negative strand but not the positive strand of SARS-CoV-2 and formed successive Dicer-spliced pattern at both strands. Similar characteristics of vsRNAs were observed in IFN-deficient cell lines infected with Sindbis and Zika viruses. Together, these findings indicate that host cell may deploy RNAi pathway to combat SARS-CoV-2 infection in IFN-deficient cells, informing the alternative antiviral strategies to be developed for patients or tissues with IFN deficiency.

Potential functional pathways of plant RNA virus-derived small RNAs in a vector insect.

During infection, RNA viruses can produce two types of virus-derived small RNAs (vsRNAs), small interfering RNA (siRNA) and microRNA (miRNA), that play a key role in RNA silencing-mediated antiviral mechanisms in various hosts by associating with different Argonaute (Ago) proteins. Ago1 has been widely identified as an essential part of the miRNA pathway, while Ago2 is required for the siRNA pathway. Thus, analysis of the interaction between vsRNAs and Ago proteins can provide a clue about which pathway the vsRNA may be involved in. In this study, using rice stripe virus (RSV)-small brown planthoppers (Laodelphax striatellus, Fallen) as an infection model, the interactions of eight vsRNAs derived from four viral genomic RNA fragments and Ago1 or Ago2 were detected via the RNA immunoprecipitation (RIP) method. vsRNA4-1 and vsRNA4-2 derived from RSV RNA4 were significantly enriched in Ago1-immunoprecipitated complexes, whereas vsRNA2-1 and vsRNA3-2 seemed enriched in Ago2-immunoprecipitated complexes. vsRNA1-2 and vsRNA2-2 were detected in both of the two Ago-immunoprecipitated complexes. In contrast, vsRNA1-1 and vsRNA3-1 did not accumulate in either Ago1- or Ago2-immunoprecipitated complexes, indicating that regulatory pathways other than miRNA or siRNA pathways might be employed. In addition, two conserved L. striatellus miRNAs were analysed via the RIP method. Both miRNAs accumulated in Ago1-immunoprecipitated complexes, which was consistent with previous studies, suggesting that our experimental system can be widely used. In conclusion, our study provides an accurate and convenient detection system to determine the potential pathway of vsRNAs, and this method may also be suitable for studying other sRNAs.

Profiling of small RNAs derived from cucumber mosaic virus in infected Nicotiana benthamiana plants by deep sequencing.

In plants, RNA silencing is a conserved mechanism underlying antiviral immunity. To investigate antiviral responses in Nicotiana benthamiana, we analyzed the profiles of the virus-derived small RNAs (vsRNAs) in wild-type N. benthamiana and NbRDR6 mutant plants infected with the cucumber mosaic virus (CMV) 2b-deficient mutant. We observed that NbRDR6 regulates RNA silencing by producing vsRNAs that trigger an effective antiviral response, while NbRDR1 may nonredundantly and synergistically function with NbRDR6 to mediate immune responses. The vsRNAs in N. benthamiana and NbRDR6 mutant plants mainly comprised 21 or 22 nucleotides, and mostly consisted of a 5'-terminal adenine. Additionally, NbAGO2 expression was significantly up-regulated in N. benthamiana and NbRDR6 mutant plants, suggesting that NbAGO2 is closely associated with the antiviral activities of vsRNAs. The distribution of vsRNAs in the CMV genome was biased toward RNA sense strands in both N. benthamiana and NbRDR6 mutant plants. These findings indicate the specific and conserved antiviral immunity in Nicotiana benthamiana.

Different Virus-Derived siRNAs Profiles between Leaves and Fruits in Cucumber Green Mottle Mosaic Virus-Infected Lagenaria siceraria Plants.

RNA silencing is an evolutionarily conserved antiviral mechanism, through which virus-derived small interfering RNAs (vsiRNAs) playing roles in host antiviral defense are produced in virus-infected plant. Deep sequencing technology has revolutionized the study on the interaction between virus and plant host through the analysis of vsiRNAs profile. However, comparison of vsiRNA profiles in different tissues from a same host plant has been rarely reported. In this study, the profiles of vsiRNAs from leaves and fruits of Lagenaria siceraria plants infected with Cucumber green mottle mosaic virus (CGMMV) were comprehensively characterized and compared. Many more vsiRNAs were present in infected leaves than in fruits. vsiRNAs from both leaves and fruits were mostly 21- and 22-nt in size as previously described in other virus-infected plants. Interestingly, vsiRNAs were predominantly produced from the viral positive strand RNAs in infected leaves, whereas in infected fruits they were derived equally from the positive and negative strands. Many leaf-specific positive vsiRNAs with lengths of 21-nt (2058) or 22-nt (3996) were identified but only six (21-nt) and one (22-nt) positive vsiRNAs were found to be specific to fruits. vsiRNAs hotspots were only present in the 5'-terminal and 3'-terminal of viral positive strand in fruits, while multiple hotspots were identified in leaves. Differences in GC content and 5'-terminal nucleotide of vsiRNAs were also observed in the two organs. To our knowledge, this provides the first high-resolution comparison of vsiRNA profiles between different tissues of the same host plant.

Biogenesis, Function, and Applications of Virus-Derived Small RNAs in Plants.

RNA silencing, an evolutionarily conserved and sequence-specific gene-inactivation system, has a pivotal role in antiviral defense in most eukaryotic organisms. In plants, a class of exogenous small RNAs (sRNAs) originating from the infecting virus called virus-derived small interfering RNAs (vsiRNAs) are predominantly responsible for RNA silencing-mediated antiviral immunity. Nowadays, the process of vsiRNA formation and the role of vsiRNAs in plant viral defense have been revealed through deep sequencing of sRNAs and diverse genetic analysis. The biogenesis of vsiRNAs is analogous to that of endogenous sRNAs, which require diverse essential components including dicer-like (DCL), argonaute (AGO), and RNA-dependent RNA polymerase (RDR) proteins. vsiRNAs trigger antiviral defense through post-transcriptional gene silencing (PTGS) or transcriptional gene silencing (TGS) of viral RNA, and they hijack the host RNA silencing system to target complementary host transcripts. Additionally, several applications that take advantage of the current knowledge of vsiRNAs research are being used, such as breeding antiviral plants through genetic engineering technology, reconstructing of viral genomes, and surveying viral ecology and populations. Here, we will provide an overview of vsiRNA pathways, with a primary focus on the advances in vsiRNA biogenesis and function, and discuss their potential applications as well as the future challenges in vsiRNAs research.

Complete genomic sequence of a Rubus yellow net virus isolate and detection of genome-wide pararetrovirus-derived small RNAs.

Rubus yellow net virus (RYNV) was cloned and sequenced from a red raspberry (Rubus idaeus L.) plant exhibiting symptoms of mosaic and mottling in the leaves. Its genomic sequence indicates that it is a distinct member of the genus Badnavirus, with 7932bp and seven ORFs, the first three corresponding in size and location to the ORFs found in the type member Commelina yellow mottle virus. Bioinformatic analysis of the genomic sequence detected several features including nucleic acid binding motifs, multiple zinc finger-like sequences and domains associated with cellular signaling. Subsequent sequencing of the small RNAs (sRNAs) from RYNV-infected R. idaeus leaf tissue was used to determine any RYNV sequences targeted by RNA silencing and identified abundant virus-derived small RNAs (vsRNAs). The majority of the vsRNAs were 22-nt in length. We observed a highly uneven genome-wide distribution of vsRNAs with strong clustering to small defined regions distributed over both strands of the RYNV genome. Together, our data show that sequences of the aphid-transmitted pararetrovirus RYNV are targeted in red raspberry by the interfering RNA pathway, a predominant antiviral defense mechanism in plants.