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 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

Expression levels of the selected miRNAs in developmental stages of <i>S. litura</i>.

<p>The absolute expression levels of the selected miRNAs with respect to U6snRNA (endogenous control) were analyzed by TaqMan miRNA assays using the total RNA isolated from each stage of the insect, <i>S. litura</i>. The bar graphs represent the expression level of each miRNA at a particular stage in comparison to the expression level in 1^st instar larvae. The statistical significance of the qRT analysis was determined by P value < 0.05 while the error bars represent the SD.</p

Analysis of novel miRNAs identified in the library.

<p>(A) Similarity between the precursor sequences of a miRNA family identified in the population. (B) (i) The largest miRNA cluster identified from the library using <i>Sf</i> draft genome assembly as a reference. (ii) Similarity in the nucleotide composition of the mature miRNA sequences present in the cluster, especially the seed region.</p

Homology analysis of <i>Spodoptera</i> miRNAs.

<p>(A) (i) Identity in the seed region of the <i>Spodoptera</i> miRNA with respect to its counterpart from other insects. (ii) Sequence conservation of the <i>Sf</i> miRNA, including the seed region over a wide range of insect species. (B) Similarity between the precursor sequences of <i>Sf</i> miRNA with its counterpart from <i>B. mori</i>. The nucleotide differences are only outside the mature region but in the stem loop sequence.</p

Gene ontology classification of the target genes for the selected miRNAs.

<p>Gene ontology (GO) term was assigned to each target gene based on the annotation and were summarized into three main GO categories (biological process, cellular component, molecular function) while only the top 10 sub categories are presented.</p

Analysis of small RNA reads and conservation of homologous miRNAs.

<p>(A) The sequence length distribution of small RNA read tags in Sf21 cells showed majority of the population at 20 nt, a typical length of mature miRNAs followed by the read lengths around 27 nt which could be putative piRNA sequences. (B) Percentage of known miRNAs from <i>Spodoptera frugiperda</i> with homologs from other insects; <i>B. mori, D. melanogaster, T. castenum, A. aegypti, A. gambie</i> and <i>C. elegans</i>.</p

Northern validation of selected miRNAs.

<p>Both the known and novel miRNAs were checked by northern analysis for their expression in Sf21 cells using the total RNA isolated from them. All the miRNAs exhibited the expected sizes from the library.</p

Tissue distribution of selected miRNAs in <i>S. litura</i>.

<p>The absolute expression levels of the selected miRNAs with respect to U6snRNA (endogenous control) were analyzed by TaqMan miRNA assays using total RNA isolated from respective tissue parts of the larvae. The bar graphs (A, C) represent the expression level of each miRNA in a particular tissue part but in comparison to that of miR-305-5p. (B) The bar graph represents the relative distribution of expression levels of each miRNA in three segments of the whole gut, i.e., fore gut, mid gut and hind gut. The statistical significance of the qRT analysis was determined by P value < 0.05 while the error bars represent the SD.</p

Enhanced autophagy in pulmonary endothelial cells on exposure to HIV-Tat and morphine: Role in HIV-related pulmonary arterial hypertension

<p>Intravenous drug use is one of the major risk factors for HIV-infection in HIV-related pulmonary arterial hypertension patients. We previously demonstrated exaggerated pulmonary vascular remodeling with enhanced apoptosis followed by increased proliferation of pulmonary endothelial cells on simultaneous exposure to both opioids and HIV protein(s). Here we hypothesize that the exacerbation of autophagy may be involved in the switching of endothelial cells from an early apoptotic state to later hyper-proliferative state. Treatment of human pulmonary microvascular endothelial cells (HPMECs) with both the HIV-protein Tat and morphine resulted in an oxidative stress-dependent increase in the expression of various markers of autophagy and formation of autophagosomes when compared to either Tat or morphine monotreatments as demonstrated by western blot, transmission electron microscopy and immunofluorescence. Autophagy flux experiments suggested increased formation rather than decreased clearance of autolysosomes. Inhibition of autophagy resulted in a significant increase in apoptosis and reduction in proliferation of HPMECs with combined morphine and Tat (M+T) treatment compared to monotreatments whereas stimulation of autophagy resulted in opposite effects. Significant increases in the expression of autophagy markers as well as the number of autophagosomes and autolysosomes was observed in the lungs of SIV-infected macaques and HIV-infected humans exposed to opioids. Overall our findings indicate that morphine in combination with viral protein(s) results in the induction of autophagy in pulmonary endothelial cells that may lead to an increase in severity of angio-proliferative remodeling of the pulmonary vasculature on simian and human immunodeficiency virus infection in the presence of opioids.</p