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.

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.

Arginine rich short linear motif of HIV-1 regulatory proteins inhibits dicer dependent RNA interference.

BACKGROUND: Arginine Rich Motif (ARM) of HIV-1 Tat and Rev are extensively studied linear motifs (LMs). They are already established as an inefficient bipartite nuclear localisation signal (NLS). The unusual passive diffusion of HIV-1 NLS tagged reporter proteins across the nucleus is due to an unknown competing functionality of ARM. Recent findings about the role of retroviral proteins as a suppressor of RNA interference (RNAi) involving their basic residues hint an interesting answer to this alternate functionality. The present work explores the role of HIV-1 ARM as a uniquely evolved viral motif to combat Dicer dependent RNAi. RESULTS: We show that RNA binding ARM of both HIV-1 Tat and Rev is a LM with a pattern RXXRRXRRR unique to viruses. Extending the in silico results to wet lab, we proved both HIV-1 Tat and Rev can suppress Dicer dependent RNA silencing process involving ARM. We show, HIV-1 Tat and Rev and their corresponding ARM can bind the RISC loading complex (RLC) components TRBP and PACT confirming ARM as an independent RNAi suppression motif. Enhancement of RNAi in infection scenario through enoxacin increases HIV-1 replication as indicated by p24 levels. Except Dicer, all other cytoplasmic RNAi components enhance HIV-1 replication, indicating crucial role of Dicer independent (Ago2 dependent) RNAi pathway in HIV-1 infection. Sequence and structural analysis of endo/exo-microRNA precursors known to be regulated in HIV-1 infection highlights differential features of microRNA biogenesis. One such set of miRNA is viral TAR encoded HIV-1-miR-TAR-5p (Tar1) and HIV-1-miR-TAR-3p (Tar2) that are known to be present throughout the HIV-1 life cycle. Our qPCR results showed that enoxacin increases Tar2 miRNA level which is interesting as Tar2 precursor shows Ago2 dependent processing features. CONCLUSIONS: We establish HIV-1 ARM as a novel viral motif evolved to target the Dicer dependent RNAi pathway. The conservation of such motif in other viral proteins possibly explains the potent suppression of Dicer dependent RNAi. Our model argues that HIV-1 suppress the processing of siRNAs through inhibition of Dicer while at the same time manipulates the RNAi machinery to process miRNA involved in HIV-1 replication from Dicer independent pathways.

Identification of glutamate ABC-Transporter component in Clostridium perfringens as a putative drug target.

Clostridium perfringens is an anaerobic pathogen known to cause vast number of diseases in mammals and birds. Various toxins and hydrolysing enzymes released by the organism are responsible for the necrosis of soft tissues. Due to serious safety issues associated with current vaccines against C. perfringens, there is a need for new drug or vaccine targets. C. perfringens is extremely dependent on its host for nutrition which can be targeted for vaccine development or drug design. Therefore, it is of interest to identify the unique transport systems used by C. perfringens involved in uptake of essential amino acids that are synthesized by the host, so that therapeutic agents can be designed to target the specific transport systems. Use of bioinformatics tools resulted in the identification of a protein component of the glutamate transport system that is not present in the host. Analysis of the conservation profile of the protein domain indicated it to be a glutamate binding protein which also stimulates the ATPase activity of ATP Binding Cassettes (ABC) transporters. Homology modelling of the protein showed two distinct lobes, which is a characteristic of substrate binding proteins. This suggests that the carboxylates of glutamate might be stabilized by electrostatic interactions with basic residues as is observed with other binding proteins. Hence, the homology model of this potential drug target can be employed for in silico docking studies by suitable inhibitors.

Cellular oncomiR orthologue in EBV oncogenesis.

MicroRNAs are small non-coding RNAs that regulate gene expression at multiple levels. The discovery of virally encoded miRNAs attracted immense attention towards their role in viral replication and pathogenesis. Kaposi's-sarcoma-associated herpes virus encodes miRNA that functions as an orthologue of human cellular miRNA, i.e., hsa-miR-155. Keeping the same view we extended the miRNA-homology search between the miRNAs of humans and Epstein-Barr virus. The In silico analyses shows that EBV encoded miR-BART-5 has a significant 'seed' sequence homology to hsa-miR-18 of humans. Further, the mRNA transcripts of the human genes involved in cellular growth could potentially be targeted by both viral as well as human miRNAs. The known etiological role of hsa-miR-18 as an oncomiR suggests that miR-BART-5 may function as viral oncomiR as observed in EBV-positive gastric carcinoma patients.