The targeting of a cellular co-factor, rather than the HIV-1-specific RNAs, by small interfering RNAs holds promise as the rapid mutational ability of the HIV-1 genome may obviate the potential clinical use of RNAi against this virus. The DEAD-box RNA helicase DDX3 is an essential Rev co-factor in the CRM1-Rev-RRE complex that promotes the export of unspliced and single-spliced HIV-1 RNAs from the nucleus to cytoplasm. In this report, human DDX3 was targeted by specific short hairpin RNAs, and the down-regulation of cell's endogenous DDX3 suppressed the nuclear export of unspliced HIV-1 RNAs but did not affect the cell viability. We further showed that the knockdown of cellular DDX3 could effectively inhibit the replication of HIV-1. Therefore, the current results suggest that the RNA helicase DDX3 may become a potential target by RNAi for future genetic therapy of HIV/AIDS.

DEAD-box RNA helicases constitute the largest family of RNA helicases and are involved in many aspects of RNA metabolism. In this study, we identified RelA (p65), a subunit of nuclear factor-kappaB (NF-kappaB), as a cellular co-factor of DEAD-box RNA helicase DDX1, through mammalian two hybrid system and co-immunoprecipitation assay. Additionally, confocal microscopy and chromatin immunoprecipitation assays confirmed this interaction. In NF-kappaB dependent reporter gene assay, DDX1 acted as a co-activator to enhance NF-kappaB-mediated transcription activation. The functional domains involved were mapped to the carboxy terminal transactivation domain of RelA and the amino terminal ATPase/helicase domain of DDX1. The DDX1 trans-dominant negative mutant lacking ATP-dependent RNA helicase activity lost it transcriptional inducer activity. Moreover, depletion of endogenous DDX1 by specific small interfering RNAs significantly reduced NF-kappaB-dependent transcription. Taken together, the results suggest that DDX1 may play an important role in NF-kappaB-mediated transactivation, and revelation of this regulatory pathway may help to explore the novel mechanisms for regulating NF-kappaB transcriptional activity.

Artificial microRNA (amiRNA) has recently become an important RNA interference (RNAi) technology for gene therapy and gene function studies. Here nine expression strategies were employed to construct plasmid vectors expressing amiRNA (amiR-Fluc) against firefly luciferase (Fluc). Our results indicate that all nine vectors can successfully produce mature amiR-Fluc and specifically suppress the expression of Fluc, although the RNAi efficiency in different mammalian cells displays obvious differences. Among these nine vectors, three can efficiently co-express DsRed reporter gene linked with amiR-Fluc cassette. Moreover, the recommended number of concatenated amiRNAs in a multi-amiRNA expression vector should not be more than four, and the relative position of an amiRNA in the multi-amiRNA expression vector has no apparent influence on its RNAi activity. In summary, all these results described here provide valuable information for the rational design and application of amiRNA expression vector.

Generation of mammalian cells stably expressing multiple exogenous genes is currently difficult. Here we provide a strategy to facilitate this process. First, a helper vector p2A containing three coding sequences for viral 2A peptides was constructed. Three reporter genes coding for red fluorescent protein (DsRed), firefly luciferase (Fluc) and enhanced green fluorescent protein (EGFP) were then inserted into p2A to form a fusion open reading frame that was subsequently subcloned into a lentiviral vector. After transduction, EGFP-positive 293T cells were selected by fluorescence activated cell sorting. The expression of exogenous genes in selected cells was stable for more than 15 passages, and EGFP-positive cells were over 95%. The efficient cleavages of 2A-peptide mediated polyprotein were also observed and all three reporter proteins were functional. Thus, a stable DsRed/Fluc/EGFP-coexpressing cell line was readily established within a short time. The strategy could be useful for basic research and protein production.

Abstract:Recently, artificial microRNA (amiRNA) has become a promising RNA interference (RNAi) technology. Here, we describe a flexible and reliable method for constructing both single-and multi-amiRNA expression vectors. Two universal primers, together with two specific primers carrying the encoding sequence of amiRNA were designed and utilized to synthesize the functional amiRNA cassette through a one-step PCR. With appropriate restriction sites, the synthesized amiRNA cassettes can be cloned into any site of different destination vectors. Using the method, we constructed both singleand multi-amiRNA expression vectors to target three reporter genes, which code firefly luciferase (Fluc), enhanced green fluorescent protein (EGFP) and β-galactosidase (LacZ), respectively. The expressions of three genes were all specifically inhibited by either the corresponding single-or the multi-amiRNA expression vector in 293T cells. And the RNAi efficiency of each amiRNA produced by both single-and multi-amiRNA expression vectors was comparable.