Dataset Information

RNAi-mediated inhibition of HIV-1 by targeting partially complementary viral sequences.

ABSTRACT: Potent antiviral RNAi can be induced by intracellular expression of short hairpin RNAs (shRNAs) and artificial microRNAs (miRNAs). Expression of shRNA and miRNA results in target mRNA degradation (perfect base pairing) or translational repression (partial base pairing). Although efficient inhibition can be obtained, error-prone viruses such as human immunodeficiency virus type 1 (HIV-1) can escape from RNAi-mediated inhibition by mutating the target sequence. Recently, artificial miRNAs have been shown to be potent RNAi inducers due to their efficient processing by the RNAi machinery. Furthermore, miRNAs may be more proficient in suppressing imperfect targets than shRNAs. In this study, we tested the knockdown efficiency of miRNAs and shRNAs against wild-type and RNAi-escape HIV-1 variants with one or two mutations in the target sequence. ShRNAs and miRNAs can significantly inhibit the production of HIV-1 variants with mutated target sequences in the open reading frame. More pronounced mutation-tolerance was measured for targets in the 3' untranslated region (3' UTR). Partially complementary sequences within the 3' UTR of the HIV-1 RNA genome efficiently act as target sites for miRNAs and shRNAs. These data suggest that targeting imperfect target sites by antiviral miRNAs or shRNAs provides an alternative RNAi approach for inhibition of pathogenic viruses.

SUBMITTER: Liu YP

PROVIDER: S-EPMC2764431 | biostudies-literature | 2009 Oct

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

RNAi-mediated inhibition of HIV-1 by targeting partially complementary viral sequences.

Liu Ying Poi YP Gruber Jens J Gruber Jens J Haasnoot Joost J Konstantinova Pavlina P Berkhout Ben B

Nucleic acids research 20090805 18

Potent antiviral RNAi can be induced by intracellular expression of short hairpin RNAs (shRNAs) and artificial microRNAs (miRNAs). Expression of shRNA and miRNA results in target mRNA degradation (perfect base pairing) or translational repression (partial base pairing). Although efficient inhibition can be obtained, error-prone viruses such as human immunodeficiency virus type 1 (HIV-1) can escape from RNAi-mediated inhibition by mutating the target sequence. Recently, artificial miRNAs have bee ...[more]

PMID: 19656954

Similar Datasets

Project description:Phylogeneticists have developed several statistical methods to infer recombination among molecular sequences that are evolutionarily related. Of these methods, Markov change-point models currently provide the most coherent framework. Yet, the Markov assumption is faulty in that the inferred relatedness of homologous sequences across regions divided by recombinant events is not independent, particularly for nonrecombinant sequences as they share the same history. To correct this limitation, we introduce a novel random tips (RT) model. The model springs from the idea that a recombinant sequence inherits its characters from an unknown number of ancestral full-length sequences, of which one only observes the incomplete portions. The RT model decomposes recombinant sequences into their ancestral portions and then augments each portion onto the data set as unique partially observed sequences. This data augmentation generates a random number of sequences related to each other through a single inferable tree with the same random number of tips. While intuitively pleasing, this single tree corrects the independence assumptions plaguing previous methods while permitting the detection of recombination. The single tree also allows for inference of the relative times of recombination events and generalizes to incorporate multiple recombinant sequences. This generalization answers important questions with which previous models struggle. For example, we demonstrate that a group of human immunodeficiency type 1 recombinant viruses from Argentina, previously thought to have the same recombinant history, actually consist of 2 groups: one, a clonal expansion of a reference sequence and another that predates the formation of the reference sequence. In another example, we demonstrate that 2 hepatitis B virus recombinant strains share similar splicing locations, suggesting a common descent of the 2 viruses. We implement and run both examples in a software package called StepBrothers, freely available to interested parties.

Project description:Since RNA interference (RNAi) has the potential to discriminate between single nucleotide changes, there is growing interest in the use of RNAi as a promising therapeutical approach to target dominant disease-associated alleles. Mutations in the leucine-rich repeat kinase 2 (LRRK2) gene have been linked to dominantly inherited Parkinson's disease (PD). We focused on three LRRK2 mutations (R1441G/C and the more prevalent G2109S) hoping to identify shRNAs that would both recognize and efficiently silence the mutated alleles preferentially over the wild-type alleles. Using a luciferase-based reporter system, we identified shRNAs that were able to specifically target the R1441G and R1441C alleles with 80% silencing efficiency. The same shRNAs were able to silence specifically mRNAs encoding either partial or full-length mutant LRRK2 fusion proteins, while having a minimal effect on endogenous wild-type LRRK2 expression when transfected in 293FT cells. Shifting of the mutant recognition site (MRS) from position 11 to other sites (4 and 16, within the 19-mer window of our shRNA design) reduced specificity and overall silencing efficiency. Developing an allele-specific RNAi of G2019S was problematic. Placement of the MRS at position 10 resulted in efficient silencing of reporters (75-80%), but failed to discriminate between mutant and wild-type alleles. Shifting of the MRS to positions 4, 5, 15, 16 increased the specificity of the shRNAs, but reduced the overall silencing efficiency. Consistent with previous reports, these data confirm that MRS placement influences both allele-specificity and silencing strength of shRNAs, while further modification to hairpin design or MRS position may lead to the development of effective G2019S shRNAs. In summary, the effective shRNA against LRRK2 R1441 alleles described herein suggests that RNAi-based therapy of inherited Parkinson's disease is a viable approach towards developing effective therapeutic interventions for this serious neurodegenerative disease.

Dataset Information

RNAi-mediated inhibition of HIV-1 by targeting partially complementary viral sequences.

Publications

RNAi-mediated inhibition of HIV-1 by targeting partially complementary viral sequences.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets