Berkhout Lab - Ben Berkhout - HIV AIDS research Laboratory - HIV-1 replication by RNAi using long hairpin RNA

Inhibition of HIV-1 replication by RNAi using long hairpin RNAs (lhRNAs)

RNA interference (RNAi) is initiated via small interfering RNAs (siRNAs) of approximately 21-23 base pairs (bp), which are cleaved from double-stranded precursor RNAs by the RnaseIII-like enzyme Dicer. These siRNAs are incorporated into a multiprotein complex called RNA-induced silencing complex (RISC) that targets endogenous transcripts, eventually leading to destruction of the target mRNA. RNAi is an ancient sequence-specific gene silencing mechanism in eukaryotes, which is believed to function as a defence against viruses and transposons. Inhibition of virus replication by means of induced RNAi has now been reported for several important human pathogens such as human immunodeficiency virus type 1 (HIV-1), hepatitis C virus (HCV), hepatitis B virus (HBV), dengue virus, poliovirus and influenza virus A. Current preventive measures against HIV-1 are not sufficient to stop the AIDS epidemic. Additionally, treatment of infected patients results in the emergence of drug-resistant virus variants. Therefore we are developing an RNAi based gene therapy as an alternative strategy to block HIV-1 replication (Fig. 1).

RNAi-mediated interference of gene expression is accomplished using synthetic siRNAs or gene constructs expressing short hairpin RNAs (shRNAs). Although efficient in activating RNAi, their use as a therapeutic antiviral is limited because of the emergence of virus escape mutants (Das et al., 2004). The use of lhRNAs (>40 bp) to induce RNAi in mammalian cells has been problematic since it may activate the antiviral interferon response. However, intracellularly expressed long double stranded RNAs (dsRNAs) may be readily processed to siRNAs, thus avoiding induction of the interferon response.

Expression of lhRNAs against HIV-1 was put under the control of constitutive or inducible promoters, and a mild inhibition of virus replication was observed. A modified HIV-1 genome (Maxvector or HIV-lhRNA) was constructed containing the large hairpin inserted in the nef gene of the HIV LAI strain. Co-transfection of the Maxvector with HIV strongly decreased virus production. The Maxvector could initially not replicate, but prolonged culturing resulted in replication competent variants, in which the large hairpin was truncated, leaving shorter hairpin structures. Moreover, replication of wtHIV was inhibited upon co-infection with the replicating Maxvectors. Although the mechanisms of this inhibition are not understood, it is highly effective and will be studied in further detail.

Westerhout EM, Ooms M, Vink M, Das AT, Berkhout B. HIV-1 can escape from RNA interference by evolving an alternative structure in its RNA genome. Nucleic Acids Res. 2005 Feb 01;33(2):796-804.

Das AT, Brummelkamp TR, Westerhout EM, Vink M, Madiredjo M, Bernards R, Berkhout B. Human immunodeficiency virus type 1 escapes from RNA interference-mediated inhibition. J Virol. 2004 Mar;78(5):2601-5.

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Inhibition of HIV-1 replication by RNAi using long hairpin RNAs (lhRNAs) and microRNAs (miRNSs)

Inhibition of HIV-1 replication by RNAi with long hairpin RNAs (lhRNAs) and microRNAs (miRNAs)
by Pavlina Konstantinova and Ying Poi Liu RNAi has been employed to inhibit replication of a wide range of viruses including the human immunodeficiency virus type 1 (HIV-1), hepatitis C virus (HCV), hepatitis B virus (HBV), dengue virus, poliovirus, influenza virus A, coronaviruses, herpesviruses and picornaviruses. RNAi-mediated suppression of HIV-1 replication has been accomplished by synthetic siRNAs in a transient manner and by shRNA-expression vectors in stably transfected cells. Despite potent inhibition, the therapeutic use of a single antiviral siRNA or shRNA targetting viral sequences is limited because of the rapid emergence of HIV-1 escape mutants. Minor sequence changes in the target sequence, sometimes even a single point mutation, are sufficient to overcome the RNAi-mediated inhibition. Strategies to reduce the chance of viral escape include the simultaneous use of multiple siRNAs in a combinatorial RNAi approach, which increases the genetic barrier for viral escape (1). However, expression of these shRNAs necessitates multiple expression cassettes and the construction of rather complex vectors that will not easily provide equimolar shRNA expression levels. Furthermore, when the same promoter is reiterated in a lentiviral vector, recombination is likely to occur with high frequency on repeat sequences.
Alternative anti-escape strategies include the use of gene constructs encoding HIV-1-specific long hairpin RNAs that produce multiple siRNAs. We have described efficient RNAi induction by lhRNAs against human deficiency virus 1 (2). Importantly, we show that lhRNA expression does not seem to induce non-specific type 1 interferon responses in cells, which may occur when dsRNA longer than 30 bp is introduced in mammalian cells. We further expressed a antiviral lhRNA from the HIV-1 genome and observed a potent trans-inhibition of the wild-type virus (3, 4).

A new approach to silence gene expression is the use of microRNA-like transcripts expressed from a polymerase II promoter, which do not require perfect sequence complementarity with the target for inhibition. Depending on the degree of sequence complementarity, target RNA cleavage or translational repression can be induced. Most precursor miRNAs (pre-miRNA) are synthesized by polymerase II as part of longer primary RNA transcripts (pri-miRNAs). The pri-miRNAs are cleaved by the nuclear Drosha-DGCR8 complex to produce a miRNA precursor (pre-miRNA) of ~70 nt. Pre-miRNAs are transported to the cytoplasm by Exportin-5, where they are cleaved by the cytoplasmic RNase III enzyme Dicer to produce the double-stranded miRNA duplex. The 22-24 nt mature miRNA, which is one strand of the duplex, guides the RNA-induced silencing complex (RISC) for mRNA cleavage (perfect complementarity) or translational repression (incomplete complementarity).
A number of miRNAs are encoded in genomic clusters that are transcribed as polycistronic pri-miRNAs, allowing the production of multiple miRNAs from a single transcription unit. One of the largest miRNA polycistron is mir-17-92 or Oncomir-1, which encodes 7 mature miRNAs: miR-17-5p, miR-17-3p, miR-18, miR-19a, miR-20, miR-19b and miR-92. Such a polycistron seems an ideal candidate for the expression of multiple antiviral siRNAs to target escape-prone RNA viruses. In our studies, we demonstrate that HIV-1 replication can be efficiently inhibited by simultaneous expression of up to 4 anti-HIV siRNAs in this mir-17-92 backbone. Furthermore, we show that the expression of individual miRNAs is significantly enhanced in multi hairpin transcripts. Most importantly we demonstrate that antiviral miRNAs are more tolerable to target mismatches than regular shRNA inhibitors, which implies that a single nt-substitution does not suffice for viral escape. The combined results indicate that multi-miRNA strategy may be particularly suited for the attack on escape-prone viral pathogens.

1. Ter Brake,O., Konstantinova,P., Ceylan,M., & Berkhout,B. Silencing of HIV-1 with RNA interference: a multiple shRNA approach. Mol. Ther. 14, 883-892 (2006).

2. Konstantinova,P., de Vries,W., Haasnoot,J., Ter Brake,O., de Haan,P., & Berkhout,B. Inhibition of human immunodeficiency virus type 1 by RNA interference using long-hairpin RNA. Gene Ther. 13, 1403-1413 (2006).

4. P. Konstantinova, Olivier ter Brake, Joost Haasnoot, Peter de Haan and Ben Berkhout (2007) Trans-inhibition of HIV-1 by a long hairpin RNA expressed within the viral genome. Retrovirology 1 March (Epub ahead of print)