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. 2006 Sep 4:3:57.
doi: 10.1186/1742-4690-3-57.

The virion-associated incoming HIV-1 RNA genome is not targeted by RNA interference

Ellen M Westerhout  1 , Olivier ter Brake, Ben Berkhout
Affiliations

The virion-associated incoming HIV-1 RNA genome is not targeted by RNA interference

Ellen M Westerhout et al. Retrovirology. .

Abstract

Background: RNA interference (RNAi) has proven to be a powerful tool to suppress gene expression and can be used as a therapeutic strategy against human pathogenic viruses such as human immunodeficiency virus type 1 (HIV-1). Theoretically, RNAi-mediated inhibition can occur at two points in the replication cycle, upon viral entry before reverse transcription of the RNA genome, and on the newly transcribed viral RNA transcripts. There have been conflicting results on whether RNAi can target the RNA genome of infecting HIV-1 particles. We have addressed this issue with HIV-1-based lentiviral vectors.

Results: We determined the transduction efficiency of a lentiviral vector, as measured by GFP expressing cells, which reflects the number of successful integration events in a cell line stably expressing shNef. We did not observe a difference in the transduction efficiency comparing lentiviral vectors with or without the Nef target sequence in their genome. The results were similar with particles pseudotyped with either the VSV-G or HIV-1 envelope. Additionally, no reduced transduction efficiencies were observed with multiple other shRNAs targeting the vector genome or with synthetic siNef when transiently transfected prior to transduction.

Conclusion: Our findings indicate that the incoming HIV-1 RNA genome is not targeted by RNAi, probably due to inaccessibility to the RNAi machinery. Thus, therapeutic RNAi strategies aimed at preventing proviral integration should be targeting cellular receptors or co-factors involved in pre-integration events.

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Figures

Figure 1
Figure 1
The lentiviral vector and packaging constructs. The lentiviral vector JS1 is a third generation self-inactivating vector [39], which contains a GFP reporter gene expressed from the phosphoglycate kinase promoter (PGK) with the posttranscriptional regulatory element (pre) from hepatitis B virus. The vector genome is expressed from the Rous sarcoma promoter (RSV) and transcription starts with the R and U5 regions of the HIV-1 long terminal repeat (LTR), the packaging signal (ψ) and part of the gag open reading frame (gag). It contains the rev responsive element (RRE), central polypurine tract (cPPT) and the 3' LTR, which has a deletion in the U3 region (ΔU3). The HIV-1 sequences are tinted gray. Transcription of the vector genome and GFP reporter terminates at the HIV-1 polyA within the 3'LTR. The Nef target sequence (wild type or mutant) was cloned into the multiple cloning site (MCS). The three packaging constructs encode the trans-acting proteins required for the production of infectious virus (HIV-1 sequences in gray).
Figure 2
Figure 2
Sequence-specific inhibition of lentiviral production by RNAi. a) Schematic of lentiviral production. When an shNef-expression plasmid is co-transfected during lentiviral vector production, the lentiviral vector RNA genome containing the Nef target (gray box) can be targeted by RNAi (dark arrow). b) Lentiviral vector stocks (JS1, JS1-Nef and JS1-R2) were produced in 293T cells in the absence (-shNef) or presence (+shNef) of an shNef-expression plasmid and were titrated on SupT1 cells. Transduced cells were analyzed by GFP-FACS. The mean values of three independent experiments are shown. The control values (-shNef) were set at 100% for each lentiviral vector.
Figure 3
Figure 3
No sequence-specific inhibition of lentiviral transduction by RNAi. a) Schematic of lentiviral transduction. When shNef is stably produced in the target cells, the question is whether the incoming vector genome with the shNef target sequence is targeted by RNAi (dark arrow with question mark). b) SupT1 cells stably expressing shNef (+ shNef) or control SupT1 cells (- shNef) were transduced at an m.o.i. of 0.03, 0.3 or 1.0 with the control vector (JS1) or vectors containing a complete (JS1-Nef) or mutated (JS1-R2) shNef target sequence. Infected cells were analyzed by GFP-FACS. The control values (- shNef) were set at 100% for each lentiviral vector. The mean values of three experiments are shown.
Figure 4
Figure 4
Sequence-specific inhibition in shNef-expressing cells. a) Schematic of RNAi-mediated targeting of mRNA with the shNef target sequence (gray box) in shNef-expressing SupT1 cells. b) SupT1 cells stably expressing shNef (+ shNef) or control SupT1 cells (- shNef) were transfected with luciferase reporter constructs that contain the complete shNef target sequence (pGL3-Nef) or not (pGL3-R2). The mean values obtained in two independent experiments are shown. Values measured in the control transfection (- shNef) were set at 100% for each reporter construct.
Figure 5
Figure 5
No inhibition of lentiviral transduction with virions containing the HIV-1 Envelope. SupT1 cells stably expressing shNef (+ shNef) or control SupT1 cells (- shNef) were transduced at an m.o.i. of 0.03, 0.2 or 0.5 with either the control (JS1) or the shNef target sequence containing (wt-Nef) lentiviral vector with an HIV-1 envelope protein. Infected cells were analyzed by GFP-FACS. The control values (- shNef) were set at 100% for each infection. The mean values of two independent experiments are shown.
Figure 6
Figure 6
No inhibition of lentiviral transduction in cells transfected with different shRNA plasmids or siRNA. a) Map of the JS1-Nef genome with the positions targeted by the shRNA inhibitors. b) 293T cells were mock transfected (-) or transfected with siNef or plasmids expressing the indicated shRNAs. The cells were subsequently transfected with luciferase reporter constructs containing the target sequences and relative luciferase expression was measured. The mean values obtained in two independent experiments are shown. The control value (-) was set at 100% for each luciferase reporter. c) 293T cells were mock transfected (-) or transfected with the control pBS, siNef or plasmids expressing the indicated shRNA. The cells were subsequently transduced with the JS1-Nef vector. Transduction efficiency was determined by GFP-FACS. The mean values obtained in two independent experiments are shown. The transduction efficiency for the control experiment (-) was set at 100%.
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References

    1. Hammond SM, Bernstein E, Beach D, Hannon GJ. An RNA-directed nuclease mediates post-transcriptional gene silencing in Drosophila cells. Nature. 2000;404:293–296. doi: 10.1038/35005107. - DOI - PubMed
    1. Fire A, Xu S, Montgomery MK, Kostas SA, Driver SE, Mello CC. Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature. 1998;391:806–811. doi: 10.1038/35888. - DOI - PubMed
    1. Elbashir SM, Lendeckel W, Tuschl T. RNA interference is mediated by 21- and 22-nucleotide RNAs. Genes Dev. 2001;15:188–200. doi: 10.1101/gad.862301. - DOI - PMC - PubMed
    1. Zamore PD, Tuschl T, Sharp PA, Bartel DP. RNAi: double-stranded RNA directs the ATP-dependent cleavage of mRNA at 21 to 23 nucleotide intervals. Cell. 2000;101:25–33. doi: 10.1016/S0092-8674(00)80620-0. - DOI - PubMed
    1. Nykanen A, Haley B, Zamore PD. ATP requirements and small interfering RNA structure in the RNA interference pathway. Cell. 2001;107:309–321. doi: 10.1016/S0092-8674(01)00547-5. - DOI - PubMed

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