Molecular Biology and Retroviral Genetics Group, Division of Nutritional Sciences, Graduate School of Pharmaceutical Sciences, Nagoya City University, Nagoya 467-8603, Japan
Correspondence
Yoichi R. Fujii
fatfuji{at}hotmail.com
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ABSTRACT |
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INTRODUCTION |
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RNA interference (RNAi) is a defence mechanism against aberrant transcripts that may be produced during viral infection and transposon mobilization (Fire et al., 1998; Ketting et al., 1999
; Tabara et al., 1999
; Aravin et al., 2001
; Baulcombe, 2001
; Sijen & Plasterk, 2003
), and is becoming a powerful tool for development of therapeutic agents against specific pathogens such as HIV-1 (Jacque et al., 2002
; Yamamoto et al., 2002
; Brisibe et al., 2003
; Das et al., 2004
; Nishitsuji et al., 2004
). Small interfering RNAs (siRNAs) are short RNA duplexes that direct the degradation of homologous mRNA (Elbashir et al., 2001
). In contrast, microRNAs (miRNAs) bind to the 3' untranslated regions (UTR) of mRNA producing translational repression with or without target degradation (Zeng et al., 2003
; Yekta et al., 2004
). Mature miRNA (
25 nt in size) is produced by the processing of
70 nt precursor stemloop hairpin RNAs (pre-miRNA) by Dicer (Lee et al., 2002
, 2003
). Animal miRNAs are thought to bind to cognate DNA sites in 3'-UTRs to produce RNA heteroduplexes that inhibit translation of target RNA by an unknown mechanism (Hobert, 2004
). On the other hand, Cullen et al. (1984)
, earlier than a report by Fire et al. (1998)
, reported that in avian leukosis viruses (ALVs) an extensive overlap between 5'- and 3'-LTRs reduced the ability of the downstream 3'-LTR to act as an efficient promoter by interfering with initiation of transcription, a phenomenon designated transcriptional overlap interference. It has also been reported that cis expression of mutated F12-HIV-1 nef inhibits replication of the highly productive NL4-3-HIV-1 strain (D'Aloja et al., 1998
; Olivetta et al., 2000
). Our previous studies have shown that defective variants of nef dsRNA containing the 3'-LTR regions, isolated from LTNP AIDS patients, inhibited HIV-1 transcription (Yamamoto et al., 2002
). Furthermore, HIV-1 nef encoding miRNA, miR-N367, has been identified in HIV-1-infected cells (Omoto et al., 2004b
), but the detailed function of miR-N367 and its relation to promoter interference have not been elucidated. The results presented here show that miR-N367 can efficiently downregulate viral transcription through the U3 region negative responsive element (NRE), possibly indicating that HIV-1 may regulate its own transcription and replication by using miR-N367.
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METHODS |
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Preparation of a full-length HIV-1 SF2 LTR-Luc plasmid (pLTRSF2) was described previously (Yamamoto et al., 2002). For construction of pLTRSF2 deletion mutants, the LTR fragment was amplified by PCR with primers E (5'-GGGGTACCGGGACTTTCCGCTGGGGACTTTCC-3') and F (5'-CCGCTCGAGTGCTAGAGATTTTTCCAACACTGAC-3') using pLTRSF2 as template. The amplified fragments were digested with KpnI and XhoI and then ligated into a pGL3-Basic vector (Promega) that had been digested with KpnI and XhoI (pLTRSF2-105). For preparation of pHILL(+) or () plasmids, the full-length SF2 nef fragment was amplified by PCR with primers G (5'-GCTCTAGAATGGGTGGCAAGTGGTC-3') and H (5'-GCTCTAGATCAGCAGTCTTTGTAGTACTCC-3') using plasmid SF2 as template. After digestion with XbaI, the fragment was inserted into the pLTRSF2, which had been digested with XbaI, in the (+) or () orientation. Preparation of pPFV/nef, pPFV LTR-Luc and pCD-Tat was described previously (Omoto et al., 2004a
, 2004b
).
Cells and vector production.
Jurkat and MT-4 T cells were cultured in RPMI 1640 medium (Gibco) supplemented with 10 % fetal bovine serum (FBS) and antibiotics. For preparation of STYLE vectors, CRFK cells were grown in Iscove's Modified Dulbecco's Medium (Gibco) with 10 % FBS and antibiotics, and BHK cells were also grown in Dulbecco's modified Eagle Medium (Gibco) supplemented with 10 % FBS and antibiotics. The STYLE and PFV/nef vectors were prepared as described previously (Omoto et al., 2004b).
Luciferase (Luc) assay.
Jurkat T or MT-4 T cells were seeded at 2x106 cells per well in 6-well plates. The next day, cells were transfected with 4 µg DNA and 6 µl DMRIE-C Reagent (Invitrogen) per well according to the manufacturer's instruction. For transfection with HIV-1 or PFV/nef-infected cells, each transfection mixture contained 3·6 µg reporter construct and 0·4 µg pCMV. The experiments using pHILL reporter system were performed with 1 µg reporter plasmid, 2 µg pH1, 0·5 µg pCD-Tat and 0·5 µg pCMV
. At 48 h post-transfection, firefly Luc assays were performed as described previously (Yamamoto et al., 2002
).
Northern blot analysis.
Total RNAs were extracted from PFV/nef, HIV-1 IIIB or mock-infected MT-4 T cells using TRIzol reagent (Invitrogen). Approximately 40 µg total RNA was treated with or without RNase A and T1 (Sigma) as described previously (Yamamoto et al., 2002), then subjected to electrophoresis on a 15 % polyacrylamide-7 M urea gel and electroblotted to Hybond-N+ (Pharmacia) for 4 h at 400 mA. RNAs were immobilized by UV cross-linking and baking for 1 h at 80 °C. Hybridization was done with an ECL Direct kit (Pharmacia). The sequence for synthetic DNA probe is (5'-TTGAAGCACCATCCAAAGGTCAGT-3').
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RESULTS |
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Suppression of HIV-1 LTR-driven transcription by miR-N367
A possible mechanism by which miR-N367 might reduce HIV-1 transcription is promoter interference, but whether this mechanism occurs remains to be determined, whereas the plant miRNAs have targets in the 5'-UTR (Sunkar & Zhu, 2004). Therefore, to determine the activity of miR-N367, we cloned miRNA into pHILL and screened for an antiviral effect. Jurkat T cells were transfected with pH1-miR-N367, pCD-Tat and an HIV-1 LTR-Luc plasmid with a full-length (pLTRSF2) or an NRE deletion in the U3 of the 5'-LTR region (pLTRSF2-105), and Luc assays were performed 48 h post-transfection (Fig. 3
). Expression of miR-N367 reduced transcriptional activity of full-length HIV-1 LTR containing NRE by about 40 %. However, transcriptional activity of the NRE-deleted LTR plasmid (pLTRSF2-105) was not statistically affected by miR-N367 expression (Fig. 3
).
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DISCUSSION |
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Promoter interference has been observed in retrovirus LTRs, the PL promoter of phage, prokaryotic operons and yeast (Cullen et al., 1984
). The fact that miR-N367 can repress HIV-1 promoter activity may be one of the mechanisms of promoter interference in retroviruses. Small regulatory RNAs (sRNAs) abound in bacteria and these have intriguing similarities to animal miRNAs (Gottesman, 2002
; Hobert, 2004
). Therefore, a small fraction of miRNA might be transported to the nucleus and gain access to target DNA sequences. Although further studies are needed to clarify the precise mechanisms of promoter interference, these results suggest that miRNAs produced in HIV-1-infected cells may efficiently downregulate HIV-1 transcription through both post-transcriptional pathway and transcriptional neo-pathway, causing low pathogenic infections to be latent.
We have also provided data on the inhibition of HIV-1 nef expression by an shRNA and miRNA expressing FV-based STYLE vector. The apathogenic FVs have proven to be an effective means of targeting various cell types (Hill et al., 1999). HIV and lentivector-mediated transfer of an shRNA-expression cassette have been shown to result in efficient silencing of a target gene (Miyagishi et al., 2004
), and led to the conclusion that RNA viruses could not become a target for siRNAs, although the HIV vector had a deletion in the LTR of the U3 nef region. Our results showed retrovirus RNA was accessible to siRNA and miRNA. The target sites in the LTR may not form a tight secondary structure, according to the siRNA Target Finder website (http://www.ambion.com/techlib/misc/siRNA_finder.html) (Vella et al., 2004
). Further, high expression levels of shRNAs in lentivectors were cytotoxic and not stable (Fish & Kruithof, 2004
). As mir-273 is also a rare miRNA in Caenorhabditis elegans and was not found by cDNA cloning (Grad et al., 2003
), small accumulations and miRNAs from the 3'-LTR U3 might be predicted, from the results above, to maintain the persistent infection state.
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ACKNOWLEDGEMENTS |
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Received 16 July 2004;
accepted 7 December 2004.