Regulation of human immunodeficiency virus 1 transcription by nef microRNA

Shinya Omoto and Yoichi R. Fujii

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


   ABSTRACT
Top
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
MicroRNAs (miRNAs) are ~21–25 nt long and interact with mRNAs to lead to either translational repression or RNA cleavage through RNA interference. A previous study showed that human immunodeficiency virus 1 (HIV-1) nef dsRNA from AIDS patients who are long-term non-progressors inhibited HIV-1 transcription. In the study reported here, nef-derived miRNAs in HIV-1-infected and nef transduced cells were identified, and showed that HIV-1 transcription was suppressed by nef-expressing miRNA, miR-N367, in human T cells. The miR-N367 could reduce HIV-1 LTR promoter activity through the negative responsive element of the U3 region in the 5'-LTR. Therefore, nef miRNA produced in HIV-1-infected cells may downregulate HIV-1 transcription through both a post-transcriptional pathway and a transcriptional neo-pathway.


   INTRODUCTION
Top
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
The human immunodeficiency virus type 1 (HIV-1) nef gene is a major virulence determinant in primate lentivirus, is uniquely conserved in HIV-1, HIV-2 and simian immunodeficiency virus (SIV), and is important, but not essential, for virus replication in vivo (Kestler et al., 1991). The nef gene is located at the 3' end of the viral genome, partially overlaps the 3'-long terminal repeat (LTR), is expressed during HIV-1 infection, and often accounts for up to 80 % of HIV-1-specific RNA transcripts during the early stages of virus replication (Robert-Guroff et al., 1990). A long-term non-progressor (LTNP) AIDS patient infected with HIV-1 with deletions in the nef gene and the 3'-LTR has been reported (Deacon et al., 1995; Kirchhoff et al., 1995). However, three of six members of the Sydney blood bank cohort harbouring nef-deleted viruses did progress to AIDS (Learmont et al., 1999). Further study is needed to evaluate these conflicting data and establish the relationship between nef gene function and AIDS progression.

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 stem–loop 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.


   METHODS
Top
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Plasmid construction.
The short hairpin RNA (shRNA)-expressing feline foamy virus (FFV)-based STYLE vector system has been described previously (Omoto et al., 2004b). For construction of an miR-N367 (SF2 nef nt 379–449)-expressing plasmid, pSTYLE-miR-N367, sense A (5'-ACTAGCTAGCATTGGCAGAATTACACACCAGGGCCAGGGATCAGATATCCACTGACCTTTGGATGGTGCTTCAAGCTAGTTTTTTCCGCGGGGAT-3') and antisense B (5'-ATCCCCGCGGAAAAAACTAGCTTGAAGCACCATCCAAAGGTCAGTGGATATCTGATCCCTGGCCCTGGTGTGTAATTCTGCCAATGCTAGCTAGT-3') oligonucleotides containing NheI and SacII linker sequences (underlined) were annealed, digested with NheI and SacII and cloned into plasmid pH1 digested with NheI and SacII (pH1-miR-N367). For insertion of the human H1 promoter plus shRNAs fragment into plasmid pSKY3.0 (Hatama et al., 2001), the fragment was PCR-amplified by primers C (5'-ACGCGTCGACTCATCCTGACTGACGTCATCAACCCGCTCC-3') and D (5'-TAATACGACTCACTATAGGG-3'). The amplified fragment was blunt-ended with T4 DNA polymerase, digested with SalI, and ligated into pSKY3.0 after digestion with SalI and HpaI (pSTYLE-miR-N367). Plasmid pSTYLE containing a shRNA expression cassette against the nef gene (pSTYLE-176, 190, 367) was described previously (Omoto et al., 2004b).

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{beta}. 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{beta}. 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').


   RESULTS
Top
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Detection of miRNAs in HIV-1-infected cells and nef transduced cells
Epstein–Barr virus (EBV)-encoded miRNAs have been recently reported. To investigate miRNA expression in PFV/nef transduced and HIV-1 persistently infected T cells, cultured for 12 months and of which about 98 % cells were Env-positive as described previously (Kawai et al., 2003; Omoto et al., 2004b), we extracted total RNA from PFV/nef-infected Jurkat T cells and carried out Northern blot analyses using probes for nef miR-N367. Analyses using an antisense miR-N367 probe detected small RNA molecules ~25 nt in size (miR), putative precursor bands (miR-L) and PFV/nef major transcripts (12, 6, 4·1, 2·7 and 2·4 kb) (Fig. 1a). The experiments using the miR-N367 probe were then carried out with total RNA from HIV-1 IIIB-infected MT-4 T cells. The miR ~25 nt in size and miR-L were detected together with HIV-1 major transcripts (9·1, 4·3 and 1·8 kb) (Fig. 1b). Similar results were obtained with total RNA from HIV-1 SF2 strain-infected MT-4 T cells (data not shown). RNA samples that were treated with a mixture of single-stranded RNA-specific RNases A and T1 retained the ~25 nt RNAs, but the PFV/nef and HIV-1 major transcripts were not detected (Fig. 1a and b), indicating that the miR could be miRNAs.



View larger version (66K):
[in this window]
[in a new window]
 
Fig. 1. Detection of nef-derived miRNAs produced by PFV/nef or HIV-1. (a) Northern blot analysis of nef miRNAs from total RNA of PFV/nef transduced or untransduced Jurkat T cells. (b) Northern blot analysis of nef miRNAs in HIV-1-infected MT-4 T cells. In some experiments, total RNAs were treated or untreated with a mixture of RNase A and T1. The approximate sizes of the PFV/nef or HIV-1 transcripts and miRNAs are indicated on the right as determined using Decade Markers (Ambion). The probe was antisense miR-N367. The positions of mature miRNAs (miR) and their predicted fold-back precursors (miR-L) are indicated on the left. The loading control (bottom panel) was rRNA stained with ethidium bromide.

 
Inhibition of viral transcription by RNAi
Mutations in the shRNA sense-strand might affect the unmutated 21 nt stem–loop (Miyagishi et al., 2004). In addition, miRNA hairpins are more effective inhibitors of targeted gene expression than conventional shRNAs (Boden et al., 2004). Therefore, we investigated whether HIV-1 nef miRNA, miR-N367, could reduce PFV/nef and HIV-1 transcription more effectively than nef shRNAs or not (Fig. 2). An shRNA against the egfp or luc gene (STYLE-siegfp or -luc) was used as a positive or negative control. The inhibitory effects of STYLE on PFV/nef transcription were evaluated by Luc assays in Jurkat T cells infected with PFV/nef. STYLE-transduced cells were cultured for 72 h followed by transfection with pPFV LTR-Luc and culture for another 48 h. STYLE-miR-N367 showed the greatest reduction of Luc activity compared with STYLE-176, 190 and 367 (Fig. 2).



View larger version (13K):
[in this window]
[in a new window]
 
Fig. 2. Inhibition of PFV/nef and HIV-1 transcription by miR-N367. To study the inhibitory effects of STYLE on PFV/nef transcription, PFV/nef-infected Jurkat T cells at an m.o.i. of 1 were transduced with STYLEs at an m.o.i. of 0·1 and, 72 h later, transfected with the pPFV LTR-Luc reporter plasmid and pCMV{beta} {beta}-gal expressing control plasmid. For HIV-1 transcription, HIV-1-infected MT-4 T cells were transfected with the pLTRSF2 reporter plasmid and pCMV{beta} plasmid 72 h after transduction with STYLEs. Each transfection mixture contained 3·6 µg reporter construct and 0·4 µg pCMV{beta}. At 48 h post-transfection, Luc activity was measured and calculated as Luc/{beta}-gal values. The mean levels of Luc activity in the sample transduced with STYLE(–) and transfected with PFV LTR-Luc or HIV-1 LTR-Luc plasmids were 43 831±2394 or 539 204±42 293 light units, respectively. The data are presented as relative Luc activities, with the percentage of positive cells in samples infected with STYLE(–) scored as 100 %. The data are mean±SD of three independent experiments. Bars, SD.

 
The inhibitory effects of miR-N367 against HIV-1 transcription were evaluated by Luc assays in MT-4 T cells infected with HIV-1 IIIB. Cultivation of the STYLE-transduced cells for 72 h followed by transfection with the pLTRSF2 and culture for another 48 h showed that miR-N367 suppressed HIV-1 LTR-driven Luc activity compared with nef shRNAs and controls (Fig. 2). These data suggested that nef shRNA has more potential to inhibit HIV-1 transcription than PFV/nef.

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).



View larger version (17K):
[in this window]
[in a new window]
 
Fig. 3. Effect of miR-N367 on HIV-1 5'-LTR promoter and nef sequences at the 3'-UTR of target genes. The maps (left) show the full-length (pLTRSF2) and NRE deleted (pLTRSF2-105) 5'-LTR reporter plasmids, and pHILL(+) and (–). Jurkat T cells were co-transfected with each reporter plasmid and Tat-expressing plasmid in the presence or absence of the miR-N367-expressing plasmid. Each transfection mixture contained 1 µg reporter plasmid, 2 µg pH1, 0·5 µg pCD-Tat and 0·5 µg pCMV{beta}. Luc activity was measured 48 h post-transfection and calculated as Luc/{beta}-gal values. The mean Luc activity in the sample of full-length HIV-1 LTR-Luc pH1 plasmid was 138 293±9394 light units. The experiments were performed at least three times and similar results were obtained. Data are given as mean±SD of three independent experiments. Bars, SD.

 
To examine further the effect of miR-N367 on post-transcriptional control by nef at the 3'-UTR, we constructed pHILL(+), which contains the nef gene downstream of the luc gene. As a negative control, we constructed pHILL(–), which contains the nef gene in an inverted orientation. Luc activity was reduced by about 60 % in pHILL(+) transfected cells compared with control pLTRSF2 transfected cells in the absence of miR-N367 (Fig. 3). Expression of miR-N367 reduced Luc activity of pHILL(+) by about 80 %, although miR-N367 expression in pHILL(–) control cells did not significantly change the Luc activity. These data suggest that miR-N367 can suppress HIV-1 LTR promoter activity via NRE in the U3 of the 5'-LTR and nef sequences located at the 3'-UTR of targeted regions.


   DISCUSSION
Top
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
In these studies, we have shown that nef-derived miR-N367 was produced in HIV-1-infected and nef transduced cells. Furthermore, miR-N367 can downregulate HIV-1 transcription via the NRE of the 5'-LTR U3 region and nef sequences at the 3'-UTR. We found that miR-N367 suppresses gene expression at the transcriptional level, which is a different mechanism than a previous report of a post-transcriptional event via a dsRNA-induced silencing complex (RISC) (Brisibe et al., 2003). Recent studies have shown that miRNAs and siRNAs can block mRNA expression by similar mechanisms (Zeng et al., 2003), that siRNAs can function as miRNAs (Doench et al., 2003), and that EBV-encoded miRNAs have been found (Pfeffer et al., 2004). The results reported here are consistent with these observations and showed that miRNA from the 3'-LTR can regulate viral transcription.

Promoter interference has been observed in retrovirus LTRs, the PL promoter of {lambda} 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.


   ACKNOWLEDGEMENTS
 
We thank E. A. Brisibe, M. Ito, S. Hatama, T. Yamamoto, R. Shimizu, M. Sugiyama, Y. Mitsuki, Y. Yasui and K. Otake for excellent technical assistance; H. Okuyama, Y. Ichikawa, T. Kawamura, N. Okada and H. Okada for financial support. English in the manuscript was proofread by Network Assistance International (NAI), Inc. (Kanagawa, Japan). The authors have declared that no conflicts of interest exist.


   REFERENCES
Top
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Aravin, A. A., Naumova, N. M., Tulin, A. V., Vagin, V. V., Rozovsky, Y. M. & Gvozdev, V. A. (2001). Double-stranded RNA-mediated silencing of genomic tandem repeats and transposable elements in the D. melanogaster germline. Curr Biol 11, 1017–1027.[CrossRef][Medline]

Baulcombe, D. C. (2001). RNA silencing. Diced defence. Nature 409, 295–296.[CrossRef][Medline]

Boden, D., Pusch, O., Silbermann, R., Lee, F., Tucker, L. & Ramratnam, B. (2004). Enhanced gene silencing of HIV-1 specific siRNA using microRNA designed hairpins. Nucleic Acids Res 32, 1154–1158.[Abstract/Free Full Text]

Brisibe, E. A., Okada, N., Mizukami, H., Okuyama, H. & Fujii, Y. R. (2003). RNA interference: potentials for the prevention of HIV infections and the challenges ahead. Trends Biotechnol 21, 306–311.[CrossRef][Medline]

Cullen, B. R., Lomedico, P. T. & Ju, G. (1984). Transcriptional interference in avian retrovirus – implication for the promoter insertion model of leukaemogenesis. Nature 307, 241–245.[Medline]

D'Aloja, P., Olivetta, E., Bona, R., Nappi, F., Pedacchia, D., Pugliese, K., Ferrari, G., Verani, P. & Federico, M. (1998). gag, vif, and nef genes contribute to the homologous viral interference induced by a nonproducer human immunodeficiency virus type 1 (HIV-1) variant: identification of novel HIV-1-inhibiting viral protein mutants. J Virol 72, 4308–4319.[Abstract/Free Full Text]

Das, A. T., Brummelkamp, T. R., Westerhout, E. M., Vink, M., Madiredjo, M., Bernards, R. & Berkhout, B. (2004). Human immunodeficiency virus type 1 escapes from RNA interference-mediated inhibition. J Virol 78, 2601–2605.[Abstract/Free Full Text]

Deacon, N. J., Tsykin, A., Solomon, A. & 17 other authors (1995). Genomic structure of an attenuated quasi species of HIV-1 from a blood transfusion donor and recipients. Science 270, 988–991.[Abstract]

Doench, J. G., Petersen, C. P. & Sharp, P. A. (2003). siRNAs can function as miRNAs. Genes Dev 17, 438–442.[Abstract/Free Full Text]

Elbashir, S. M., Lendeckel, W. & Tuschl, T. (2001). RNA interference is mediated by 21- and 22-nucleotide RNAs. Genes Dev 15, 188–200.[Abstract/Free Full Text]

Fire, A., Xu, S., Montgomery, M. K., Kostas, S. A., Driver, S. E. & Mello, C. C. (1998). Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature 391, 806–811.[CrossRef][Medline]

Fish, R. J. & Kruithof, E. K. (2004). Short-term cytotoxic effects and long-term instability of RNAi delivered using lentiviral vectors. BMC Mol Biol 5, 9.[CrossRef][Medline]

Gottesman, S. (2002). Stealth regulation: biological circuits with small RNA switches. Genes Dev 16, 2829–2842.[Free Full Text]

Grad, Y., Aach, J., Hayes, G. D., Reinhart, B. J., Church, G. M., Ruvkun, G. & Kim, J. (2003). Computational and experimental identification of C. elegans microRNAs. Mol Cell 11, 1253–1263.[Medline]

Hatama, S., Otake, K., Omoto, S., Murase, Y., Ikemoto, A., Mochizuki, M., Takahashi, E., Okuyama, H. & Fujii, Y. R. (2001). Isolation and sequencing of infectious clones of feline foamy virus and human/feline foamy virus Env chimera. J Gen Virol 82, 2999–3004.[Abstract/Free Full Text]

Hill, C. L., Bieniasz, P. D. & McClure, M. O. (1999). Properties of human foamy virus relevant to its development as a vector for gene therapy. J Gen Virol 80, 2003–2009.[Abstract/Free Full Text]

Hobert, O. (2004). Common logic of transcription factor and microRNA action. Trends Biochem Sci 29, 462–468.[CrossRef][Medline]

Jacque, J. M., Triques, K. & Stevenson, M. (2002). Modulation of HIV-1 replication by RNA interference. Nature 418, 435–438.[CrossRef][Medline]

Kawai, M., He, L., Kawamura, T., Omoto, S., Fujii, Y. R. & Okada, N. (2003). Chimeric human/murine monoclonal IgM antibodies to HIV-1 Nef antigen expressed on chronically infected cells. Microbiol Immunol 47, 247–253.[Medline]

Kestler, H. W., III, Ringler, D. J., Mori, K., Panicali, D. L., Sehgal, P. K., Daniel, M. D. & Desrosiers, R. C. (1991). Importance of the nef gene for maintenance of high virus loads and for development of AIDS. Cell 65, 651–662.[Medline]

Ketting, R. F., Haverkamp, T. H., van Luenen, H. G. & Plasterk, R. H. (1999). Mut-7 of C. elegans, required for transposon silencing and RNA interference, is a homolog of Werner syndrome helicase and RNaseD. Cell 99, 133–141.[Medline]

Kirchhoff, F., Greenough, T. G., Brettler, D. B., Sullivan, J. L. & Desrosiers, R. C. (1995). Brief report: absence of intact nef sequences in a long-term survivor with nonprogressive HIV-1 infection. N Engl J Med 332, 228–232.[Free Full Text]

Learmont, J. C., Geczy, A. F., Mills, J. & 9 other authors (1999). Immunologic and virologic status after 14 to 18 years of infection with an attenuated strain of HIV-1. A report from the Sydney Blood Bank Cohort. N Engl J Med 340, 1715–1722.[Abstract/Free Full Text]

Lee, Y., Jeon, K., Lee, J. T., Kim, S. & Kim, V. N. (2002). MicroRNA maturation: stepwise processing and subcellular localization. EMBO J 21, 4663–4670.[Abstract/Free Full Text]

Lee, Y., Ahn, C., Han, J. & 8 other authors (2003). The nuclear RNase III Drosha initiates microRNA processing. Nature 425, 415–419.[CrossRef][Medline]

Miyagishi, M., Sumimoto, H., Miyoshi, H., Kawakami, Y. & Taira, K. (2004). Optimization of an siRNA-expression system with an improved hairpin and its significant suppressive effects in mammalian cells. J Gene Med 6, 715–723.[CrossRef][Medline]

Nishitsuji, H., Ikeda, T., Miyoshi, H., Ohashi, T., Kannagi, M. & Masuda, T. (2004). Expression of small hairpin RNA by lentivirus-based vector confers efficient and stable gene-suppression of HIV-1 on human cells including primary non-dividing cells. Microbes Infect 6, 76–85.[CrossRef][Medline]

Olivetta, E., Pugliese, K., Bona, R., D'Aloja, P., Ferrantelli, F., Santarcangelo, A. C., Mattia, G., Verani, P. & Federico, M. (2000). cis expression of the F12 human immunodeficiency virus (HIV) Nef allele transforms the highly productive NL4-3 HIV type 1 to a replication-defective strain: involvement of both Env gp41 and CD4 intracytoplasmic tails. J Virol 74, 483–492.[Abstract/Free Full Text]

Omoto, S., Brisibe, E. A., Okuyama, H. & Fujii, Y. R. (2004a). Feline foamy virus Tas protein is a DNA-binding transactivator. J Gen Virol 85, 2931–2935.[Abstract/Free Full Text]

Omoto, S., Ito, M., Tsutsumi, Y., Ichikawa, Y., Okuyama, H., Brisibe, E. A., Saksena, N. K. & Fujii, Y. R. (2004b). HIV-1 nef suppression by virally encoded microRNA. Retrovirology 1, 44.[CrossRef][Medline]

Pfeffer, S., Zavolan, M., Grasser, F. A. & 8 other authors (2004). Identification of virus-encoded microRNAs. Science 304, 734–736.[Abstract/Free Full Text]

Robert-Guroff, M., Popovic, M., Gartner, S., Markham, P., Gallo, R. C. & Reitz, M. S. (1990). Structure and expression of tat-, rev-, and nef-specific transcripts of human immunodeficiency virus type 1 in infected lymphocytes and macrophages. J Virol 64, 3391–3398.[Medline]

Sijen, T. & Plasterk, R. H. (2003). Transposon silencing in the Caenorhabditis elegans germ line by natural RNAi. Nature 426, 310–314.[CrossRef][Medline]

Sunkar, R. & Zhu, J. K. (2004). Novel and stress-regulated microRNAs and other small RNAs from Arabidopsis. Plant Cell 16, 2001–2019.[Abstract/Free Full Text]

Tabara, H., Sarkissian, M., Kelly, W. G., Fleenor, J., Grishok, A., Timmons, L., Fire, A. & Mello, C. C. (1999). The rde-1 gene, RNA interference, and transposon silencing in C. elegans. Cell 99, 123–132.[Medline]

Vella, M. C., Choi, E. Y., Lin, S. Y., Reinert, K. & Slack, F. J. (2004). The C. elegans microRNA let-7 binds to imperfect let-7 complementary sites from the lin-41 3'UTR. Genes Dev 18, 132–137.[Abstract/Free Full Text]

Yamamoto, T., Omoto, S., Mizuguchi, M. & 7 other authors (2002). Double-stranded nef RNA interferes with human immunodeficiency virus type 1 replication. Microbiol Immunol 46, 809–817.[Medline]

Yekta, S., Shih, I. & Bartel, D. P. (2004). MicroRNA-directed cleavage of HOXB8 mRNA. Science 304, 594–596.[Abstract/Free Full Text]

Zeng, Y., Yi, R. & Cullen, B. R. (2003). MicroRNAs and small interfering RNAs can inhibit mRNA expression by similar mechanisms. Proc Natl Acad Sci U S A 100, 9779–9784.[Abstract/Free Full Text]

Received 16 July 2004; accepted 7 December 2004.