Background MicroRNAs (miRNAs) are 21~25-nucleotides (nt) long and interact with mRNAs to trigger either translational repression or RNA cleavage through RNA interference (RNAi), depending on the degree of complementarity with the target mRNAs. Our recent study has shown that HIV-1  nef  dsRNA from AIDS patients who are long-term non-progressors (LTNPs) inhibited the transcription of HIV-1.
Background
MicroRNAs (miRNAs) are 21~25-nucleotides (nt) long and interact with mRNAs to trigger either translational repression or RNA cleavage through RNA interference (RNAi), depending on the degree of complementarity with the target mRNAs. Our recent study has shown that HIV-1  nef  dsRNA from AIDS patients who are long-term non-progressors (LTNPs) inhibited the transcription of HIV-1.nefResults Here, we show the possibility that  nef -derived miRNAs are produced in HIV-1 persistently infected cells. Furthermore,  nef  short hairpin RNA (shRNA) that corresponded to a predicted  nef  miRNA (~25 nt, miR-N367) can block HIV-1 Nef expression  in vitro  and the suppression by shRNA/miR-N367 would be related with low viremia in an LTNP (15-2-2). In the 15-2-2 model mice, the weight loss, which may be rendered by  nef  was also inhibited by shRNA/miR-N367 corresponding to suppression of  nef  expression  in vivo .
Results
Here, we show the possibility that  nef -derived miRNAs are produced in HIV-1 persistently infected cells. Furthermore,  nef  short hairpin RNA (shRNA) that corresponded to a predicted  nef  miRNA (~25 nt, miR-N367) can block HIV-1 Nef expression  in vitro  and the suppression by shRNA/miR-N367 would be related with low viremia in an LTNP (15-2-2). In the 15-2-2 model mice, the weight loss, which may be rendered by  nef  was also inhibited by shRNA/miR-N367 corresponding to suppression of  nef  expression  in vivo .nefnefnefin vitronefnefin vivoConclusions These data suggest that  nef /U3 miRNAs produced in HIV-1-infected cells may suppress both Nef function and HIV-1 virulence through the RNAi pathway.
Conclusions
These data suggest that  nef /U3 miRNAs produced in HIV-1-infected cells may suppress both Nef function and HIV-1 virulence through the RNAi pathway.nef

Background The human immunodeficiency virus (HIV), which infect humans cause acquired immunodeficiency syndrome (AIDS), which has reached pandemic levels in some societies, especially those in Southern Africa and Southeast Asia [ 1 ]. Given the immensity of HIV pandemic, the development of a rather safe and cheap, effective therapeutics, has become the main focus [ 2 ]. Several strategies attempted to control the spread of AIDS have not shown major breakthrough and the vaccines have shown little promise as far as their efficacy is concerned. However, one approach used extensively in other diploid organisms, which now has tremendous potential to encourage antiviral defense against HIV appears to be double stranded RNA-dependent post-transcriptional gene silencing or RNA interference (RNAi). RNAi is a defense mechanism against aberrant transcripts that may be produced during viral infection and mobilization of transposons [ 3 , 4 ]. The RNAi pathway has been implicated in silencing transposons in the  C. elegans  germline [ 5 , 6 ], silencing stellate repeats in the  Drosophila  germline, and the response against invading viruses in plants [ 7 ]. Post-transcriptional regulation by RNAi is mediated by small non-coding RNAs (~25-nucleotides; nt). Small interfering RNAs (siRNAs) are short RNA duplexes that direct the degradation of homologous transcripts [ 8 ]. In contrast, the single stranded microRNAs (miRNAs) bind to 3' untranslated regions of mRNA with complementarity of 50 to 85% to give translational repression without target degradation [ 9 ]. The mature miRNA (~25-nt) is produced by processing of ~70-nt precursor stem-loop hairpin RNAs (Pre-miRNA) by Dicer [ 10 , 11 ]. At the moment several human diseases, including spinal muscular atrophy (Paushkin  et al ., 2002), fragile X mental retardation [ 13 , 14 ] and chronic lymphocytic leukemia [ 15 ] have been identified as illnesses in which miRNAs or their machinery might be implicated. However, up until now there has been no clear-cut scientific proof that establishes the exact correlation between miRNAs and human infectious diseases such as AIDS. One of the human immunodeficiency virus type 1 (HIV-1) coding accessory genes,  nef , is located at the 3' end of the viral genome and partially overlaps the 3'-long terminal repeat (LTR). The  nef  gene is uniquely conserved in HIV-1, HIV-2 and simian immunodeficiency virus (SIV) and is not essential but important for viral replication  in vivo  [ 16 ]. The  nef  gene is expressed during HIV infection and often accounts for up to 80% of HIV-1 specific RNA transcripts during the early stages of viral replication [ 2 ]. Our own investigations have shown that defective variants of  nef  dsRNA containing the 3'-LTR regions, obtained from long-term non-progressor (LTNP) AIDS patients, actually inhibited the transcription of HIV-1 [ 17 ]. Furthermore,  cis -expression of mutated F12-HIV-1  nef  inhibits replication of highly productive NL43-HIV-1 strain, which is not related to down-regulation of CD4 [ 18 , 19 ]. It has been demonstrated that F12  nef  gene cloned from the provirus of naturally occurring HUT-78 T cells infected with the supernatant of the peripheral blood mononuclear cells (PBMCs) of an HIV-seropositive non-producer patient, induces a block of viral replication [ 19 ]. Thus, it has been suggested that  nef  RNAs may be a  cis -regulatory factor for HIV-1 replication [ 20 ]. In the current study, we have established the link between miRNAs and HIV infections by demonstrating that  nef -derived miRNAs are produced in HIV-1-infected cells. The results presented here show that  nef  short hairpin RNAs (shRNAs) corresponding to the  nef  miRNAs efficiently block RNA stability or mRNA translation, perhaps an indication that HIV-1 regulates its own replication by using  nef  miRNAs.
Background
The human immunodeficiency virus (HIV), which infect humans cause acquired immunodeficiency syndrome (AIDS), which has reached pandemic levels in some societies, especially those in Southern Africa and Southeast Asia [ 1 ]. Given the immensity of HIV pandemic, the development of a rather safe and cheap, effective therapeutics, has become the main focus [ 2 ]. Several strategies attempted to control the spread of AIDS have not shown major breakthrough and the vaccines have shown little promise as far as their efficacy is concerned. However, one approach used extensively in other diploid organisms, which now has tremendous potential to encourage antiviral defense against HIV appears to be double stranded RNA-dependent post-transcriptional gene silencing or RNA interference (RNAi).RNAi is a defense mechanism against aberrant transcripts that may be produced during viral infection and mobilization of transposons [ 3 , 4 ]. The RNAi pathway has been implicated in silencing transposons in the  C. elegans  germline [ 5 , 6 ], silencing stellate repeats in the  Drosophila  germline, and the response against invading viruses in plants [ 7 ]. Post-transcriptional regulation by RNAi is mediated by small non-coding RNAs (~25-nucleotides; nt). Small interfering RNAs (siRNAs) are short RNA duplexes that direct the degradation of homologous transcripts [ 8 ]. In contrast, the single stranded microRNAs (miRNAs) bind to 3' untranslated regions of mRNA with complementarity of 50 to 85% to give translational repression without target degradation [ 9 ]. The mature miRNA (~25-nt) is produced by processing of ~70-nt precursor stem-loop hairpin RNAs (Pre-miRNA) by Dicer [ 10 , 11 ]. At the moment several human diseases, including spinal muscular atrophy (Paushkin  et al ., 2002), fragile X mental retardation [ 13 , 14 ] and chronic lymphocytic leukemia [ 15 ] have been identified as illnesses in which miRNAs or their machinery might be implicated. However, up until now there has been no clear-cut scientific proof that establishes the exact correlation between miRNAs and human infectious diseases such as AIDS.C. elegansDrosophilaet alOne of the human immunodeficiency virus type 1 (HIV-1) coding accessory genes,  nef , is located at the 3' end of the viral genome and partially overlaps the 3'-long terminal repeat (LTR). The  nef  gene is uniquely conserved in HIV-1, HIV-2 and simian immunodeficiency virus (SIV) and is not essential but important for viral replication  in vivo  [ 16 ]. The  nef  gene is expressed during HIV infection and often accounts for up to 80% of HIV-1 specific RNA transcripts during the early stages of viral replication [ 2 ]. Our own investigations have shown that defective variants of  nef  dsRNA containing the 3'-LTR regions, obtained from long-term non-progressor (LTNP) AIDS patients, actually inhibited the transcription of HIV-1 [ 17 ]. Furthermore,  cis -expression of mutated F12-HIV-1  nef  inhibits replication of highly productive NL43-HIV-1 strain, which is not related to down-regulation of CD4 [ 18 , 19 ]. It has been demonstrated that F12  nef  gene cloned from the provirus of naturally occurring HUT-78 T cells infected with the supernatant of the peripheral blood mononuclear cells (PBMCs) of an HIV-seropositive non-producer patient, induces a block of viral replication [ 19 ]. Thus, it has been suggested that  nef  RNAs may be a  cis -regulatory factor for HIV-1 replication [ 20 ].nefnefin vivonefnefcisnefnefnefcisIn the current study, we have established the link between miRNAs and HIV infections by demonstrating that  nef -derived miRNAs are produced in HIV-1-infected cells. The results presented here show that  nef  short hairpin RNAs (shRNAs) corresponding to the  nef  miRNAs efficiently block RNA stability or mRNA translation, perhaps an indication that HIV-1 regulates its own replication by using  nef  miRNAs.nefnefnefnefResults and Discussion Identification of a candidate of miRNAs in HIV-1-infected cells Very recently, the Epstein-Barr virus (EBV)-encoded miRNAs were identified. Thus, during the preliminary stages of this study, our curiosity was fixed on the need to find out if indeed there was any relationship between  nef  miRNAs and HIV-1-infected cells. To achieve this purpose, we extracted total RNA from HIV-1 IIIB strain persistently infected MT-4 T cells and northern blot analysis was performed using eight probes against the  nef  coding region, as shown in Figure  1A . Analyses using several anti-sense probes, small RNA molecules approximately ~25-nt in size were detected as well as HIV-1 major transcripts, 9.1, 4.3 and 1.8 kb bands (Fig.  1B ). Similar results were obtained with total RNA from HIV-1 SF2 strain infected MT-4 T cells (data not shown). RNA samples treated with a mixture of the single stranded specific RNases A and T1 also generated ~25-nt RNAs that hybridized in northern blots with the sense probes against the same  nef  region. However HIV-1 major transcripts were not detected (data not shown), indicating that the structure of the small RNA molecules could be double-stranded RNAs (miRNAs). Some variability was observed when the quantity of the miRNAs was compared with the total of the major transcripts. A maximum of 3.2% of miRNAs was detected by using #367 probe when compared with total HIV-1 transcripts, and the minimum of 0.3% was detected by using probe# 90 (Fig.  1B ). Figure 1 Detection of HIV-1  nef  miRNAs and inhibition of Nef expression by  nef  RNAs. ( A ) Schematic representation of HIV-1  nef  in its genome and synthetic DNA probes (#) used in this study. ( B ) Total RNAs were extracted from MT-4 T cells persistently infected with HIV-1 IIIB, separated on a 15% polyacrylamide-7 M urea gel, and subjected to northern blot analysis. The approximate sizes of the three classes of HIV-1 transcripts and small RNAs are indicated on the right. The loading control was rRNA stained with ethidium bromide. Relative expression (%) of  nef  small RNAs to the three classes of HIV-1 transcripts is at the bottom of figure. ( C ) Schematic representation of effector plasmids ( E ) H1 promoter-driven shRNA expression plasmids. Reporter (R) Nef-EGFP expression plasmid (pYM2.2) is also shown.  (D)  Inhibition by sinefs in pH1 plamids of Nef-EGFP expression. Either sinef, siluc or siegfp in each plasmid was transfected into Jurkat T cells in the presence of either pYM2.2 or control pEGFP-N1. At 36 h after transfection EGFP-positive cells were counted by flow cytometry. Data represent the relative activity of EGFP-positive cells where the percentage of positive cells in the sample transfected with pYM2.2 or pEGFP-N1 plus pcDNA3.1 or si(-) in pH1 plasmid was scored as 100%. Data are averages of three independent experiments + SD. Bars, SD. ( E ) Immunoblot analysis showing inhibition of YM2.2 expression by different  nef  shRNAs. Jurakat T cells were transfected with pYM2.2 and pH1/sinefs or siluc plasmid, cellular lysates were prepared 48 h after transfection, and immunoblotted with rabbit serum against Nef (upper panel) and anti-β-actin antibody ( lower panel ). The β-actin expression shows equal loading of all samples. To randomly clone the  nef  miRNA, ~25-nt RNAs were gel purified, cloned and sequenced. The sequences from the  nef  miRNA clones were 5'-acugaccuuuggauggugcuucaa-3' or similar ones, corresponded to the nucleotides approximately 420 to 443 conserved region of  nef  (miR-N367). The most notable feature of this analysis is that it has proven beyond reasonable doubts that  nef -derived miRNAs are produced in HIV-1 infected cells. Inhibition of Nef by plasmids-encoding siRNA/miRNA To examine inhibition of  nef  expression by the  nef  miRNA, we constructed eight shRNAs homologous to the native miRNA or probes used in Figure  1A [ 21 ]. Although it has been reported that three to four mutations in the sense strand derived from miRNA could have the potential to control unmutated 21-nt stem loop [ 22 ], we investigated whether the native shRNA-expressing plasmid can effectively reduce  nef  gene expression or not (Fig.  1C  and  1D ). We used  egfp  or  luc  gene (pH1/siegfp or luc) as a positive or negative control. All the shRNA-expressing plasmids including the controls were co-transfected into Jurkat T cells with pYM2.2 and cell fluorescence resulting from the expression of EGFP reporter gene was quantified by flow cytometry. The sinef176, 190, 367/miR-N367 and control siegfp all showed efficient reduction, but the sinef 007, 084, 299, 468 and 580 constructs gave only modest reductions, and no suppression was observed with si(-) and luc (Fig.  1D  and Table  1 ). Immunoblot analysis using anti-Nef rabbit serum also confirmed the inhibition of Nef-EGFP expression by sinef 176, 190 and 367/miR-N367 (Fig.  1E ). Table 1 Relation between AIDS clinical courses and  nef  dsRNA or siRNA/miRNA in suppression of  nef  gene expression Nef inhibitor Nef expressed by: Target region Clinical courses Nef-EGFP HIV-1 IIIB PFV/nef dsRNA * Human SF2 ++  † ++ ND ‡ Full-length (including miR-N367) ND 1-3-3 ++ - ND U3 deleted Rapid progressor, Died within 3 years 4-2-1 ND + ND U3 region (including miR-N367) Rapid progressor, Died within 3 years 15-2-2 ++ +++ ND U3 region (including miR-N367) Non progressor with low plasma viremia 16-1-1 ND + ND U3 deleted Non progressor with low plasma viremia jw95-1 ND + ND U3 region (including miR-N367) Non progressor with undetectable viremia siRNA 15-2-2 model mouse dsnef + + + Full-length ND sinef007 + ND ND Upstream of U3 region ND sinef084 + ND ND Upstream of U3 region ND sinef176 +++ +++ ++ Upstream of U3 region § ND sinef190 +++ +++ +++ Upstream of U3 region ND sinef299 + ND ND U3 region (aa 83–135) ND sinef367/miR-N367 ++ +++ ++ U3 region No weight loss sinef468 + ND ND U3 region ND sinef580 + ND ND U3 region ND *dsRNA to LTNPs'  nef  has been described (Yamamoto  et al ., 2002). †  -, negative; +, 0 to 50 % inhibition; ++, 50 to 75% inhibition; +++, 75 to 100 % inhibition. ‡  ND, not done. Inhibition of Nef expression by STYLE vector-encoding nef siRNA/miR-N367 To assess the effects of  nef  miR-N367  in vivo , we constructed a prototype foamy virus (PFV)-based live vector. The PFV vector expressed HIV-1 SF2  nef  gene as a reporter and the STYLE vector expressed shRNA as effectors. The full-length  nef  gene was inserted into the  bel-2  portion of a PFV clone (22) in frame to obtain pPFV/nef (Fig.  2A ). The pPFV/nef was transfected into BHK cells and treated with the histone deacetylase inhibitor, trichostatin A (TA) [ 23 ]. The viral supernatant, which contained approximately 5 × 10 6  infectious units (IFU), was collected at 72 h after transfection. For preparation of the STYLE vectors to deliver the shRNAs, the  env  gene portion of pSKY3.0 was replaced with the shRNA expression cassette under the control of the H1 promoter. The pSTYLE was produced (Fig.  2A ), and transfected into the FFV envelope-expressing packaging cells, CRFK sugi clone # 6, in the presence of TA. The transfected CRFKsugi clone #6 had 99% FFV Env positive cells when analyzed by flow cytometry. The viral supernatant with a titer of ca. 1 × 10 5  IFU was collected at 72 h post-transfection. Figure 2 Inhibition of  nef  expression in human T cells by  nef  siRNAs. ( A ) Schematic representation of shRNA-expressing STYLE vector ( E ) and HIV-1 SF2  nef  gene expressing pPFV/nef vector (R). The helper plasmid, pFFenv expresses FFV envelope protein under the control of CMV promoter. ( B ) Detection of expression of  nef  mRNA and integration of vectors. STYLE, SKY3.0, PFV/nef, PFV or mock was used to infect Jurkat T cells and the infected cells were cultured for 2 weeks. After 2 weeks,  gag  and  nef  mRNA expression was measured by RT-PCR. Genomic DNA of LTR,  gag  and  nef  of STYLE or PFV/nef were also detected by PCR. β- actin  was used as a control. ( C ) Inhibition of Nef-EGFP expression by  nef  siRNA-expressing STYLE in Jurkat T cells. The pYM2.2 was transfected into each of the STYLE or mock-infected Jurkat T cells and EGFP-expressing cells were counted by flow cytometry at 48 h after transfection. Data represent the relative activity of EGFP-positive cells, where the percentage of positive cells in the sample transfected with pYM2.2 upon the STYLE-si(-) infected cells was scored as 100%. ( D ) Inhibition of HIV-1 transcription and replication by  nef  STYLE-367. HIV-1 IIIB persistently infected MT-4 T cells were transfected with the pLTR SF2  reporter and β-gal expressing control pCMVβ plasmids at 72 h after infection with STYLE. At 48 h post-transfection, Luc activity was measured and normalized as Luc values (Luc/β-gal). Absolute levels of Luc activity in the samples of pLTR SF2  plus SRYLE-si(-) were 16,311 + 1,253 or 783 + 87 light units for STYLE-367/miR-N367 transfectants. Data represent the relative Luc activities where the percentage of positive cells in the samples infected with the STYLE-si(-) was scored as 100%. After 48 h, p24 antigen was also measured in the cell culture supernatant of STYLE-infected Jurkat T cells. Data are averages of three independent experiments + SD. Bars, SD. ( E ) Inhibition of  nef  expression by  nef  siRNA in Jurkat T cells. Cells were infected with PFV/nef 48 h after infection with the STYLE and then subjected to semi-quantitative RT-PCR analysis. Data represent the relative expression of mRNA, where the percentage of positive cells in the sample of mock-infected cells (E: Mock) relative to the PFV/nef (R: PFV/nef) infected cells was scored as 100%. Data averages were derived from three independent experiments + SD. Bars, SD. The expression of viral mRNAs and integrated DNAs from either the PFV/nef or STYLE vectors was confirmed by infection of Jurkat T cells. The mRNAs and genomic DNA were extracted from the infected cells at 2 weeks post-infection. The PFV/nef-expressed  gag  and  nef  mRNAs and the STYLE-expressed  gag  mRNA were detected after amplification of these regions using reverse transcription (RT)-PCR. The integration of the DNAs into the genome of Jurkat T cells was also confirmed by PCR of the LTR,  gag  and/or  nef  regions (Fig.  2B ). The control SKY3.0 and PFV-infected cells were both negative for  nef  mRNA and integrated DNA (Fig.  2B ). The integrated DNA was also detected by southern blot analysis with genomic DNA of either PFV/nef or STYLE-infected cells (data not shown). Expression of shRNAs (~22-nt) was also confirmed in STYLE-infected cells by northern blot analysis (data not shown). Expression of Nef protein in PFV/nef-infected cells was also detected with specific rabbit anti-Nef serum in immunoblots (data not shown). To evaluate whether the STYLE encoding siRNA could inhibit the expression of the  nef  gene in cultured human T cells, pYM2.2 was transfected into each of the STYLE-infected Jurkat T cells (m.o.i. = ca. 0.1). The most efficient sinef176, 190 and 367/miR-N367 vectors for reduction in  nef  expression (Fig.  1D  and  1E ) were selected for this experiment. The EGFP-positive cells were counted by flow cytometry at 48 h after transfection. Expression of Nef-EGFP fusion protein was reduced drastically following treatment with either the STYLE-176 (74 + 3.2) or 190 (51 + 4.2) and also reduced with 367/miR-N367 (32 + 2.3%). Reduction was insignificant with either the STYLE-si(-) (0 + 0.7) or STYLE-luc (7 + 0.9%) controls (Fig.  2C ). The  in vitro  inhibitory effects of STYLE encoding  nef  siRNA on HIV-1-infected cells were evaluated in Luc assays and using MT-4 T cells persistently infected with HIV-1 IIIB. Cultivation of the STYLE infected cells for 72 h followed by transfection with the pLTR SF2  and culture for another 48 h showed that STYLE-176, 190 and 367/miR-N367 all significantly (p < 0.005) suppressed Luc activity when compared to controls (Fig.  2D ). HIV-1 p24 Gag was also significantly inhibited in the culture supernatant by infection with STYLE-176, 190 and 367/miR-N367 when compared to controls (p < 0.001) (Fig.  2D ). These data suggested that shRNA/miR-N367 could inhibit HIV-1 transcription and replication in intact HIV-1-infected human T cells. Jurkat T cells that had been transduced with  nef  shRNA for 48 h were infected with the PFV/nef. Semi-quantitative RT-PCR analysis revealed that while treatment with STYLE-190 dramatically reduced the expression of both  nef  and  gag  mRNAs of the PFV/nef, the expression of  nef  mRNA was also drastically suppressed by STYLE-176 and 367/miR-N367 (Fig.  2E ). However the STYLE-si(-) and luc controls showed ~10% suppression of  nef  and ~20% suppression of  gag  mRNAs (Fig.  2E ), which was probably a result of interference following super-infection. Nonetheless, both  nef  transcription and PFV/nef replication were substantially inhibited by STYLE-176, 190 and 367/miR-N367. Inhibition of Nef expression by siRNA/miR-N367 in mice Since different host gene products are required for siRNA-mediated RNAi and miRNA-mediated translational repression with  let-7  and  lin-4  in  C. elegans , the two RNAs may not have the same functions  in vivo  [ 24 ]. To test this point, we investigated the efficacy of miR-N367 using STYLE-367 in mammalian tissues. The study mice were group 1 = PFV/nef-infected (n = 6); group 2 = PFV/nef and control STYLE-luc infected (n = 6); group 3 = PFV/nef and STYLE-367-infected (n = 8); and group 4 = STYLE-367-infected (n = 6). Identical study groups were used for both Balb/c and C3H/Hej mouse strains. Nef protein expressing lymphocytes were quantified by histochemical analysis using F3 Nef monoclonal antibody (mAb) or anti-Nef rabbit serum 2 days after PFV/nef infection. Nef protein was detected by immunofluoresence assay in the subcapsular area of the spleens of groups 1 or 2 Balb/c mice, but not groups 3 or 4 (Fig.  3A  and Table  2 ). No positive cell staining was observed using normal rabbit serum as a primary antibody (Fig.  3A ). To test the expression of  nef , nested RT-PCR was also done on day 2 to evaluate the degree of  nef  mRNA expression in the spleen, liver, adipose tissues and hematopoietic cells in groups 1–4. The  nef  mRNA was significantly expressed in liver and hematopoietic cells of Balb/c mice in groups 1 and 2, but not in the group 3 animals that were STYLE-367 infected (Table  2 ). Tissues from group 4 did not show any  nef  bands after RT-PCR (data not shown). Figure 3 In vivo  effects of miR-N367. ( A ) Distribution of Nef positive staining cells in the subcapsular area of groups 2 or 3 mouse spleens at 2 days after infection with PFV/nef. Anti-Nef rabbit serum or normal rabbit serum was used as a primary antibody. ( B ) Immunofluorescence for 305 mAb positive staining cells in the subcapsular area of groups 2 or 3 mouse spleens at 2 days after infection with PFV/nef and immunoperoxidase staining by 305 mAb in cells of interfollicular area of HIV-1 uninfected human spleen and tonsillar follicle. ( C ) Short term body weights of PFV/nef-infected Balb/c mice. The body weights of the PFV/nef-infected mice (group 1, n = 6, solid circle), the PFV/nef-infected followed by the STYLE-luc-infected mice (group 2, n = 6, solid triangle), the PFV/nef-infected followed by the STYLE-367-infected mice (group 3, n = 8, open triangle) and the STYLE-367-infected mice (group 4, n = 6, open circles) were measured from days 0 to 5. ( D ) Long term body weights of PFV/nef-infected C3H/Hej mice. Treatment of each group and numbers of mice were same as ( C ). Bars, SD. *; p < 0.05, **; p < 0.01 (relative to group 3). ( E ) Immunoperoxidase staining by 305 mAb and anti-Nef rabbit serum in cells of mouse or human adipose tissue. Arrows show positively stained areas. Magnification, X 20 (A and B); X 20 and X 200 ( E ). Table 2 Histochemical detection and RT-PCR amplification of Nef from human and mouse tissues Tissues Histochemistry* RT-PCR † F3 Anti-Nef rabbit serum 305 nef gag PPARγ Human (HIV-1 uninfected) Spleen - § - + - - ‡ ND Tonsillar follicle - - + - - ND Liver - - + - - ND Adipose tissue (Salivary gland) - - + - - + Bone marrow - - + - - ND Bronchi - - + - - ND Thyroid gland - - + - - ND Heart muscle - - + - - ND Prostate gland - - + - - ND Testis - - + - - ND Colon mucosa - - + - - ND Lung - - + - - ND Adrenal gland - - + - - ND Brain (Cerebrum cortex) - - + - - ND Mouse Group 1 and 2 Spleen + + + + + ND Liver ND ND + + + ND Hematopoietic cells ND ND + + + ND Adipose tissue (Intestine) + + + - - - Mouse Group 3 Spleen - - + ± ± ND Liver ND ND + - - ND Hematopoietic cells ND ND + - - ND Adipose tissue (Intestine) - - + - - + *Histological analysis was performed with human or each group of mouse tissues by using F3 anti-Nef mAb, anti-Nef rabbit serum or 305 mAb as a primary antibody. For secondary antibody, FITC or peroxidase-conjugated antibody was used. † HIV-1  nef , PFV  gag  ( ‡ HIV-1  gag  for human tissues), and  PPARγ  mRNA expression were detected by RT-PCR with mRNA from human or each group of mouse tissues. § +, positive; -, negative; ND, not done. Because extracellular Nef is internalized into human and mouse lymphocytes and macrophages [ 25 - 27 ], we examined putative Nef receptor molecule (Ner) expression with 305 mAb [ 27 ] in both mouse and human tissues by histochemical analysis. In mice, 305 mAb positive lymphocytes were detected in the subcapsular area of the spleens by immunofluoresence assay (Fig.  3B ) and liver and hematopoietic cells (Table  2 ) in groups 1, 2 and 3, indicating that detection of antigen by 305 mAb was not altered by Nef expression. In HIV-1 uninfected humans, the 305 mAb positive cells were detected by immunoperoxidase staining in spleen (red pulp), tonsillar follicle (germinal center), liver (Kupffer cells), salivary gland (germinal center and adipose cells), bronchi (smooth muscle cells), lung (stroma cells), thyroid gland (colloid), heart muscle (smooth muscle cells), prostate gland (smooth muscle cells), colon mucosa (intestinal absorptive and muscle cells), testis (basement membrane of tubuli seminiferi), adrenal gland (adipose cells), and brain (cerebrum cortex and cortical cells) (Fig.  3B  and Table  2 ). Since Nef suppressed PPARγ expression and reduced fatty acid levels  in vitro  [ 29 - 32 ], we monitored the expression of  PPARγ  mRNA and body weights of mice. Significant PPARγ mRNA expression in intestinal adipose tissue of group 3, but not group 1 and 2, was detected on day 2 (Table  2 ). All Balb/c mice in group 1 showed sedation and a drastic loss of weight from days 1 to 3 (day 1, p = 0.003; day 2, p = 0.021; day 3, p = 0.032 relative to mice in group 3) (Fig.  3C ). Similar results were obtained in group 2 (Fig.  3C ). However, group 3 mice infected with STYLE-367 did not appear to be sedated and had no drastic loss of weight (Fig.  3C ). The group 4 animals, which were not infected with the PFV/nef but treated with STYLE-367, had no changes in either behavior or weight (Fig.  3C ). In longitudinal examinations done during the post-infection period, the animals in groups 1 and 2 had recovered the lost weight (Fig.  3D ). Similar results were obtained in group 2 from day 1 to 5 (day 1, p = 0.037; day 3, p = 0.044; day 5, p = 0.048 relative to mice in group 3) in the C3H/Hej mouse groups (Fig.  3D ). To assess the above  in vivo  results, expression of  nef  mRNA was examined in adipose tissues (Table  2 ). As shown in Table  2 , although mRNAs of  nef  and  gag  were not detected in mouse adipose tissues, 305 mAb and anti-Nef rabbit serum positive staining cells were detected in mouse group 1 and 2 adipose tissues (Fig.  3E  and Table  2 ). Considering that the 305 mAb positive staining adipocytes appeared in mouse as well as human tissues (Fig.  3E  and Table  2 ), these data suggest that the interaction between 305 and soluble Nef detected in adipose tissues may be responsible for the weight loss observed in mice. In this study, whereas siRNA has been reported to inhibit hepatitis B virus replication  in vivo  (33–34), our results show that  nef -derived miRNAs are produced in HIV-1 infected cells, and support the possibility that miRNA and siRNA may be functionally identical, at least in a retrotransposon such as HIV. Recent studies have revealed that miRNAs and siRNAs could block mRNA expression by similar mechanisms [ 9 ] and that siRNAs could function as miRNAs [ 35 ] and EBV-encoded miRNAs were found [ 36 ]. Our results reported here are consistent with these previous observations and are suggestive of the fact that  nef  miR-N367 could regulate  nef  expression even  in vivo . In our unpublished data, HIV-1 LTR promoter activity was inhibited by miR-N367 (nt number 379 to 449 of SF2  nef , 71-nt) expression, of which activity was dependent on negative responsive element (NRE) of U3 region (our unpublished data). Although no mismatch shRNA against region #367 was active, the miR-N367 from HIV-1 genome may have some mismatches and effectively inhibit HIV-1 transcription. Further the effects of siRNAs of Tc 1 , in particular those to the terminal inverted repeats derived from read-through transcription of entire transposable elements, were presented for silencing transposase gene expression by RNAi machinery in germ lines of  C. elegans , [ 37 ]. Taken together, it could be implied from these and our other results that miRNAs produced in HIV-1-infected cells can efficiently block not only Nef function but also HIV-1 replication through RNAi, which renders persistently low pathogenic infection latent as observed in an LTNP of 15-2-2 (see Table  1 ). It is equally important that although the weight loss reported here occurred only temporarily  in vivo , however the inserted  nef  gene in the foamy retrotransposon may represent miRNAs which could inhibit  nef  mRNA expression by presumably an identical mechanism to that observed of siRNAs. Thus, RNAi might serve as a new sequence-specific therapeutic arsenal in AIDS prevention and possibly treatment. Overall, our results indicate that nef shRNA transduced into T cell line inhibited HIV-1 transcription. Further, nef miRNAs could be produced from infected T cells and can block the trans-activity of Nef as well as HIV-1 replication on its own via the cis-action of nef. These functions of nef via RNAi pathways may allow persistently low pathogenic or latent infection as observed in HIV-infected non-progressors. Cumulatively, these data suggest that Nef may be involved in both viral replication and the disease progression, the findings, which may facilitate new strategies for HIV control  in vivo .
Results and Discussion
Identification of a candidate of miRNAs in HIV-1-infected cells Very recently, the Epstein-Barr virus (EBV)-encoded miRNAs were identified. Thus, during the preliminary stages of this study, our curiosity was fixed on the need to find out if indeed there was any relationship between  nef  miRNAs and HIV-1-infected cells. To achieve this purpose, we extracted total RNA from HIV-1 IIIB strain persistently infected MT-4 T cells and northern blot analysis was performed using eight probes against the  nef  coding region, as shown in Figure  1A . Analyses using several anti-sense probes, small RNA molecules approximately ~25-nt in size were detected as well as HIV-1 major transcripts, 9.1, 4.3 and 1.8 kb bands (Fig.  1B ). Similar results were obtained with total RNA from HIV-1 SF2 strain infected MT-4 T cells (data not shown). RNA samples treated with a mixture of the single stranded specific RNases A and T1 also generated ~25-nt RNAs that hybridized in northern blots with the sense probes against the same  nef  region. However HIV-1 major transcripts were not detected (data not shown), indicating that the structure of the small RNA molecules could be double-stranded RNAs (miRNAs). Some variability was observed when the quantity of the miRNAs was compared with the total of the major transcripts. A maximum of 3.2% of miRNAs was detected by using #367 probe when compared with total HIV-1 transcripts, and the minimum of 0.3% was detected by using probe# 90 (Fig.  1B ). Figure 1 Detection of HIV-1  nef  miRNAs and inhibition of Nef expression by  nef  RNAs. ( A ) Schematic representation of HIV-1  nef  in its genome and synthetic DNA probes (#) used in this study. ( B ) Total RNAs were extracted from MT-4 T cells persistently infected with HIV-1 IIIB, separated on a 15% polyacrylamide-7 M urea gel, and subjected to northern blot analysis. The approximate sizes of the three classes of HIV-1 transcripts and small RNAs are indicated on the right. The loading control was rRNA stained with ethidium bromide. Relative expression (%) of  nef  small RNAs to the three classes of HIV-1 transcripts is at the bottom of figure. ( C ) Schematic representation of effector plasmids ( E ) H1 promoter-driven shRNA expression plasmids. Reporter (R) Nef-EGFP expression plasmid (pYM2.2) is also shown.  (D)  Inhibition by sinefs in pH1 plamids of Nef-EGFP expression. Either sinef, siluc or siegfp in each plasmid was transfected into Jurkat T cells in the presence of either pYM2.2 or control pEGFP-N1. At 36 h after transfection EGFP-positive cells were counted by flow cytometry. Data represent the relative activity of EGFP-positive cells where the percentage of positive cells in the sample transfected with pYM2.2 or pEGFP-N1 plus pcDNA3.1 or si(-) in pH1 plasmid was scored as 100%. Data are averages of three independent experiments + SD. Bars, SD. ( E ) Immunoblot analysis showing inhibition of YM2.2 expression by different  nef  shRNAs. Jurakat T cells were transfected with pYM2.2 and pH1/sinefs or siluc plasmid, cellular lysates were prepared 48 h after transfection, and immunoblotted with rabbit serum against Nef (upper panel) and anti-β-actin antibody ( lower panel ). The β-actin expression shows equal loading of all samples. To randomly clone the  nef  miRNA, ~25-nt RNAs were gel purified, cloned and sequenced. The sequences from the  nef  miRNA clones were 5'-acugaccuuuggauggugcuucaa-3' or similar ones, corresponded to the nucleotides approximately 420 to 443 conserved region of  nef  (miR-N367). The most notable feature of this analysis is that it has proven beyond reasonable doubts that  nef -derived miRNAs are produced in HIV-1 infected cells.
Identification of a candidate of miRNAs in HIV-1-infected cells
Very recently, the Epstein-Barr virus (EBV)-encoded miRNAs were identified. Thus, during the preliminary stages of this study, our curiosity was fixed on the need to find out if indeed there was any relationship between  nef  miRNAs and HIV-1-infected cells. To achieve this purpose, we extracted total RNA from HIV-1 IIIB strain persistently infected MT-4 T cells and northern blot analysis was performed using eight probes against the  nef  coding region, as shown in Figure  1A . Analyses using several anti-sense probes, small RNA molecules approximately ~25-nt in size were detected as well as HIV-1 major transcripts, 9.1, 4.3 and 1.8 kb bands (Fig.  1B ). Similar results were obtained with total RNA from HIV-1 SF2 strain infected MT-4 T cells (data not shown). RNA samples treated with a mixture of the single stranded specific RNases A and T1 also generated ~25-nt RNAs that hybridized in northern blots with the sense probes against the same  nef  region. However HIV-1 major transcripts were not detected (data not shown), indicating that the structure of the small RNA molecules could be double-stranded RNAs (miRNAs). Some variability was observed when the quantity of the miRNAs was compared with the total of the major transcripts. A maximum of 3.2% of miRNAs was detected by using #367 probe when compared with total HIV-1 transcripts, and the minimum of 0.3% was detected by using probe# 90 (Fig.  1B ).nefnefnefTo randomly clone the  nef  miRNA, ~25-nt RNAs were gel purified, cloned and sequenced. The sequences from the  nef  miRNA clones were 5'-acugaccuuuggauggugcuucaa-3' or similar ones, corresponded to the nucleotides approximately 420 to 443 conserved region of  nef  (miR-N367). The most notable feature of this analysis is that it has proven beyond reasonable doubts that  nef -derived miRNAs are produced in HIV-1 infected cells.nefnefnefnefInhibition of Nef by plasmids-encoding siRNA/miRNA To examine inhibition of  nef  expression by the  nef  miRNA, we constructed eight shRNAs homologous to the native miRNA or probes used in Figure  1A [ 21 ]. Although it has been reported that three to four mutations in the sense strand derived from miRNA could have the potential to control unmutated 21-nt stem loop [ 22 ], we investigated whether the native shRNA-expressing plasmid can effectively reduce  nef  gene expression or not (Fig.  1C  and  1D ). We used  egfp  or  luc  gene (pH1/siegfp or luc) as a positive or negative control. All the shRNA-expressing plasmids including the controls were co-transfected into Jurkat T cells with pYM2.2 and cell fluorescence resulting from the expression of EGFP reporter gene was quantified by flow cytometry. The sinef176, 190, 367/miR-N367 and control siegfp all showed efficient reduction, but the sinef 007, 084, 299, 468 and 580 constructs gave only modest reductions, and no suppression was observed with si(-) and luc (Fig.  1D  and Table  1 ). Immunoblot analysis using anti-Nef rabbit serum also confirmed the inhibition of Nef-EGFP expression by sinef 176, 190 and 367/miR-N367 (Fig.  1E ). Table 1 Relation between AIDS clinical courses and  nef  dsRNA or siRNA/miRNA in suppression of  nef  gene expression Nef inhibitor Nef expressed by: Target region Clinical courses Nef-EGFP HIV-1 IIIB PFV/nef dsRNA * Human SF2 ++  † ++ ND ‡ Full-length (including miR-N367) ND 1-3-3 ++ - ND U3 deleted Rapid progressor, Died within 3 years 4-2-1 ND + ND U3 region (including miR-N367) Rapid progressor, Died within 3 years 15-2-2 ++ +++ ND U3 region (including miR-N367) Non progressor with low plasma viremia 16-1-1 ND + ND U3 deleted Non progressor with low plasma viremia jw95-1 ND + ND U3 region (including miR-N367) Non progressor with undetectable viremia siRNA 15-2-2 model mouse dsnef + + + Full-length ND sinef007 + ND ND Upstream of U3 region ND sinef084 + ND ND Upstream of U3 region ND sinef176 +++ +++ ++ Upstream of U3 region § ND sinef190 +++ +++ +++ Upstream of U3 region ND sinef299 + ND ND U3 region (aa 83–135) ND sinef367/miR-N367 ++ +++ ++ U3 region No weight loss sinef468 + ND ND U3 region ND sinef580 + ND ND U3 region ND *dsRNA to LTNPs'  nef  has been described (Yamamoto  et al ., 2002). †  -, negative; +, 0 to 50 % inhibition; ++, 50 to 75% inhibition; +++, 75 to 100 % inhibition. ‡  ND, not done.
Inhibition of Nef by plasmids-encoding siRNA/miRNA
To examine inhibition of  nef  expression by the  nef  miRNA, we constructed eight shRNAs homologous to the native miRNA or probes used in Figure  1A [ 21 ]. Although it has been reported that three to four mutations in the sense strand derived from miRNA could have the potential to control unmutated 21-nt stem loop [ 22 ], we investigated whether the native shRNA-expressing plasmid can effectively reduce  nef  gene expression or not (Fig.  1C  and  1D ). We used  egfp  or  luc  gene (pH1/siegfp or luc) as a positive or negative control. All the shRNA-expressing plasmids including the controls were co-transfected into Jurkat T cells with pYM2.2 and cell fluorescence resulting from the expression of EGFP reporter gene was quantified by flow cytometry. The sinef176, 190, 367/miR-N367 and control siegfp all showed efficient reduction, but the sinef 007, 084, 299, 468 and 580 constructs gave only modest reductions, and no suppression was observed with si(-) and luc (Fig.  1D  and Table  1 ). Immunoblot analysis using anti-Nef rabbit serum also confirmed the inhibition of Nef-EGFP expression by sinef 176, 190 and 367/miR-N367 (Fig.  1E ).nefnefnefegfplucRelation between AIDS clinical courses and  nef  dsRNA or siRNA/miRNA in suppression of  nef  gene expressionnefnefdsRNA *dsRNAHumanHumansiRNAsiRNA15-2-2 model mouse15-2-2 model mouse*dsRNA to LTNPs'  nef  has been described (Yamamoto  et al ., 2002).nefet al†  -, negative; +, 0 to 50 % inhibition; ++, 50 to 75% inhibition; +++, 75 to 100 % inhibition.‡  ND, not done.Inhibition of Nef expression by STYLE vector-encoding nef siRNA/miR-N367 To assess the effects of  nef  miR-N367  in vivo , we constructed a prototype foamy virus (PFV)-based live vector. The PFV vector expressed HIV-1 SF2  nef  gene as a reporter and the STYLE vector expressed shRNA as effectors. The full-length  nef  gene was inserted into the  bel-2  portion of a PFV clone (22) in frame to obtain pPFV/nef (Fig.  2A ). The pPFV/nef was transfected into BHK cells and treated with the histone deacetylase inhibitor, trichostatin A (TA) [ 23 ]. The viral supernatant, which contained approximately 5 × 10 6  infectious units (IFU), was collected at 72 h after transfection. For preparation of the STYLE vectors to deliver the shRNAs, the  env  gene portion of pSKY3.0 was replaced with the shRNA expression cassette under the control of the H1 promoter. The pSTYLE was produced (Fig.  2A ), and transfected into the FFV envelope-expressing packaging cells, CRFK sugi clone # 6, in the presence of TA. The transfected CRFKsugi clone #6 had 99% FFV Env positive cells when analyzed by flow cytometry. The viral supernatant with a titer of ca. 1 × 10 5  IFU was collected at 72 h post-transfection. Figure 2 Inhibition of  nef  expression in human T cells by  nef  siRNAs. ( A ) Schematic representation of shRNA-expressing STYLE vector ( E ) and HIV-1 SF2  nef  gene expressing pPFV/nef vector (R). The helper plasmid, pFFenv expresses FFV envelope protein under the control of CMV promoter. ( B ) Detection of expression of  nef  mRNA and integration of vectors. STYLE, SKY3.0, PFV/nef, PFV or mock was used to infect Jurkat T cells and the infected cells were cultured for 2 weeks. After 2 weeks,  gag  and  nef  mRNA expression was measured by RT-PCR. Genomic DNA of LTR,  gag  and  nef  of STYLE or PFV/nef were also detected by PCR. β- actin  was used as a control. ( C ) Inhibition of Nef-EGFP expression by  nef  siRNA-expressing STYLE in Jurkat T cells. The pYM2.2 was transfected into each of the STYLE or mock-infected Jurkat T cells and EGFP-expressing cells were counted by flow cytometry at 48 h after transfection. Data represent the relative activity of EGFP-positive cells, where the percentage of positive cells in the sample transfected with pYM2.2 upon the STYLE-si(-) infected cells was scored as 100%. ( D ) Inhibition of HIV-1 transcription and replication by  nef  STYLE-367. HIV-1 IIIB persistently infected MT-4 T cells were transfected with the pLTR SF2  reporter and β-gal expressing control pCMVβ plasmids at 72 h after infection with STYLE. At 48 h post-transfection, Luc activity was measured and normalized as Luc values (Luc/β-gal). Absolute levels of Luc activity in the samples of pLTR SF2  plus SRYLE-si(-) were 16,311 + 1,253 or 783 + 87 light units for STYLE-367/miR-N367 transfectants. Data represent the relative Luc activities where the percentage of positive cells in the samples infected with the STYLE-si(-) was scored as 100%. After 48 h, p24 antigen was also measured in the cell culture supernatant of STYLE-infected Jurkat T cells. Data are averages of three independent experiments + SD. Bars, SD. ( E ) Inhibition of  nef  expression by  nef  siRNA in Jurkat T cells. Cells were infected with PFV/nef 48 h after infection with the STYLE and then subjected to semi-quantitative RT-PCR analysis. Data represent the relative expression of mRNA, where the percentage of positive cells in the sample of mock-infected cells (E: Mock) relative to the PFV/nef (R: PFV/nef) infected cells was scored as 100%. Data averages were derived from three independent experiments + SD. Bars, SD. The expression of viral mRNAs and integrated DNAs from either the PFV/nef or STYLE vectors was confirmed by infection of Jurkat T cells. The mRNAs and genomic DNA were extracted from the infected cells at 2 weeks post-infection. The PFV/nef-expressed  gag  and  nef  mRNAs and the STYLE-expressed  gag  mRNA were detected after amplification of these regions using reverse transcription (RT)-PCR. The integration of the DNAs into the genome of Jurkat T cells was also confirmed by PCR of the LTR,  gag  and/or  nef  regions (Fig.  2B ). The control SKY3.0 and PFV-infected cells were both negative for  nef  mRNA and integrated DNA (Fig.  2B ). The integrated DNA was also detected by southern blot analysis with genomic DNA of either PFV/nef or STYLE-infected cells (data not shown). Expression of shRNAs (~22-nt) was also confirmed in STYLE-infected cells by northern blot analysis (data not shown). Expression of Nef protein in PFV/nef-infected cells was also detected with specific rabbit anti-Nef serum in immunoblots (data not shown). To evaluate whether the STYLE encoding siRNA could inhibit the expression of the  nef  gene in cultured human T cells, pYM2.2 was transfected into each of the STYLE-infected Jurkat T cells (m.o.i. = ca. 0.1). The most efficient sinef176, 190 and 367/miR-N367 vectors for reduction in  nef  expression (Fig.  1D  and  1E ) were selected for this experiment. The EGFP-positive cells were counted by flow cytometry at 48 h after transfection. Expression of Nef-EGFP fusion protein was reduced drastically following treatment with either the STYLE-176 (74 + 3.2) or 190 (51 + 4.2) and also reduced with 367/miR-N367 (32 + 2.3%). Reduction was insignificant with either the STYLE-si(-) (0 + 0.7) or STYLE-luc (7 + 0.9%) controls (Fig.  2C ). The  in vitro  inhibitory effects of STYLE encoding  nef  siRNA on HIV-1-infected cells were evaluated in Luc assays and using MT-4 T cells persistently infected with HIV-1 IIIB. Cultivation of the STYLE infected cells for 72 h followed by transfection with the pLTR SF2  and culture for another 48 h showed that STYLE-176, 190 and 367/miR-N367 all significantly (p < 0.005) suppressed Luc activity when compared to controls (Fig.  2D ). HIV-1 p24 Gag was also significantly inhibited in the culture supernatant by infection with STYLE-176, 190 and 367/miR-N367 when compared to controls (p < 0.001) (Fig.  2D ). These data suggested that shRNA/miR-N367 could inhibit HIV-1 transcription and replication in intact HIV-1-infected human T cells. Jurkat T cells that had been transduced with  nef  shRNA for 48 h were infected with the PFV/nef. Semi-quantitative RT-PCR analysis revealed that while treatment with STYLE-190 dramatically reduced the expression of both  nef  and  gag  mRNAs of the PFV/nef, the expression of  nef  mRNA was also drastically suppressed by STYLE-176 and 367/miR-N367 (Fig.  2E ). However the STYLE-si(-) and luc controls showed ~10% suppression of  nef  and ~20% suppression of  gag  mRNAs (Fig.  2E ), which was probably a result of interference following super-infection. Nonetheless, both  nef  transcription and PFV/nef replication were substantially inhibited by STYLE-176, 190 and 367/miR-N367.
Inhibition of Nef expression by STYLE vector-encoding nef siRNA/miR-N367
To assess the effects of  nef  miR-N367  in vivo , we constructed a prototype foamy virus (PFV)-based live vector. The PFV vector expressed HIV-1 SF2  nef  gene as a reporter and the STYLE vector expressed shRNA as effectors. The full-length  nef  gene was inserted into the  bel-2  portion of a PFV clone (22) in frame to obtain pPFV/nef (Fig.  2A ). The pPFV/nef was transfected into BHK cells and treated with the histone deacetylase inhibitor, trichostatin A (TA) [ 23 ]. The viral supernatant, which contained approximately 5 × 10 6  infectious units (IFU), was collected at 72 h after transfection. For preparation of the STYLE vectors to deliver the shRNAs, the  env  gene portion of pSKY3.0 was replaced with the shRNA expression cassette under the control of the H1 promoter. The pSTYLE was produced (Fig.  2A ), and transfected into the FFV envelope-expressing packaging cells, CRFK sugi clone # 6, in the presence of TA. The transfected CRFKsugi clone #6 had 99% FFV Env positive cells when analyzed by flow cytometry. The viral supernatant with a titer of ca. 1 × 10 5  IFU was collected at 72 h post-transfection.nefin vivonefnefbel-2envThe expression of viral mRNAs and integrated DNAs from either the PFV/nef or STYLE vectors was confirmed by infection of Jurkat T cells. The mRNAs and genomic DNA were extracted from the infected cells at 2 weeks post-infection. The PFV/nef-expressed  gag  and  nef  mRNAs and the STYLE-expressed  gag  mRNA were detected after amplification of these regions using reverse transcription (RT)-PCR. The integration of the DNAs into the genome of Jurkat T cells was also confirmed by PCR of the LTR,  gag  and/or  nef  regions (Fig.  2B ). The control SKY3.0 and PFV-infected cells were both negative for  nef  mRNA and integrated DNA (Fig.  2B ). The integrated DNA was also detected by southern blot analysis with genomic DNA of either PFV/nef or STYLE-infected cells (data not shown). Expression of shRNAs (~22-nt) was also confirmed in STYLE-infected cells by northern blot analysis (data not shown). Expression of Nef protein in PFV/nef-infected cells was also detected with specific rabbit anti-Nef serum in immunoblots (data not shown).gagnefgaggagnefnefTo evaluate whether the STYLE encoding siRNA could inhibit the expression of the  nef  gene in cultured human T cells, pYM2.2 was transfected into each of the STYLE-infected Jurkat T cells (m.o.i. = ca. 0.1). The most efficient sinef176, 190 and 367/miR-N367 vectors for reduction in  nef  expression (Fig.  1D  and  1E ) were selected for this experiment. The EGFP-positive cells were counted by flow cytometry at 48 h after transfection. Expression of Nef-EGFP fusion protein was reduced drastically following treatment with either the STYLE-176 (74 + 3.2) or 190 (51 + 4.2) and also reduced with 367/miR-N367 (32 + 2.3%). Reduction was insignificant with either the STYLE-si(-) (0 + 0.7) or STYLE-luc (7 + 0.9%) controls (Fig.  2C ).nefnefThe  in vitro  inhibitory effects of STYLE encoding  nef  siRNA on HIV-1-infected cells were evaluated in Luc assays and using MT-4 T cells persistently infected with HIV-1 IIIB. Cultivation of the STYLE infected cells for 72 h followed by transfection with the pLTR SF2  and culture for another 48 h showed that STYLE-176, 190 and 367/miR-N367 all significantly (p < 0.005) suppressed Luc activity when compared to controls (Fig.  2D ). HIV-1 p24 Gag was also significantly inhibited in the culture supernatant by infection with STYLE-176, 190 and 367/miR-N367 when compared to controls (p < 0.001) (Fig.  2D ). These data suggested that shRNA/miR-N367 could inhibit HIV-1 transcription and replication in intact HIV-1-infected human T cells.in vitronefJurkat T cells that had been transduced with  nef  shRNA for 48 h were infected with the PFV/nef. Semi-quantitative RT-PCR analysis revealed that while treatment with STYLE-190 dramatically reduced the expression of both  nef  and  gag  mRNAs of the PFV/nef, the expression of  nef  mRNA was also drastically suppressed by STYLE-176 and 367/miR-N367 (Fig.  2E ). However the STYLE-si(-) and luc controls showed ~10% suppression of  nef  and ~20% suppression of  gag  mRNAs (Fig.  2E ), which was probably a result of interference following super-infection. Nonetheless, both  nef  transcription and PFV/nef replication were substantially inhibited by STYLE-176, 190 and 367/miR-N367.nefnefgagnefnefgagnefInhibition of Nef expression by siRNA/miR-N367 in mice Since different host gene products are required for siRNA-mediated RNAi and miRNA-mediated translational repression with  let-7  and  lin-4  in  C. elegans , the two RNAs may not have the same functions  in vivo  [ 24 ]. To test this point, we investigated the efficacy of miR-N367 using STYLE-367 in mammalian tissues. The study mice were group 1 = PFV/nef-infected (n = 6); group 2 = PFV/nef and control STYLE-luc infected (n = 6); group 3 = PFV/nef and STYLE-367-infected (n = 8); and group 4 = STYLE-367-infected (n = 6). Identical study groups were used for both Balb/c and C3H/Hej mouse strains. Nef protein expressing lymphocytes were quantified by histochemical analysis using F3 Nef monoclonal antibody (mAb) or anti-Nef rabbit serum 2 days after PFV/nef infection. Nef protein was detected by immunofluoresence assay in the subcapsular area of the spleens of groups 1 or 2 Balb/c mice, but not groups 3 or 4 (Fig.  3A  and Table  2 ). No positive cell staining was observed using normal rabbit serum as a primary antibody (Fig.  3A ). To test the expression of  nef , nested RT-PCR was also done on day 2 to evaluate the degree of  nef  mRNA expression in the spleen, liver, adipose tissues and hematopoietic cells in groups 1–4. The  nef  mRNA was significantly expressed in liver and hematopoietic cells of Balb/c mice in groups 1 and 2, but not in the group 3 animals that were STYLE-367 infected (Table  2 ). Tissues from group 4 did not show any  nef  bands after RT-PCR (data not shown). Figure 3 In vivo  effects of miR-N367. ( A ) Distribution of Nef positive staining cells in the subcapsular area of groups 2 or 3 mouse spleens at 2 days after infection with PFV/nef. Anti-Nef rabbit serum or normal rabbit serum was used as a primary antibody. ( B ) Immunofluorescence for 305 mAb positive staining cells in the subcapsular area of groups 2 or 3 mouse spleens at 2 days after infection with PFV/nef and immunoperoxidase staining by 305 mAb in cells of interfollicular area of HIV-1 uninfected human spleen and tonsillar follicle. ( C ) Short term body weights of PFV/nef-infected Balb/c mice. The body weights of the PFV/nef-infected mice (group 1, n = 6, solid circle), the PFV/nef-infected followed by the STYLE-luc-infected mice (group 2, n = 6, solid triangle), the PFV/nef-infected followed by the STYLE-367-infected mice (group 3, n = 8, open triangle) and the STYLE-367-infected mice (group 4, n = 6, open circles) were measured from days 0 to 5. ( D ) Long term body weights of PFV/nef-infected C3H/Hej mice. Treatment of each group and numbers of mice were same as ( C ). Bars, SD. *; p < 0.05, **; p < 0.01 (relative to group 3). ( E ) Immunoperoxidase staining by 305 mAb and anti-Nef rabbit serum in cells of mouse or human adipose tissue. Arrows show positively stained areas. Magnification, X 20 (A and B); X 20 and X 200 ( E ). Table 2 Histochemical detection and RT-PCR amplification of Nef from human and mouse tissues Tissues Histochemistry* RT-PCR † F3 Anti-Nef rabbit serum 305 nef gag PPARγ Human (HIV-1 uninfected) Spleen - § - + - - ‡ ND Tonsillar follicle - - + - - ND Liver - - + - - ND Adipose tissue (Salivary gland) - - + - - + Bone marrow - - + - - ND Bronchi - - + - - ND Thyroid gland - - + - - ND Heart muscle - - + - - ND Prostate gland - - + - - ND Testis - - + - - ND Colon mucosa - - + - - ND Lung - - + - - ND Adrenal gland - - + - - ND Brain (Cerebrum cortex) - - + - - ND Mouse Group 1 and 2 Spleen + + + + + ND Liver ND ND + + + ND Hematopoietic cells ND ND + + + ND Adipose tissue (Intestine) + + + - - - Mouse Group 3 Spleen - - + ± ± ND Liver ND ND + - - ND Hematopoietic cells ND ND + - - ND Adipose tissue (Intestine) - - + - - + *Histological analysis was performed with human or each group of mouse tissues by using F3 anti-Nef mAb, anti-Nef rabbit serum or 305 mAb as a primary antibody. For secondary antibody, FITC or peroxidase-conjugated antibody was used. † HIV-1  nef , PFV  gag  ( ‡ HIV-1  gag  for human tissues), and  PPARγ  mRNA expression were detected by RT-PCR with mRNA from human or each group of mouse tissues. § +, positive; -, negative; ND, not done. Because extracellular Nef is internalized into human and mouse lymphocytes and macrophages [ 25 - 27 ], we examined putative Nef receptor molecule (Ner) expression with 305 mAb [ 27 ] in both mouse and human tissues by histochemical analysis. In mice, 305 mAb positive lymphocytes were detected in the subcapsular area of the spleens by immunofluoresence assay (Fig.  3B ) and liver and hematopoietic cells (Table  2 ) in groups 1, 2 and 3, indicating that detection of antigen by 305 mAb was not altered by Nef expression. In HIV-1 uninfected humans, the 305 mAb positive cells were detected by immunoperoxidase staining in spleen (red pulp), tonsillar follicle (germinal center), liver (Kupffer cells), salivary gland (germinal center and adipose cells), bronchi (smooth muscle cells), lung (stroma cells), thyroid gland (colloid), heart muscle (smooth muscle cells), prostate gland (smooth muscle cells), colon mucosa (intestinal absorptive and muscle cells), testis (basement membrane of tubuli seminiferi), adrenal gland (adipose cells), and brain (cerebrum cortex and cortical cells) (Fig.  3B  and Table  2 ). Since Nef suppressed PPARγ expression and reduced fatty acid levels  in vitro  [ 29 - 32 ], we monitored the expression of  PPARγ  mRNA and body weights of mice. Significant PPARγ mRNA expression in intestinal adipose tissue of group 3, but not group 1 and 2, was detected on day 2 (Table  2 ). All Balb/c mice in group 1 showed sedation and a drastic loss of weight from days 1 to 3 (day 1, p = 0.003; day 2, p = 0.021; day 3, p = 0.032 relative to mice in group 3) (Fig.  3C ). Similar results were obtained in group 2 (Fig.  3C ). However, group 3 mice infected with STYLE-367 did not appear to be sedated and had no drastic loss of weight (Fig.  3C ). The group 4 animals, which were not infected with the PFV/nef but treated with STYLE-367, had no changes in either behavior or weight (Fig.  3C ). In longitudinal examinations done during the post-infection period, the animals in groups 1 and 2 had recovered the lost weight (Fig.  3D ). Similar results were obtained in group 2 from day 1 to 5 (day 1, p = 0.037; day 3, p = 0.044; day 5, p = 0.048 relative to mice in group 3) in the C3H/Hej mouse groups (Fig.  3D ). To assess the above  in vivo  results, expression of  nef  mRNA was examined in adipose tissues (Table  2 ). As shown in Table  2 , although mRNAs of  nef  and  gag  were not detected in mouse adipose tissues, 305 mAb and anti-Nef rabbit serum positive staining cells were detected in mouse group 1 and 2 adipose tissues (Fig.  3E  and Table  2 ). Considering that the 305 mAb positive staining adipocytes appeared in mouse as well as human tissues (Fig.  3E  and Table  2 ), these data suggest that the interaction between 305 and soluble Nef detected in adipose tissues may be responsible for the weight loss observed in mice. In this study, whereas siRNA has been reported to inhibit hepatitis B virus replication  in vivo  (33–34), our results show that  nef -derived miRNAs are produced in HIV-1 infected cells, and support the possibility that miRNA and siRNA may be functionally identical, at least in a retrotransposon such as HIV. Recent studies have revealed that miRNAs and siRNAs could block mRNA expression by similar mechanisms [ 9 ] and that siRNAs could function as miRNAs [ 35 ] and EBV-encoded miRNAs were found [ 36 ]. Our results reported here are consistent with these previous observations and are suggestive of the fact that  nef  miR-N367 could regulate  nef  expression even  in vivo . In our unpublished data, HIV-1 LTR promoter activity was inhibited by miR-N367 (nt number 379 to 449 of SF2  nef , 71-nt) expression, of which activity was dependent on negative responsive element (NRE) of U3 region (our unpublished data). Although no mismatch shRNA against region #367 was active, the miR-N367 from HIV-1 genome may have some mismatches and effectively inhibit HIV-1 transcription. Further the effects of siRNAs of Tc 1 , in particular those to the terminal inverted repeats derived from read-through transcription of entire transposable elements, were presented for silencing transposase gene expression by RNAi machinery in germ lines of  C. elegans , [ 37 ]. Taken together, it could be implied from these and our other results that miRNAs produced in HIV-1-infected cells can efficiently block not only Nef function but also HIV-1 replication through RNAi, which renders persistently low pathogenic infection latent as observed in an LTNP of 15-2-2 (see Table  1 ). It is equally important that although the weight loss reported here occurred only temporarily  in vivo , however the inserted  nef  gene in the foamy retrotransposon may represent miRNAs which could inhibit  nef  mRNA expression by presumably an identical mechanism to that observed of siRNAs. Thus, RNAi might serve as a new sequence-specific therapeutic arsenal in AIDS prevention and possibly treatment. Overall, our results indicate that nef shRNA transduced into T cell line inhibited HIV-1 transcription. Further, nef miRNAs could be produced from infected T cells and can block the trans-activity of Nef as well as HIV-1 replication on its own via the cis-action of nef. These functions of nef via RNAi pathways may allow persistently low pathogenic or latent infection as observed in HIV-infected non-progressors. Cumulatively, these data suggest that Nef may be involved in both viral replication and the disease progression, the findings, which may facilitate new strategies for HIV control  in vivo .
Inhibition of Nef expression by siRNA/miR-N367 in mice
Since different host gene products are required for siRNA-mediated RNAi and miRNA-mediated translational repression with  let-7  and  lin-4  in  C. elegans , the two RNAs may not have the same functions  in vivo  [ 24 ]. To test this point, we investigated the efficacy of miR-N367 using STYLE-367 in mammalian tissues. The study mice were group 1 = PFV/nef-infected (n = 6); group 2 = PFV/nef and control STYLE-luc infected (n = 6); group 3 = PFV/nef and STYLE-367-infected (n = 8); and group 4 = STYLE-367-infected (n = 6). Identical study groups were used for both Balb/c and C3H/Hej mouse strains. Nef protein expressing lymphocytes were quantified by histochemical analysis using F3 Nef monoclonal antibody (mAb) or anti-Nef rabbit serum 2 days after PFV/nef infection. Nef protein was detected by immunofluoresence assay in the subcapsular area of the spleens of groups 1 or 2 Balb/c mice, but not groups 3 or 4 (Fig.  3A  and Table  2 ). No positive cell staining was observed using normal rabbit serum as a primary antibody (Fig.  3A ). To test the expression of  nef , nested RT-PCR was also done on day 2 to evaluate the degree of  nef  mRNA expression in the spleen, liver, adipose tissues and hematopoietic cells in groups 1–4. The  nef  mRNA was significantly expressed in liver and hematopoietic cells of Balb/c mice in groups 1 and 2, but not in the group 3 animals that were STYLE-367 infected (Table  2 ). Tissues from group 4 did not show any  nef  bands after RT-PCR (data not shown).let-7lin-4C. elegansin vivonefnefnefnefHistochemical detection and RT-PCR amplification of Nef from human and mouse tissuesnefgagPPARγHuman (HIV-1 uninfected)Mouse Group 1 and 2Mouse Group 3*Histological analysis was performed with human or each group of mouse tissues by using F3 anti-Nef mAb, anti-Nef rabbit serum or 305 mAb as a primary antibody. For secondary antibody, FITC or peroxidase-conjugated antibody was used.† HIV-1  nef , PFV  gag  ( ‡ HIV-1  gag  for human tissues), and  PPARγ  mRNA expression were detected by RT-PCR with mRNA from human or each group of mouse tissues.nefgaggagPPARγ§ +, positive; -, negative; ND, not done.Because extracellular Nef is internalized into human and mouse lymphocytes and macrophages [ 25 - 27 ], we examined putative Nef receptor molecule (Ner) expression with 305 mAb [ 27 ] in both mouse and human tissues by histochemical analysis. In mice, 305 mAb positive lymphocytes were detected in the subcapsular area of the spleens by immunofluoresence assay (Fig.  3B ) and liver and hematopoietic cells (Table  2 ) in groups 1, 2 and 3, indicating that detection of antigen by 305 mAb was not altered by Nef expression. In HIV-1 uninfected humans, the 305 mAb positive cells were detected by immunoperoxidase staining in spleen (red pulp), tonsillar follicle (germinal center), liver (Kupffer cells), salivary gland (germinal center and adipose cells), bronchi (smooth muscle cells), lung (stroma cells), thyroid gland (colloid), heart muscle (smooth muscle cells), prostate gland (smooth muscle cells), colon mucosa (intestinal absorptive and muscle cells), testis (basement membrane of tubuli seminiferi), adrenal gland (adipose cells), and brain (cerebrum cortex and cortical cells) (Fig.  3B  and Table  2 ).Since Nef suppressed PPARγ expression and reduced fatty acid levels  in vitro  [ 29 - 32 ], we monitored the expression of  PPARγ  mRNA and body weights of mice. Significant PPARγ mRNA expression in intestinal adipose tissue of group 3, but not group 1 and 2, was detected on day 2 (Table  2 ). All Balb/c mice in group 1 showed sedation and a drastic loss of weight from days 1 to 3 (day 1, p = 0.003; day 2, p = 0.021; day 3, p = 0.032 relative to mice in group 3) (Fig.  3C ). Similar results were obtained in group 2 (Fig.  3C ). However, group 3 mice infected with STYLE-367 did not appear to be sedated and had no drastic loss of weight (Fig.  3C ). The group 4 animals, which were not infected with the PFV/nef but treated with STYLE-367, had no changes in either behavior or weight (Fig.  3C ). In longitudinal examinations done during the post-infection period, the animals in groups 1 and 2 had recovered the lost weight (Fig.  3D ). Similar results were obtained in group 2 from day 1 to 5 (day 1, p = 0.037; day 3, p = 0.044; day 5, p = 0.048 relative to mice in group 3) in the C3H/Hej mouse groups (Fig.  3D ). To assess the above  in vivo  results, expression of  nef  mRNA was examined in adipose tissues (Table  2 ). As shown in Table  2 , although mRNAs of  nef  and  gag  were not detected in mouse adipose tissues, 305 mAb and anti-Nef rabbit serum positive staining cells were detected in mouse group 1 and 2 adipose tissues (Fig.  3E  and Table  2 ). Considering that the 305 mAb positive staining adipocytes appeared in mouse as well as human tissues (Fig.  3E  and Table  2 ), these data suggest that the interaction between 305 and soluble Nef detected in adipose tissues may be responsible for the weight loss observed in mice.in vitroPPARγin vivonefnefgagIn this study, whereas siRNA has been reported to inhibit hepatitis B virus replication  in vivo  (33–34), our results show that  nef -derived miRNAs are produced in HIV-1 infected cells, and support the possibility that miRNA and siRNA may be functionally identical, at least in a retrotransposon such as HIV. Recent studies have revealed that miRNAs and siRNAs could block mRNA expression by similar mechanisms [ 9 ] and that siRNAs could function as miRNAs [ 35 ] and EBV-encoded miRNAs were found [ 36 ]. Our results reported here are consistent with these previous observations and are suggestive of the fact that  nef  miR-N367 could regulate  nef  expression even  in vivo . In our unpublished data, HIV-1 LTR promoter activity was inhibited by miR-N367 (nt number 379 to 449 of SF2  nef , 71-nt) expression, of which activity was dependent on negative responsive element (NRE) of U3 region (our unpublished data). Although no mismatch shRNA against region #367 was active, the miR-N367 from HIV-1 genome may have some mismatches and effectively inhibit HIV-1 transcription. Further the effects of siRNAs of Tc 1 , in particular those to the terminal inverted repeats derived from read-through transcription of entire transposable elements, were presented for silencing transposase gene expression by RNAi machinery in germ lines of  C. elegans , [ 37 ]. Taken together, it could be implied from these and our other results that miRNAs produced in HIV-1-infected cells can efficiently block not only Nef function but also HIV-1 replication through RNAi, which renders persistently low pathogenic infection latent as observed in an LTNP of 15-2-2 (see Table  1 ). It is equally important that although the weight loss reported here occurred only temporarily  in vivo , however the inserted  nef  gene in the foamy retrotransposon may represent miRNAs which could inhibit  nef  mRNA expression by presumably an identical mechanism to that observed of siRNAs. Thus, RNAi might serve as a new sequence-specific therapeutic arsenal in AIDS prevention and possibly treatment.in vivonefnefnefin vivonef1C. elegansin vivonefnefOverall, our results indicate that nef shRNA transduced into T cell line inhibited HIV-1 transcription. Further, nef miRNAs could be produced from infected T cells and can block the trans-activity of Nef as well as HIV-1 replication on its own via the cis-action of nef. These functions of nef via RNAi pathways may allow persistently low pathogenic or latent infection as observed in HIV-infected non-progressors. Cumulatively, these data suggest that Nef may be involved in both viral replication and the disease progression, the findings, which may facilitate new strategies for HIV control  in vivo .in vivoMaterials and Methods Patient details Patient selection is showed in Table  1 . These SF2 (HIV-1 subtype B prototype) was included as a control  nef  sequence, because of the inclusion of viruses, which were also subtype B. The SF2 contained full-length  nef  reading frame as indicated in Table  1 . Patients 1-3-3, 4-2-1 (Table  1 ) are rapid progressors infected with HIV-1. These patients were infected in 1984–1985 and died within 3 years of primary infection with >1 × 106 viral copies and CD4+ T cell count of 75 and 110/ml blood. Patients 15-2-2 and 16-1-1 (Table  1 ) are slow progressors, who were infected in 1984 and have survived HIV-1 infection with high and stable CD4+ T cell counts (690 and 760/ml blood) with low (<5000 copies) plasma viremia. All these patients acquired virus through homosexual sex. JW95-1 (Table  1 ) is a boy who was infected from his mother via breast feeding. The child was infected in 1983 and has survived disease free with high CD4+ T cell count (890/ml blood) with undetectable viremia. Human samples were obtained from a donor after informed consent. Cells and viruses HeLa and BHK cells were grown in Dulbecco's modified Eagle Medium (DMEM) (GIBCO, Grand Island, NY) supplemented with 10% heat-inactivated fetal bovine serum (FBS) and antibiotics. CRFK cells were grown in Iscove's Modified Dulbecco's Medium (IMDM) (GIBCO) with 10% FBS and antibiotics. Jurkat T cells and MT-4 T cells persistently infected with HIV-1 IIIB strain were cultured in RPMI-1640 medium (GIBCO) supplemented with 10% FBS and antibiotics. The packaging cells (CRFKsugi) were made by transfecting CRFK cells with 10 μg of the pFFenv with Lipofectin Reagent (Invitrogen) and selecting transformants after culture for 14 days with 25 μg/ml of hygromycine B (Invitrogen). After 14 days, FFV Env protein expression was measured by flow cytometry and immunoblot analyses with FFV-infected cat B serum [ 34 ]. The pPFV/nef (10 μg) was transfected into BHK cells and pSTYLE/si (10 μg) was transfected into CRFKsugi cells with Lipofectin Reagent. The transfected cells were cultured for 72 hr, and the viral supernatant was collected and filtered through a 0.45 μm pore size Millex-GP filter (Millipore, Bedford, MA). Vector stocks were stored at -70°C prior to use. Viral titers were determined as described previously [ 21 ]. Cells were infected with PFV/nef and/or SKY/si at an m.o.i. of ca. 0.1 in the presence of 4 μg/ml of polybrene and infected cells were cultured at a density of 1 × 10 6  cells per ml for 3 days. The details of plasmid constructs and the primer sequences used in cloning strategies are shown in supplementary file (see  Additional file: 1 ). Flow cytometric analysis Flow cytometry was performed with a FACS Calibur (Becton Dickson, San Jose, CA) as described previously [ 17 ]. Luc assay and Immunoblotting Firefly Luc assay was performed using the Luciferase Assay System (Promega) as described previously [ 17 ]. Immunoblotting was performed essentially as described previously by Otake et al. [ 28 ]. P24 ELISA The concentration of p24 supernatant was determined by an antigen capture assay (Beckman Coulter, Fullerton, CA) according to the manufacturer's instructions. Confocal laser microscopy analysis Confocal laser microscopy analysis was performed as described previously [ 28 ]. Northern blot analysis Total RNAs were extracted from HIV-1 IIIB or SF2 persistently infected or uninfected MT-4 T cells using TRIzol reagent (Invitrogen). Approximately 40 μg of total RNA was treated with RNase A and T1 (Sigma, St. Louis, MO) as described previously [ 17 ], subjected to electrophoresis on a 15% polyacrylamide-7 M urea gel and electroblotted to HybondN+ (Pharmacia, Uppsala, Sweden) for 4 hr at 400 mA. RNAs were immobilized by UV crosslinking and baking for 1 hr at 80°C. Hybridization was done with an ECL direct Kit (Pharmacia). Synthetic DNA probes were labeled with horseradish peroxidase. The sequence for synthetic sense DNA probes for northern blot analysis are as follows: #007 (5'-gcgtcgacggcaagtggtcaaaacgta-3'); #084 (5'-gcgtcgacgccagcagcagatggggtg-3'); #176 (5'-gcgtcgacgtgcctggctagaagcaca-3'); #190 (5'-gcgtcgacgcacaagaggaggaga-3'); #299 (5'-gcgtcgacgactggaagggctaatttg-3'); #367 (5'-gctcgacggctacttccctgattggc-3'); #468 (5'-gcgtcgacggtagaagaggccaatgaa-3'); #580 (5'-gcgtcgacgcatttcatcacatggccc-3'). RNAs were cloned by 5'RACE System (Invitrogen, CA., USA) with a slight modification in that primers were used that were the same as the synthetic DNA probes as described above that are abbreviated as #primer. In brief, gel purified small RNAs were annealed with #primer and first strand cDNA was synthesized with SuperScript II RT (Invitrogen, CA., USA). Afrer RNase H and T1 treatment, a homopolymeric tail was added to the 3'-end of the cDNA using terminal deoxynucleotidyl transferase and dCTP. After ethanol precipitation, PCR amplification was done with abridged anchor primer and #primer. Then the PCR products were obtained using abridged universal amplification primer and #primer. The PCR fragments were digested with  Sal I and cloned into  Sal I site of pBluescript SK(-), followed by sequence analysis. The secondary structures of RNAs were predicted by GENETYX-MAC program (Software Development Co. Ltd, Tokyo, Japan). Semi-quantitative RT-PCR analysis Semi-quantitative RT-PCR analysis was performed using the ThermoScript RT-PCR System (Invitrogen, CA., USA) according to the manufacturer's protocol with the following primers: III (5'-atcatgggccaaagagaattc-3') and IV (5'-aaatttcactcaatcgagcc-3') for FFV LTR, VI (5'-aggacctgaaaggcatg-3') and VII (5'-ttgttgagatcgtcccg-3') for FFV  gag , VIII (5'-tgtggtggaatgccactag-3') and IX (5'-attgtcatggaattttgta-3') for PFV LTR, XI (5'-tcttacagaccagtaacaa-3') and XII (5'-gtcaatcattacatctgca-3') for PFV  gag , XIII (5'-aactactagtacccttcagg-3') and XIV (5'-aaaactcttgctttatggcc-3') for HIV-1  gag , XV (5'-atgggtggcaagtcaaaacg-3') and XVI (5'-tcagcagtctttgtagtactccg-3') for HIV-1  nef , XVII (5'-gttatgggtgaaactctgggagat-3') and XVIII (5'-atgttcctgaacataatcgtc-3') for  PPARγ , XIX (5'-gacaacggctccggcatgtgcaaag-3') and XX (5'-ttcacggttggccttagggttcag-3') for β- actin , respectively. The nested PCR followed RT reaction was performed as described previously [ 34 ]. PCR products were quantified with the NIH image program. Relative mRNA expression was calculated as percentage expression using the following formula: integrating number of  nef  or  gag  bands/integrating number of β- actin  X 100. In vivo studies and tissue analyses Balb/c and C3H/Hej mice were raised under specific pathogen-free (SPF) conditions. Mice were infected with 1 ml of 10 5  IFU of PFV/nef and STYLE-367 by intravenous (i.v.) injection. RT-PCR analyses were performed 2 days after infection. For histological analysis, cryostat sections were prepared from both human and mouse tissues. The fixed sections were rinsed with PBS and incubated with 5% BSA for at least 1 hr to inhibit nonspecific binding of antibodies. Sections were incubated overnight at 4°C with anti-Nef rabbit serum, F3 or 305 mAb, and incubated with peroxidase or FITC conjugated secondary antibodies. The washed sections were incubated in 0.03% 3,3'diaminobenzidine (Sigma) solution in 0.05 M Tris buffer with 0.01% H 2 O 2  for development of peroxidase activity. After counterstaining with hematoxylin or methylgreen, the sections were dehydrated and mounted. Statistical methods Data were analysed using a one-way ANOVA analysis with a post-hoc Fisher's test. P values of 0.05 or more were determined for that of cut off.
Materials and Methods
Patient details Patient selection is showed in Table  1 . These SF2 (HIV-1 subtype B prototype) was included as a control  nef  sequence, because of the inclusion of viruses, which were also subtype B. The SF2 contained full-length  nef  reading frame as indicated in Table  1 . Patients 1-3-3, 4-2-1 (Table  1 ) are rapid progressors infected with HIV-1. These patients were infected in 1984–1985 and died within 3 years of primary infection with >1 × 106 viral copies and CD4+ T cell count of 75 and 110/ml blood. Patients 15-2-2 and 16-1-1 (Table  1 ) are slow progressors, who were infected in 1984 and have survived HIV-1 infection with high and stable CD4+ T cell counts (690 and 760/ml blood) with low (<5000 copies) plasma viremia. All these patients acquired virus through homosexual sex. JW95-1 (Table  1 ) is a boy who was infected from his mother via breast feeding. The child was infected in 1983 and has survived disease free with high CD4+ T cell count (890/ml blood) with undetectable viremia. Human samples were obtained from a donor after informed consent.
Patient details
Patient selection is showed in Table  1 . These SF2 (HIV-1 subtype B prototype) was included as a control  nef  sequence, because of the inclusion of viruses, which were also subtype B. The SF2 contained full-length  nef  reading frame as indicated in Table  1 . Patients 1-3-3, 4-2-1 (Table  1 ) are rapid progressors infected with HIV-1. These patients were infected in 1984–1985 and died within 3 years of primary infection with >1 × 106 viral copies and CD4+ T cell count of 75 and 110/ml blood. Patients 15-2-2 and 16-1-1 (Table  1 ) are slow progressors, who were infected in 1984 and have survived HIV-1 infection with high and stable CD4+ T cell counts (690 and 760/ml blood) with low (<5000 copies) plasma viremia. All these patients acquired virus through homosexual sex. JW95-1 (Table  1 ) is a boy who was infected from his mother via breast feeding. The child was infected in 1983 and has survived disease free with high CD4+ T cell count (890/ml blood) with undetectable viremia. Human samples were obtained from a donor after informed consent.nefnefCells and viruses HeLa and BHK cells were grown in Dulbecco's modified Eagle Medium (DMEM) (GIBCO, Grand Island, NY) supplemented with 10% heat-inactivated fetal bovine serum (FBS) and antibiotics. CRFK cells were grown in Iscove's Modified Dulbecco's Medium (IMDM) (GIBCO) with 10% FBS and antibiotics. Jurkat T cells and MT-4 T cells persistently infected with HIV-1 IIIB strain were cultured in RPMI-1640 medium (GIBCO) supplemented with 10% FBS and antibiotics. The packaging cells (CRFKsugi) were made by transfecting CRFK cells with 10 μg of the pFFenv with Lipofectin Reagent (Invitrogen) and selecting transformants after culture for 14 days with 25 μg/ml of hygromycine B (Invitrogen). After 14 days, FFV Env protein expression was measured by flow cytometry and immunoblot analyses with FFV-infected cat B serum [ 34 ]. The pPFV/nef (10 μg) was transfected into BHK cells and pSTYLE/si (10 μg) was transfected into CRFKsugi cells with Lipofectin Reagent. The transfected cells were cultured for 72 hr, and the viral supernatant was collected and filtered through a 0.45 μm pore size Millex-GP filter (Millipore, Bedford, MA). Vector stocks were stored at -70°C prior to use. Viral titers were determined as described previously [ 21 ]. Cells were infected with PFV/nef and/or SKY/si at an m.o.i. of ca. 0.1 in the presence of 4 μg/ml of polybrene and infected cells were cultured at a density of 1 × 10 6  cells per ml for 3 days. The details of plasmid constructs and the primer sequences used in cloning strategies are shown in supplementary file (see  Additional file: 1 ).
Cells and viruses
HeLa and BHK cells were grown in Dulbecco's modified Eagle Medium (DMEM) (GIBCO, Grand Island, NY) supplemented with 10% heat-inactivated fetal bovine serum (FBS) and antibiotics. CRFK cells were grown in Iscove's Modified Dulbecco's Medium (IMDM) (GIBCO) with 10% FBS and antibiotics. Jurkat T cells and MT-4 T cells persistently infected with HIV-1 IIIB strain were cultured in RPMI-1640 medium (GIBCO) supplemented with 10% FBS and antibiotics. The packaging cells (CRFKsugi) were made by transfecting CRFK cells with 10 μg of the pFFenv with Lipofectin Reagent (Invitrogen) and selecting transformants after culture for 14 days with 25 μg/ml of hygromycine B (Invitrogen). After 14 days, FFV Env protein expression was measured by flow cytometry and immunoblot analyses with FFV-infected cat B serum [ 34 ]. The pPFV/nef (10 μg) was transfected into BHK cells and pSTYLE/si (10 μg) was transfected into CRFKsugi cells with Lipofectin Reagent. The transfected cells were cultured for 72 hr, and the viral supernatant was collected and filtered through a 0.45 μm pore size Millex-GP filter (Millipore, Bedford, MA). Vector stocks were stored at -70°C prior to use. Viral titers were determined as described previously [ 21 ]. Cells were infected with PFV/nef and/or SKY/si at an m.o.i. of ca. 0.1 in the presence of 4 μg/ml of polybrene and infected cells were cultured at a density of 1 × 10 6  cells per ml for 3 days.The details of plasmid constructs and the primer sequences used in cloning strategies are shown in supplementary file (see  Additional file: 1 ).Flow cytometric analysis Flow cytometry was performed with a FACS Calibur (Becton Dickson, San Jose, CA) as described previously [ 17 ].
Flow cytometric analysis
Flow cytometry was performed with a FACS Calibur (Becton Dickson, San Jose, CA) as described previously [ 17 ].Luc assay and Immunoblotting Firefly Luc assay was performed using the Luciferase Assay System (Promega) as described previously [ 17 ]. Immunoblotting was performed essentially as described previously by Otake et al. [ 28 ].
Luc assay and Immunoblotting
Firefly Luc assay was performed using the Luciferase Assay System (Promega) as described previously [ 17 ]. Immunoblotting was performed essentially as described previously by Otake et al. [ 28 ].P24 ELISA The concentration of p24 supernatant was determined by an antigen capture assay (Beckman Coulter, Fullerton, CA) according to the manufacturer's instructions.
P24 ELISA
The concentration of p24 supernatant was determined by an antigen capture assay (Beckman Coulter, Fullerton, CA) according to the manufacturer's instructions.Confocal laser microscopy analysis Confocal laser microscopy analysis was performed as described previously [ 28 ].
Confocal laser microscopy analysis
Confocal laser microscopy analysis was performed as described previously [ 28 ].Northern blot analysis Total RNAs were extracted from HIV-1 IIIB or SF2 persistently infected or uninfected MT-4 T cells using TRIzol reagent (Invitrogen). Approximately 40 μg of total RNA was treated with RNase A and T1 (Sigma, St. Louis, MO) as described previously [ 17 ], subjected to electrophoresis on a 15% polyacrylamide-7 M urea gel and electroblotted to HybondN+ (Pharmacia, Uppsala, Sweden) for 4 hr at 400 mA. RNAs were immobilized by UV crosslinking and baking for 1 hr at 80°C. Hybridization was done with an ECL direct Kit (Pharmacia). Synthetic DNA probes were labeled with horseradish peroxidase. The sequence for synthetic sense DNA probes for northern blot analysis are as follows: #007 (5'-gcgtcgacggcaagtggtcaaaacgta-3'); #084 (5'-gcgtcgacgccagcagcagatggggtg-3'); #176 (5'-gcgtcgacgtgcctggctagaagcaca-3'); #190 (5'-gcgtcgacgcacaagaggaggaga-3'); #299 (5'-gcgtcgacgactggaagggctaatttg-3'); #367 (5'-gctcgacggctacttccctgattggc-3'); #468 (5'-gcgtcgacggtagaagaggccaatgaa-3'); #580 (5'-gcgtcgacgcatttcatcacatggccc-3'). RNAs were cloned by 5'RACE System (Invitrogen, CA., USA) with a slight modification in that primers were used that were the same as the synthetic DNA probes as described above that are abbreviated as #primer. In brief, gel purified small RNAs were annealed with #primer and first strand cDNA was synthesized with SuperScript II RT (Invitrogen, CA., USA). Afrer RNase H and T1 treatment, a homopolymeric tail was added to the 3'-end of the cDNA using terminal deoxynucleotidyl transferase and dCTP. After ethanol precipitation, PCR amplification was done with abridged anchor primer and #primer. Then the PCR products were obtained using abridged universal amplification primer and #primer. The PCR fragments were digested with  Sal I and cloned into  Sal I site of pBluescript SK(-), followed by sequence analysis. The secondary structures of RNAs were predicted by GENETYX-MAC program (Software Development Co. Ltd, Tokyo, Japan).
Northern blot analysis
Total RNAs were extracted from HIV-1 IIIB or SF2 persistently infected or uninfected MT-4 T cells using TRIzol reagent (Invitrogen). Approximately 40 μg of total RNA was treated with RNase A and T1 (Sigma, St. Louis, MO) as described previously [ 17 ], subjected to electrophoresis on a 15% polyacrylamide-7 M urea gel and electroblotted to HybondN+ (Pharmacia, Uppsala, Sweden) for 4 hr at 400 mA. RNAs were immobilized by UV crosslinking and baking for 1 hr at 80°C. Hybridization was done with an ECL direct Kit (Pharmacia). Synthetic DNA probes were labeled with horseradish peroxidase. The sequence for synthetic sense DNA probes for northern blot analysis are as follows: #007 (5'-gcgtcgacggcaagtggtcaaaacgta-3'); #084 (5'-gcgtcgacgccagcagcagatggggtg-3'); #176 (5'-gcgtcgacgtgcctggctagaagcaca-3'); #190 (5'-gcgtcgacgcacaagaggaggaga-3'); #299 (5'-gcgtcgacgactggaagggctaatttg-3'); #367 (5'-gctcgacggctacttccctgattggc-3'); #468 (5'-gcgtcgacggtagaagaggccaatgaa-3'); #580 (5'-gcgtcgacgcatttcatcacatggccc-3'). RNAs were cloned by 5'RACE System (Invitrogen, CA., USA) with a slight modification in that primers were used that were the same as the synthetic DNA probes as described above that are abbreviated as #primer. In brief, gel purified small RNAs were annealed with #primer and first strand cDNA was synthesized with SuperScript II RT (Invitrogen, CA., USA). Afrer RNase H and T1 treatment, a homopolymeric tail was added to the 3'-end of the cDNA using terminal deoxynucleotidyl transferase and dCTP. After ethanol precipitation, PCR amplification was done with abridged anchor primer and #primer. Then the PCR products were obtained using abridged universal amplification primer and #primer. The PCR fragments were digested with  Sal I and cloned into  Sal I site of pBluescript SK(-), followed by sequence analysis. The secondary structures of RNAs were predicted by GENETYX-MAC program (Software Development Co. Ltd, Tokyo, Japan).SalSalSemi-quantitative RT-PCR analysis Semi-quantitative RT-PCR analysis was performed using the ThermoScript RT-PCR System (Invitrogen, CA., USA) according to the manufacturer's protocol with the following primers: III (5'-atcatgggccaaagagaattc-3') and IV (5'-aaatttcactcaatcgagcc-3') for FFV LTR, VI (5'-aggacctgaaaggcatg-3') and VII (5'-ttgttgagatcgtcccg-3') for FFV  gag , VIII (5'-tgtggtggaatgccactag-3') and IX (5'-attgtcatggaattttgta-3') for PFV LTR, XI (5'-tcttacagaccagtaacaa-3') and XII (5'-gtcaatcattacatctgca-3') for PFV  gag , XIII (5'-aactactagtacccttcagg-3') and XIV (5'-aaaactcttgctttatggcc-3') for HIV-1  gag , XV (5'-atgggtggcaagtcaaaacg-3') and XVI (5'-tcagcagtctttgtagtactccg-3') for HIV-1  nef , XVII (5'-gttatgggtgaaactctgggagat-3') and XVIII (5'-atgttcctgaacataatcgtc-3') for  PPARγ , XIX (5'-gacaacggctccggcatgtgcaaag-3') and XX (5'-ttcacggttggccttagggttcag-3') for β- actin , respectively. The nested PCR followed RT reaction was performed as described previously [ 34 ]. PCR products were quantified with the NIH image program. Relative mRNA expression was calculated as percentage expression using the following formula: integrating number of  nef  or  gag  bands/integrating number of β- actin  X 100.
Semi-quantitative RT-PCR analysis
Semi-quantitative RT-PCR analysis was performed using the ThermoScript RT-PCR System (Invitrogen, CA., USA) according to the manufacturer's protocol with the following primers: III (5'-atcatgggccaaagagaattc-3') and IV (5'-aaatttcactcaatcgagcc-3') for FFV LTR, VI (5'-aggacctgaaaggcatg-3') and VII (5'-ttgttgagatcgtcccg-3') for FFV  gag , VIII (5'-tgtggtggaatgccactag-3') and IX (5'-attgtcatggaattttgta-3') for PFV LTR, XI (5'-tcttacagaccagtaacaa-3') and XII (5'-gtcaatcattacatctgca-3') for PFV  gag , XIII (5'-aactactagtacccttcagg-3') and XIV (5'-aaaactcttgctttatggcc-3') for HIV-1  gag , XV (5'-atgggtggcaagtcaaaacg-3') and XVI (5'-tcagcagtctttgtagtactccg-3') for HIV-1  nef , XVII (5'-gttatgggtgaaactctgggagat-3') and XVIII (5'-atgttcctgaacataatcgtc-3') for  PPARγ , XIX (5'-gacaacggctccggcatgtgcaaag-3') and XX (5'-ttcacggttggccttagggttcag-3') for β- actin , respectively. The nested PCR followed RT reaction was performed as described previously [ 34 ]. PCR products were quantified with the NIH image program. Relative mRNA expression was calculated as percentage expression using the following formula: integrating number of  nef  or  gag  bands/integrating number of β- actin  X 100.gaggaggagnefPPARγactinnefgagactinIn vivo studies and tissue analyses Balb/c and C3H/Hej mice were raised under specific pathogen-free (SPF) conditions. Mice were infected with 1 ml of 10 5  IFU of PFV/nef and STYLE-367 by intravenous (i.v.) injection. RT-PCR analyses were performed 2 days after infection. For histological analysis, cryostat sections were prepared from both human and mouse tissues. The fixed sections were rinsed with PBS and incubated with 5% BSA for at least 1 hr to inhibit nonspecific binding of antibodies. Sections were incubated overnight at 4°C with anti-Nef rabbit serum, F3 or 305 mAb, and incubated with peroxidase or FITC conjugated secondary antibodies. The washed sections were incubated in 0.03% 3,3'diaminobenzidine (Sigma) solution in 0.05 M Tris buffer with 0.01% H 2 O 2  for development of peroxidase activity. After counterstaining with hematoxylin or methylgreen, the sections were dehydrated and mounted.
In vivo studies and tissue analyses
Balb/c and C3H/Hej mice were raised under specific pathogen-free (SPF) conditions. Mice were infected with 1 ml of 10 5  IFU of PFV/nef and STYLE-367 by intravenous (i.v.) injection. RT-PCR analyses were performed 2 days after infection. For histological analysis, cryostat sections were prepared from both human and mouse tissues. The fixed sections were rinsed with PBS and incubated with 5% BSA for at least 1 hr to inhibit nonspecific binding of antibodies. Sections were incubated overnight at 4°C with anti-Nef rabbit serum, F3 or 305 mAb, and incubated with peroxidase or FITC conjugated secondary antibodies. The washed sections were incubated in 0.03% 3,3'diaminobenzidine (Sigma) solution in 0.05 M Tris buffer with 0.01% H 2 O 2  for development of peroxidase activity. After counterstaining with hematoxylin or methylgreen, the sections were dehydrated and mounted.Statistical methods Data were analysed using a one-way ANOVA analysis with a post-hoc Fisher's test. P values of 0.05 or more were determined for that of cut off.
Statistical methods
Data were analysed using a one-way ANOVA analysis with a post-hoc Fisher's test. P values of 0.05 or more were determined for that of cut off.List of abbreviations used HIV-1 human immunodeficiency virus type 1 miRNA microRNA nt, nucleotides LTNP long-term non-progressors shRNA short hairpin RNA AIDS acquired immunodeficiency syndrome RNAi RNA interference siRNA small interfering RNA LTR long terminal repeat SIV simian immunodeficiency virus PBMCs peripheral blood mononuclear cells; EBV Epstein-Barr virus; EGFP enhanced green fluorescence protein PFV prototype foamy virus TA trichostatin A IFU infectious units FFV feline foamy virus RT reverse transcription m.o.i., multiplicity of infectionm Ab monoclonal antibody; Ner Nef receptor molecule; NRE negative responsive element.
List of abbreviations used
HIV-1 human immunodeficiency virus type 1miRNA microRNAnt, nucleotidesLTNP long-term non-progressorsshRNA short hairpin RNAAIDS acquired immunodeficiency syndromeRNAi RNA interferencesiRNA small interfering RNALTR long terminal repeatSIV simian immunodeficiency virusPBMCs peripheral blood mononuclear cells;EBV Epstein-Barr virus;EGFP enhanced green fluorescence proteinPFV prototype foamy virusTA trichostatinA IFU infectious unitsFFV feline foamy virusRT reverse transcriptionm.o.i., multiplicity of infectionmAb monoclonal antibody;Ner Nef receptor molecule;NRE negative responsive element.Competing interests The authors declare that they have no competing interests.
Competing interests
The authors declare that they have no competing interests.Authors' contributions S.O. carried out northern analyses, immunoblot analyses, RNAi assays and was involved in the construction of plasmids. M.I. and Y.T. participated in  in vivo  studies and tissue analyses. Y.I. and H.O. participated in data validation and overall experimental design. E.A.B. and N.K.S. carried out the clinical, sequencing, and virological studies and the writing of the manuscript. Y.R.F. participated in the design of the study and coordinated it. All authors read and approved the final manuscript.
Authors' contributions
S.O. carried out northern analyses, immunoblot analyses, RNAi assays and was involved in the construction of plasmids. M.I. and Y.T. participated in  in vivo  studies and tissue analyses. Y.I. and H.O. participated in data validation and overall experimental design. E.A.B. and N.K.S. carried out the clinical, sequencing, and virological studies and the writing of the manuscript. Y.R.F. participated in the design of the study and coordinated it. All authors read and approved the final manuscript.in vivoSupplementary Material Additional file 1 Click here for file
Supplementary Material
Additional file 1
Click here for file