Modulating HIV-1 replication by RNA interference directed against human transcription elongation factor SPT5
© Ping et al; licensee BioMed Central Ltd. 2004
Received: 17 December 2004
Accepted: 27 December 2004
Published: 27 December 2004
Several cellular positive and negative elongation factors are involved in regulating RNA polymerase II processivity during transcription elongation in human cells. In recruiting several of these regulatory factors to the 5' long terminal repeat (LTR) promoter during transcription elongation, HIV-1 modulates replication of its genome in a process mediated by the virus-encoded transactivator Tat. One particular cellular regulatory factor, DSIF subunit human SPT5 (hSpt5), has been implicated in both positively and negatively regulating transcriptional elongation but its role in Tat transactivation in vivo and in HIV-1 replication has not been completely elucidated.
To understand the in vivo function of hSpt5 and define its role in Tat transactivation and HIV-1 replication, we used RNA interference (RNAi) to specifically knockdown hSpt5 expression by degrading hSpt5 mRNA. Short-interfering RNA (siRNA) designed to target hSpt5 for RNAi successfully resulted in knockdown of both hSpt5 mRNA and protein levels, and did not significantly affect cell viability. In contrast to hSpt5 knockdown, siRNA-mediated silencing of human mRNA capping enzyme, a functionally important hSpt5-interacting cellular protein, was lethal and showed a significant increase in cell death over the course of the knockdown experiment. In addition, hSpt5 knockdown led to significant decreases in Tat transactivation and inhibited HIV-1 replication, indicating that hSpt5 was required for mediating Tat transactivation and HIV-1 replication.
The findings presented here showed that hSpt5 is a bona fide positive regulator of Tat transactivation and HIV-1 replication in vivo. These results also suggest that hSpt5 function in transcription regulation and mRNA capping is essential for a subset of cellular and viral genes and may not be required for global gene expression.
The elongation phase of transcription is often a critical juncture for regulating gene expression [1, 2] and a number of genes including c-myc, c-fms, hsp70, and those encoded by HIV-1 are regulated at this stage of transcription [3–6]. During transcription elongation, shortly after successful initiation of RNA synthesis, RNA polymerase II (RNA pol II) can pause, arrest, pass through terminator sequences, or terminate transcription. The varying processivity of RNA pol II prior to entering productive elongation is controlled by the action of both negative and positive transcription elongation factors (N-TEFs and P-TEFs, respectively). The function of P-TEFs is to reduce the barrier of N-TEFs and promote the release of RNA pol II from the transition state that can cause termination of transcription . Three elongation regulatory factors, P-TEFb (p ositive t ranscription e longation f actor b), DSIF (D RB (5,6-d ichloro-1-β-D-r ibofuranosylb enzimidazole) s ensitivity-i nducing f actor) and NELF (n egative e longation f actor), have been identified using DRB as a transcription inhibitor [8–10] and function together to regulate transcription elongation.
Modulation of HIV-1 gene expression provides one fundamental example of how transcription elongation can be controlled by such regulatory factors [11–14]. Tat, an HIV-1 regulatory protein, is required for synthesis of viral mRNA and increases the efficiency of transcription elongation from the HIV-1 promoter. In the presence of Tat, the processivity of RNA Pol II complexes that initiate transcription in the HIV-1 5' long terminal repeat (5' LTR) region becomes greatly enhanced. For this increased processivity to occur, Tat binds with a nascent leader RNA element, trans-activation responsive (TAR) RNA, located at the 5' end of all HIV-1 transcripts . Cellular factors in association with Tat and TAR are then recruited to the 5' LTR, stimulating RNA pol II processivity during elongation. More specifically, the C-terminal domain (CTD) of RNA pol II is proposed to be hyperphosphorylated by P-TEFb during Tat transactivation to promote elongation [12–14]. Composed of cyclin-dependent kinase CDK9 and Cyclin T1, P-TEFb has been shown to bind the activation domain of Tat and TAR RNA loop sequence and phosphorylate the CTD of RNA pol II [16–18]. Tat transactivation is postulated to involve Tat-TAR interactions that then give rise to the recruitment of P-TEFb to RNA pol II complexes at the 5' LTR. This recruitment is necessary to enhance the processivity of RNA Pol II from the HIV-1 5' LTR promoter [7, 14, 17, 19]. Thus, TAR RNA provides a scaffold for Tat and P-TEFb to bind and assemble a regulatory switch during HIV replication .
Human DSIF consists of subunits hSpt5 and hSPT4 and was originally discovered as a negative elongation factor that binds to RNA pol II . In conjunction with NELF, DSIF represses transcriptional elongation at positions proximal to promoters [9, 10]. Escape from transcriptional repression imposed by DSIF and NELF requires P-TEFb, which has been shown in vitro to phosphorylate both hSpt5 and CTD [7, 10, 21–29]. Interestingly, hSpt5 is conserved among eukaryotes and is a dual transcriptional regulator that can function as both a negative and positive elongation factor [30–32]. Currently, it is postulated that phosphorylation of hSpt5 and RNA pol II by P-TEFb is the key event during which hSpt5 functionally switches from a negative barrier to a positive elongation factor during transcription in human cells. Methylation of SPT5 also has been shown to regulate its interaction with RNA pol II and this posttranslational modification of SPT5 may alter transcriptional elongation functions in response to viral and cellular factors .
Although hSpt5's role in transcription regulation in association with P-TEFb has been established, its involvement in Tat transactivation and HIV-1 replication continues to be elucidated. Several in vitro studies have shown that hSpt5 is required for Tat transactivation and that both hSpt5 and RNA pol II phosphorylation is stimulated after recruitment of P-TEFb by Tat [25, 29, 34]. hSpt5 may also play a positive role in Tat transactivation through its association with human mRNA capping enzyme (HCE), which is a bifunctional triphosphatase-guanylyltransferase required for capping mRNA (reviewed in [1, 35]), since SPT5, Tat, and CTD associate with the capping apparatus to stimulate capping [36–43]. However, studies in a recent report have suggested that only P-TEFb hyperphosphorylation of the RNA pol II CTD is directly required for Tat transactivation, precluding a direct role for hSpt5 in RNA pol II processivity during HIV-1 replication . Therefore, hSpt5 role in Tat transactivation and HIV-1 replication in vivo remains unclear.
Here, we used RNA interference (RNAi) to address whether hSpt5 is required for Tat transactivation and thus HIV-1 replication in vivo and further defined hSpt5 cellular functions. RNAi is a remarkably efficient process whereby double-stranded RNA (dsRNA) induces sequence-specific degradation of homologous mRNA in animal and plant cells (reviewed in ref. ). In mammalian cells, RNAi can be triggered by 21-nucleotide (nt) small interfering RNA (siRNA) duplexes and a few dsRNA molecules are sufficient to inactivate a continuously transcribed target mRNA for an observable period of time [45, 46]. RNAi has recently been used to successfully knockdown the expression of a number of HIV genes, including p24, reverse transcriptase, vif, nef, tat, and rev, and has led to pre- and post-integrative HIV-1 RNA degradation and reduced HIV infectivity [47–52]. These results suggested that targeting viral factors required for the HIV life cycle with siRNAs including those required for HIV replication is a viable method for treating HIV infections. Other groups have targeted cellular factors implicated in supporting the HIV life cycle, including T-cell co-receptors CD4, CXCR4, CCR5, and CD8 [50, 52–54] and transcription factor NF-κB , which has a role in HIV transcription initiation. Knockdown of the co-receptors reduced HIV infectivity, effectively blocking HIV entry into cells . RNAi has become one of the leading methodologies for studying gene knockdown in human cells. During RNAi, a double-stranded 21-nucleotide (nt) short-interfering RNA (siRNA) targets a specific, complementary mRNA for degradation, resulting in significantly decreased expression, or knockdown, of the targeted gene (reviewed in [56, 57]). In this report, siRNA designed to target hSpt5 successfully silenced hSpt5 as observed by decreased hSpt5 mRNA and protein expression. In addition, RNAi directed against hSpt5 did not significantly affect cell viability. hSpt5 knockdown led to significant decreases in Tat transactivation and inhibited HIV replication, indicating that hSpt5 was required for Tat transactivation and HIV replication in vivo. Taken together, silencing of hSpt5 by RNAi firmly established that the regulation of HIV-1 gene expression requires both Tat-TAR-P-TEFb interactions and interactions between RNA pol II transcription complexes and hSpt5.
Specific silencing of hSpt5 expression by siRNA in HeLa cells
Kinetics of hSpt5 knockdown by RNAi
Knockdown of hSpt5 is not lethal to human cells
hSpt5 has been shown to interact with the human mRNA capping enzyme (HCE) and this interaction enhances capping enzyme guanylylation and mRNA capping several fold . Since Spt5, Tat, and CTD associate with the capping apparatus to stimulate capping [36, 37, 39–42], we planned to separately define the role of HCE and hSpt5 in Tat transactivation by using RNAi to specifically knockdown HCE expression. HCE knockdown was confirmed by RT-PCR (data not shown). In contrast to hSpt5 knockdown cells, HCE knockdown cells showed a significant increase in cell death (Figure 3, red line) over the course of the knockdown experiment. These results indicated that HCE is an essential enzyme for cell viability and growth. Simialr findings showing that RNA capping was essential for metazoan viability have also been previously reported using RNAi in C. elegans . These results indicated that hSpt5 knockdown was not lethal to human cells, while a much more stringent requirement for HCE expression was essential for cell viability.
Role of hSpt5 on HIV-1 Tat Transactivation
To examine whether hSpt5 was required for HIV-1 Tat transactivation in vivo, Tat transactivation during hSpt5 knockdown in Magi cells was monitored. Magi cells are a HeLa cell line harboring a stably integrated single copy of the HIV-1 5' LTR-β-galactosidase gene. These cells are also genetically programmed to express the CD4 receptor for HIV-1 infection (; see below). In this experiment, Magi cells were co-transfected with Tat expression plasmid pTat-RFP and hSpt5 duplex siRNA. Co-transfection with Tat siRNA was used as a positive control for inhibition of Tat transactivation while single-stranded antisense hSpt5 siRNA and mismatched siRNA were used as negative controls. Tat transactivation and protein levels were evaluated by harvesting cells 48 h post transfection, which was within the timeframe that hSpt5 knockdown peaked. Expression of HIV-1 Tat-RFP under the control of the CMV early promoter was confirmed by western blot using anti-RFP antibody and by measuring RFP fluorescence using a fluorescence spectrophotometer (data not shown). In addition, immunoblot analysis confirmed that hSpt5 siRNA specifically inhibited hSpt5 protein expression in the absence and presence of HIV-1 Tat protein in Magi cells (data not shown).
hSpt5 knockdown inhibits HIV-1 replication
To evaluate the effect of hSpt5 knockdown on HIV-1 replication, a double siRNA transfection protocol was used to maximize the knockdown efficiency of hSpt5 during HIV-1 infection. Magi cells were transfected with siRNA directed against hSpt5. Cells mock transfected without siRNA, or transfected with single-stranded antisense hSpt5 siRNA or mismatch hSpt5 siRNA were used as negative controls. Transfection with Vif or Nef siRNAs was used as a positive control . 24 h after the first transfection, a second siRNA transfection identical to the first was performed. 24 h later, doubly transfected cells were infected with various concentrations of HIVNL-GFP, an infectious molecular clone of HIV-1. Knockdown of hSpt5 protein levels was then evaluated 48 h post infection in doubly transfected cells. An even larger decrease in hSpt5 protein levels was observed in doubly transfected cells (~95% knockdown) as compared to singly transfected cells (~85–90% knockdown; Supplementary Figure 1, compare lanes 4 and 10), suggesting that more robust knockdown of gene expression can be achieved using this double transfection method.
hSpt5, as part of the DSIF complex, was originally discovered as a negative elongation factor required for conferring DRB sensitivity to transcription elongation complexes thereby inhibiting transcription . This negative barrier provided by hSpt5 was thought to be relieved through P-TEFb phosphorylation of both hSpt5 and RNA pol II CTD, which results in increased processivity of RNA pol II complexes [7, 10, 21–29]. Increased processivity has also been linked to the phosphorylated form of hSpt5 conferring a positive effect on transcription elongation [25, 29, 34]. Recently, however, it has been shown that Tat is able to enhance transcription elongation in vitro in the absence of hSpt5 . These results appeared to indicate that P-TEFb phosphorylation of RNA pol II was the sole event that directly led to Tat transactivation and increased RNA pol II processivity . Thus, from the results of all of these in vitro studies collectively, the requirement for hSpt5 in positively regulating transcription elongation during Tat transactivation has remained unclear.
Here, we studied the role of hSpt5 in vivo using RNAi and established that hSpt5 played a positive role in Tat transactivation and HIV-1 replication. Knockdown of hSpt5 provided insight into several functional aspects of the hSpt5 protein. First, knockdown of hSpt5 was not lethal in Magi cells, indicating that hSpt5 was not required for cell viability. This was an interesting result because studies of SPT5 mutants in yeast and zebrafish and RNAi of SPT5 in C. elegans have shown that SPT5 was essential for growth and/or embryonic development in those organisms [30, 31, 60]. It seems likely that hSpt5 holds similar essential functions in human cells during embryonic development but may not be absolutely required in adult cells. Alternatively, hSpt5 knockdown may have led to decreased levels of expression that were still sufficient for hSpt5 to carry out its essential functions. Our results support the notion of using RNAi against hSpt5 as a potential therapeutic strategy for fighting HIV-1 infection since there is the potential that HIV-1 functions could be targeted for inhibition without significantly interfering with host cell functions.
Conceivably, hSpt5 functions in transcription elongation as a stabilization factor that enhances the stability of RNA pol II elongation complexes formed after P-TEFb hyperphosphorylation of the CTD. This type of role would also support hSpt5 function as an antiterminator factor as described previously . Another important positive function for hSpt5 during Tat transactivation may involve hSpt5 and Tat interactions with the capping machinery [40–42]. Phosphorylation of hSpt5 by P-TEFb may stabilize hSpt5 interactions with HCE thereby stabilizing Tat and CTD interactions with the capping machinery to promote capping and successful production of stable HIV transcripts (see model in Figure 6). Due to the highly structured nature of TAR, capping of the 5' end of HIV transcripts is not very efficient in the absence of Tat [41, 42] and Tat stimulated capping may require the presence of hSpt5 for greater access to the 5' end or to stabilize and kinetically arrest the elongation complex. Capping of HIV transcripts has also been shown to occur more proficiently when elongation is paused and not continuous , suggesting that DSIF/NELF-dependent pausing of early stage elongation complexes is representative of an elongation checkpoint. One function of this checkpoint may be to allow time for the recruitment of capping machinery and subsequent capping of HIV RNA to stabilize nascent transcripts prior to further elongation. In the absence of hSpt5, pausing may no longer occur during elongation since neither NELF nor hSPT4 binds to RNA pol II without hSpt5 [62, 63]. Thus, the window for the capping apparatus to be recruited by Tat and/or stimulate capping may be severely shortened or lost altogether without hSpt5. Any resulting uncapped HIV transcripts would be prone to degradation, accounting for the lower level of Tat transactivation and HIV replication observed during hSpt5 knockdown. The potential roles for phosphorylated hSpt5 in stabilizing RNA pol II processive elongation complexes or with respect to capping during Tat transactivation are not mutually exclusive as shown in Figure 6. hSpt5 may indeed have multi-functional roles as a positive regulator during HIV-1 replication.
The in vitro and in vivo approaches taken to address the importance of hSpt5 function all shed light on the multi-faceted nature of Tat transactivation. Accordingly, these studies altogether support important roles for both P-TEFb and hSpt5 in mediating transcription elongation during HIV-1 replication in vivo. The dual function of hSpt5 as a negative and positive transcription elongation factor also demonstrates the complexity associated with transcriptional regulation during transcription elongation and HIV-1 Tat transactivation. It is likely that posttranslational modifications of hSpt5 dictate functions of Spt5 at various promoters. Further studies will be required to elucidate how various modifications of hSpt5 such as phosphorylation and methylation control transcription elongation of both cellular and viral genes.
Twenty-one nucleotide siRNAs were chemically synthesized as 2' bis(acetoxyethoxy)-methyl ether-protected oligos (Dharmacon, Lafayette, CO). Synthetic RNAs were deprotected, annealed and purified using standard protocols provided by the manufacturer. Formation of duplex RNA was confirmed by 20% non-denaturing polyacrylamide gel electrophoresis (PAGE). Sequences of siRNA duplexes were designed as described previously  and subjected to a BLAST search against the NCBI EST library to ensure that only the desired genes were targeted.
Culture and transfection of cells
Magi (m ultinucleate a ctivation of g alactosidase i ndicator) cells carrying the endogenous HIV-5'LTR β-galactosidase gene were maintained at 37°C in Dulbecco's modified Eagle's medium (DMEM, Invitrogen) supplemented with 10% fetal bovine serum (FBS), 0.2 mg/ml of Geneticin (G418) and 0.1 mg/ml Hygromycin B (Roche Molecular Biochemicals). Cells were passaged at sub-confluence and plated at 70% confluency for transfection. Reporter plasmids and siRNA were co-transfected into Magi cells using Lipofectamine (Invitrogen) in duplicate 6-well plates (Falcon). A transfection mixture containing 25–150 nM siRNA and 9 μl of Lipofectamine in 1 ml of serum-reduced OPTI-MEM (Invitrogen) was added to each well. For high efficiency knockdown experiments, 150 nM siRNA was used. After incubating at 37°C for 6 h, cells were cultured in antibiotic-free DMEM. For further analysis, transfected cells were harvested at various time intervals, washed twice with phosphate buffered saline (PBS, Invitrogen), flash frozen in liquid nitrogen, and stored at -80°C.
RT-PCR for amplification of hSpt5 and hCycT1 mRNA
Total cellular mRNA was prepared from HeLa cells transfected without siRNA or with hSpt5 or control siRNAs using a Qiagen RNA mini kit, followed by an oligotex mRNA mini kit (Qiagen). RT-PCR was performed using a SuperScript One-Step RT-PCR kit with platinum Taq (Invitrogen) and 40 cycles of amplification. Each RT-PCR reaction included 100 ng total cellular mRNA, gene-specific primer sets for hSpt5 and hCycT1 amplification (0.5 μM for each primer), 200 μM dNTP, 1.2 mM MgSO4 and 1U of RT/platinum Taq mix. Primer sets for hSpt5 produced 2.6 kb products while hCycT1 produced 1.8 kb products. RT-PCR products were resolved on a 1% agarose gel and viewed by ethidium bromide staining. Forward and reverse primer sequence for amplifying SPT5 were 5'-ATGTCGGACAGCGAGGACAGC-3' (nts 1–21) and 5'-TGTACATGGCCGGCGTCCC-3' (nts 2638–2656), respectively. Forward and reverse primer sequences for amplifying hCycT1 are 5'-GCAACAAGTTCAAGATCTGGTCAT-3' (nts 381–404) and 5'-CCCGGGGGATCC TTACTTAGGAAGGGGTGGAAGTGG-3' (nts 2158–2200); underlined sequences represent restriction enzyme sites), respectively.
siRNA treated cells were harvested as described above and lysed in 1X reporter lysis buffer (Promega). After centrifugation to remove cellular debris, concentrations of proteins in lysates were determined using a Dc protein assay kit (Bio-Rad). Proteins in 30 μg of total cell lysates were fractionated by 10% SDS-PAGE, transferred onto a polyvinylidene difluoride membrane (PVDF membrane, Bio-Rad), and immunoblotted with antibodies against hSpt5 (Pharmingen) and hCycT1 (Santa Cruz Biotech). Protein content was visualized by a BM chemiluminescence Blotting Kit (Roche Molecular Biochemicals). The blots were exposed to X-ray film (Kodak MR-1) for various times (between 1 s and 5 min).
β-galactosidase enzyme assay
Magi cells were harvested 48 h after transfection with Tat-RFP plasmids in the absence or presence of siRNAs. Cell lysates were prepared and quantified as described above. To perform standard β-galactosidase assays, 120 μg of cell lysates were mixed in 150 μl of 1X reporter lysis buffer and 150 μl of 2X β-galactosidase assay buffer (Promega), and incubated at 37°C for 30 min. To stop the reaction, 500 μl of 1 M sodium carbonate was added to the mixture and mixed well by vortexing briefly. Absorbance of the reaction mixture was read immediately at 420 nm. The amount of Tat-RFP protein was determined using a fluorescence spectrophotometer (Photon Technology International). 300 μg of cell lysates was subjected to the spectrophotometer with slit widths set at 4 nm for both excitation and emission wavelengths as described previously [46, 64]. Fluorescence of Tat-RFP in the cell lysate was detected by exciting at 558 nm and recording the emission spectrum from 568 nm to 650 nm; the spectrum peak at 583 nm represents the maximum fluorescence intensity of Tat-RFP. Tat transactivation was determined by calculating the ratio of β-galactosidase activity (absorbance at 420 nm) of the pTat-RFP transfected cells to that of cells without pTat-RFP plasmid treatment. The inhibitory effect of siRNA treatment was determined by normalizing Tat-transactivation activity to the amount of Tat-RFP protein (represented by RFP fluorescence intensity) in the presence and absence of siRNA.
Magi infectivity assay
HeLa-CD4-LTR/β-gal indicator (Magi) cells  were plated in 24-well plates (7.5 × 105 cells per well) and transfected with siRNAs as previously described . siRNA (60 pmol) was transfected into cells using oligofectamine (2 μl, Invitrogen) for 3 h in serum-free DMEM (GIBCO). Cells were rinsed twice and top-layered in 500 μl of DMEM-10% FBS. 24 h after the first transfection, a second identical siRNA transfection was performed. 24 h after the second transfection, cells were trypsinized and seeded in 96-well microtiter plates (4 × 104 cells per well), incubated 3 h and infected with HIVNL-GFP, an infectious molecular clone of HIV-1. HIV-1 virions (normalized to RT activity in cpm) were added in doubling dilutions to duplicate wells. 48 h post infection, cells were harvested to quantify β-galactosidase activity and protein levels.
We thank Stewart Shuman, Bryan Cullen, Jon Karn, and B. Matija Peterlin for helpful discussions. We also thank Stewart Shuman for valuable reagents and Tamara J. Richman for editorial assistance, and acknowledge assay support provided by the University of Massachusetts Center for AIDS Research. This work was supported by grants from the NIH to T.M.R. (AI43198) and M.S (AI32890 and RR11489).
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