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Department of Neuroscience, Center for Neurovirology, Temple University School of Medicine, 1900 North 12th Street, 015-96, Room 203, Philadelphia, PA 19122, USA
Correspondence
Martyn K. White
martyn.white{at}temple.edu
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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B p65 subunit to a B motif near the 3' end of BKVE. In addition, a sequence within the 5' UTR of BKVE transcripts (BKVE-TAR) was identified that is identical to the HIV-1 transactivation response (TAR) element. The BKVE-TAR sequence bound TAT in RNA EMSA assays and deletion of the BKVE-TAR sequence eliminated Tat transactivation of BKVE transcription. Thus, Tat positively affected BKVE transcription by a dual mechanism and this may be important in diseases involving BKV reactivation in AIDS patients. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). Occasionally, BKV can reactivate in healthy individuals and virus becomes detectable in the urine by PCR (Polo et al., 2004). In the context of the immunosuppressive therapy received by kidney allograft recipients, reactivation of latent BKV can occur in the kidney and causes renal stenosis and interstitial nephritis in >5 % of kidney transplant patients (Hirsch & Steiger, 2003; Trofe et al., 2004). Factors that control the balance between viral latency and reactivation in humans are closely tied to the individual's immune status. In recent years, opportunistic BKV reactivation has been shown to be involved in a growing list of complications in individuals infected with human immunodeficiency virus type 1 (HIV-1) including nephritis due to BKV reactivation in the kidney (Nebuloni et al., 1999; Smith et al., 1998), haemorrhagic cystitis (Barouch et al., 2002; Gluck et al., 1994), pneumonitis (Cubukcu-Dimopulo et al., 2000), encephalitis (Garavelli & Boldorini, 2002; Gray et al., 2003; Lesprit et al., 2001), atypical retinitis (Hedquist et al., 1999) and disseminated infection involving multiple organs (Bratt et al., 1999; Vallbracht et al., 1993). Furthermore, several studies have shown increased levels of BKV in AIDS patients (Knowles et al., 1999; Markowitz et al., 1993; Pietropaolo et al., 2003). In addition, BKV reactivation correlates with the severity of immunodeficiency (Behzad-Behbahani et al., 2004; Knowles et al., 1999; Markowitz et al., 1993). These observations indicate an interaction between HIV-1 and BKV and suggest that BKV may be an emerging AIDS-associated pathogen.
BKV is a polyomavirus, a family of small DNA tumour viruses, and is closely related to Simian virus 40 and polyomavirus JC (JCV), the aetiological agent of progressive multifocal leukoencephalopathy (Khalili et al., 2004; White & Khalili, 2004, 2005). The genome of BKV comprises a circular dsDNA of approximately 5.2 kb. BKV contains a variable bidirectional promoter/enhancer region of 
500 bp, known as the non-coding control region (NCCR), which, in one direction, controls transcription of the early genes encoding large T and small t antigens. In the opposite direction, the BKV NCCR initiates transcription of the viral late genes for the viral capsid proteins VP1, VP2 and VP3 and the small auxiliary protein, agnoprotein, after the onset of viral DNA replication. Sequences within the NCCR determine the level of early gene expression and thus are also referred to as the BKV early promoter (BKVE). The replicative phase of BKV infection absolutely requires the viral early protein T antigen, which is a component of the multiprotein viral DNA replication complex. Thus, regulation of T antigen expression by BKVE is a key determinant of the balance between latency and lytic infection.
The NCCR of each BKV strain displays a high degree of variability due to mutations, duplications, deletions and rearrangements (Moens & van Ghelue, 2005; Shah, 1996). Rearrangement of the NCCR can occur in patients or during the propagation of virus in tissue culture and is not well understood. The NCCR of archetype BKV, which predominates in urine and is the transmissible form of the virus, is highly conserved and consists of a true palindrome, two inverted repeats (IR1 and IR2), an approximately 20 bp AT-rich region and several blocks designated by the letters P (68 bp), Q (39 bp), R (63 bp) and S (63 bp). Each BKV strain isolated from patient samples contains a unique arrangement of deleted, duplicated and/or rearranged P-Q-R-S segments. For example, the BKV Dunlop NCCR consists of the arrangement P1–68-P1–7;26–68-P1–64-S1–63 in which the Q and R segments are deleted, P is triplicated and an 18 bp segment is deleted from the middle P segment (Fig. 1a; Moens & Rekvig, 2001). Previous studies using linker scan analysis and DNase protection assays have identified binding sites for transcription factors NF-1 and Sp1 within the NCCR (Deyerle & Subramani, 1988; Ferguson & Subramani, 1994; Markowitz & Dynan, 1988; Moens & Rekvig, 2001). The NF-1 and Sp1 sites are required for efficient early gene transcription (Deyerle & Subramani, 1988). In addition to NF-1 and Sp1 sites, the BKV Dunlop NCCR contains an AP-1 site at each P block junction, which is not present in the BKV archetype. Recently, we found that co-operative interaction of the NF-B p65 subunit and C/EBP
transcription factors potently stimulates BKVE, suggesting that NF-B signalling is involved in BKV reactivation (Gorrill & Khalili, 2005).
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B. This may play a role in diseases involving BKV reactivation in AIDS patients. |
METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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), which contains the chloramphenicol acetyltransferase (CAT) reporter gene, in the early (BKVE) or late (BKVL) orientation, respectively. The following plasmids have been described previously: BKVE-
B-CAT (Gorrill & Khalili, 2005), pCMV-Tat, pGST, pGST-Tat, pCMV-Vpr (Sawaya et al., 2000), pCMV-p50, pCMV-p65 (Safak et al., 1999), CMV-IB
and CMV-IBN (Ansari et al., 2001). BKVE-TAR-CAT was made by site-directed mutagenesis of the transactivation response element (TAR)-like region of BKVE-CAT using primers 5'-CCAAATAGTTTTGCTAGGCCAAAAGCCTCCAC-3' and 5'-GGCCTAGCAAAACTAAAAGGGGAAATC-3' and the GeneTailor Site-directed Mutagenesis System (Invitrogen). Plasmid pBL3CAT(–450/+80) contains the HIV-1 long terminal repeat (LTR) and plasmid pBL3CAT(–450/+3) contains the HIV-1 LTR minus the TAR. A PCR fragment corresponding to the 5' untranslated region (UTR) (nt 1–110) of BKV Dunlop was cloned downstream of the LTR of plasmid pBL3CAT(–450/+3) to generate the plasmid pBL3CAT(–450/+3+BKV5'UTR).
For experiments involving the NCCR of the archetypal (WW) strain of BKV (Rubinstein et al., 1987), a plasmid containing the WW NCCR in pBlueScript (Stratagene) was kindly provided by Dr Hans Hirsch (University of Basel, Basel, Switzerland). The WW NCCR was cut out with SacI and recloned into the pGL3-basic luciferase reporter vector (Promega) in the early orientation and designated pWWBKVE-LUC.
Antibodies.
Rabbit anti-p65 (C-20) and goat anti-p50 (C-19) were from Santa Cruz Biotechnology. Anti-GRB2 was from BD Transduction Laboratories.
Cell culture, transfection and CAT and luciferase assays.
Cell lines were maintained in Dulbecco's modified Eagle's medium supplemented with 10 % FBS (Life Technologies). For transfection of Vero cells, 3x105 cells were transfected with 1.0 µg reporter plasmid (BKVE-CAT, BKVE-B-CAT, BKVE-TAR-CAT or BKVL-CAT) with or without 0.5 µg of the following plasmids as indicated in the figure legends: pCMV-Tat, pCMV-Vpr, pCMV-p50, pCMV-p65, CMV-IB or CMV-IBN. The Fugene6 transfection reagent (Roche) was used according to the manufacturer's instructions. At 48 h after transfection, protein was harvested and CAT activity was determined as described previously (Coyle-Rink et al., 2002). For CV-1, HeLa and U87-MG cells, transfection was performed as follows. CV-1 cells were transfected with 1.0 µg BKVE-CAT reporter alone or with 0.5 µg pCMV-Tat using Fugene6. HeLa and U87-MG cells were transfected with 3 µg BKVE-CAT reporter alone or in combination with 0.5 µg pCMV-Tat using the calcium phosphate precipitation method (Graham & van der Eb, 1973). CAT and luciferase assays were performed using assay kits (Promega) according to the protocols provided by the manufacturer.
Primer extension analysis.
Primer extension analysis to identify the BKVE transcription initiation site directed by HIV-1 Tat was performed as follows. CV-1 cells (1x106) were seeded on to 100 mm dishes. After 24 h, cells were transfected with 1 µg BKVE-CAT reporter alone or in combination with 0.5 µg pCMV-Tat. At 48 h after transfection, total RNA was isolated using the TRIzol reagent (Invitrogen). Total RNA (50 µg) was hybridized with 106 c.p.m. 5'-labelled primer (5'-TCCAGTGATTTTTTTCTCCAT-3', which anneals to the 5' end of the CAT gene) overnight in hybridization buffer [400 mM NaCl, 40 mM PIPES/NaOH (pH 6.4), 1 mM EDTA (pH 8) and 80 % formamide (pH 6.1)]. Hybridized samples were precipitated and incubated in a reaction containing 50 U avian myeloblastosis virus reverse transcriptase (Roche) at 42 °C for 40 min in 50 mM Tris/HCl (pH 8), 5 mM MgCl2, 5 mM DTT, 50 mM KCl, 50 mg BSA ml–1 and 160 mM each dNTP. Samples were extracted with phenol/chloroform and resolved on a 6 % polyacrylamide gel.
In vivo and in vitro production of proteins.
GST and GST–Tat fusion protein were prokaryotically expressed and purified as follows. Overnight cultures of bacteria expressing recombinant GST–Tat or GST alone were diluted 10-fold. Cultures were grown at 37 °C to an OD600 of 0.6 and induced to express GST fusion protein by adding 0.1 mM IPTG for 3–4 h. Cells were pelleted by centrifugation and resuspended in EBC-DTT buffer [50 mM Tris/HCl (pH 8), 120 mM NaCl, 0.5 % IGEPAL and 5 mM DTT]. After brief sonication and centrifugation, the supernatant was incubated with glutathione–Sepharose beads (Amersham Biosciences) at 4 °C for 30 min. Beads were washed extensively with fresh EBC-DTT buffer and GST fusion protein was collected by incubation in GST elution buffer [100 mM Tris/HCl, 2 mM DTT, 20 mM free glutathione (Sigma)]. The integrity and purity of the GST fusion protein was analysed by SDS-PAGE followed by Coomassie blue staining. Known amounts of BSA were included on the same gel for determination of the yield of the full-length protein.
Treatment of HL3T1 cells with GST and GST–Tat.
HL3T1 cells (a stable HeLa-derived cell line that contains several integrated copies of the CAT gene under the control of the HIV-1 LTR) were treated with GST or GST–Tat protein as follows. HL3T1 cells (3x105) were plated on 60 mm dishes. After 24 h, cells were transfected with GST–Tat or GST. Transfections were prepared by the method of Demarchi et al. (1996). GST or GST–Tat (5 µg) was combined with 300 µl Optimem medium (Invitrogen). Similarly, 25 µl lipofectin (Invitrogen) was combined with 300 µl Optimem medium. Both mixtures were combined and incubated at room temperature for 10 min. HL3T1 cells were rinsed once with Optimem and 2.4 ml Optimem was added to the cells. After the 10 min incubation, lipofectin/GST protein mixtures were added to the cells. After 4 h, the Optimem/transfection mixture was replaced with fresh DMEM with 10 % FBS. At 24 h post-transfection, protein was collected and CAT activity was determined.
Electrophoretic mobility shift assay (EMSA).
An EMSA was performed as follows to assess the binding of NF-B to the BKVE B motif in response to Tat. HeLa cells (1x106) were seeded on 100 mm dishes. After 24 h, cells were transfected with GST or GST–Tat as described above. At 5 h post-transfection, nuclear extracts were collected according to the method of Andrews & Faller (1991). As a positive control for activation of NF-B, HeLa cells were treated with 100 ng phorbol 12-myristate 13-acetate (PMA; Sigma) ml–1 for 30 min. Nuclear extract (10 µg) from untreated, PMA-treated, GST-treated and GST–Tat-treated cells was incubated with 50 000 c.p.m. of a 32P-labelled double-stranded oligonucleotide probe containing the BKVE B motif (5'-TTGCAAAAATTGCAAAAGAATAGGGATTCCCCAAATA-3'), as described previously (Safak et al., 1999).
RNA interference with p65 small interfering (si) RNA.
Transient knock-down of p65 was performed with an siRNA specific for p65 (5'-GCCCUAUCCCUUUACGUCAdTdT-3'; Dharmacon Research).
Cells (3x105) were plated on 60 mm dishes. After 24 h, cells were rinsed once with Optimem. siRNA was added to a final concentration of 50 nM by the method of Surabhi & Gaynor (2002). At 24 h after siRNA transfection, cells were transfected as described above and CAT activity was determined. Western blot analysis of protein extracts from untransfected cells or cells transfected with p65-specific siRNA using anti-p65 was used to ascertain p65 knock-down, with anti-Grb2 antibody used as a loading control. Control non-targeting siRNA was also obtained from Dharmacon.
Tat RNA EMSA.
The oligoribonucleotide 5'-GUGGAGGCUUUUUCUGAGGCCUAGC-3' (BKV TAR), which corresponds to nt 70–46 of the BKV genome, was end-labelled using [32P]ATP with T4 polynucleotide kinase. The labelled BKV TAR was incubated in a reaction mixture with recombinant Tat using the method of Wei et al. (1998). To obtain recombinant Tat, Tat was cleaved from GST beads by cleavage in buffer [50 mM Tris/HCl (pH 7.6), 20 mM KCl, 1 mM DTT] containing thrombin.
PMA treatment.
For experiments to measure the effect of PMA on BKVE transcription, cells were transfected as described above and treated 24 h later with 100 ng PMA ml–1. After a further 24 h, cells were harvested for the CAT assay.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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) was fused in the early (BKVE) or late (BKVL) orientation into the BamHI site of pBLCAT3 to give the BKVE-CAT and BKVL-CAT reporter plasmids, respectively. These constructs were introduced into the Vero monkey kidney cell line, alone or in combination with expression constructs for the HIV-1 proteins Vpr and Tat, and the transcriptional activity was determined. As can be seen in Fig. 1(b), expression of Tat significantly activated transcription from the BKVE promoter. Neither Vpr nor Tat had a significant effect on the BKVL promoter. The effect of Tat on BKVE transcriptional activity was also evident in several other cell lines (Fig. 1c), suggesting that the observed event was not cell-type specific. Since others have shown that the BKVE transcription initiation site may vary (Moens & Rekvig, 2001), we next determined the site of Tat-induced transcription initiation. Vero cells were transfected with the BKVE-CAT reporter alone or in combination with the Tat expression construct and the transcription initiation site was determined by primer extension analysis by annealing total RNA to a primer specific for the 5' end of the CAT gene. As shown by an arrow in Fig. 1(d), the initiation site for Tat-directed transcription was located at nt 93, which has been reported by others to be the major transcription start site for BKVE transcription (Deyerle et al., 1987; Ferguson & Subramani, 1994; Moens & Rekvig, 2001; Seif et al., 1979). An A/G sequence ladder was run on the same gel and used to determine the size of the primer extension product (Fig. 1d).
In order to determine whether the observed Tat stimulation was peculiar to the Dunlop strain of BKV or might be a general feature of the virus, we constructed a reporter plasmid in which the archetypal, non-rearranged form of the BKV NCCR (WW) drives luciferase expression in the early orientation (pWWBKVE-LUC). Tat significantly activated transcription from the archetypal BKVE promoter (Fig. 1e). All subsequent experiments were performed with the Dunlop promoter.
The BKVE promoter has a B motif downstream from the transcription initiation site at nt 25–34 (shown in Fig. 1a). As the HIV-1 Tat protein has been shown to activate NF-B-dependent transcription (Demarchi et al., 1996), we investigated next whether Tat transactivation of the BKVE promoter depended on this site. Vero cells were transfected with the BKVE reporter alone or with the indicated combinations of expression constructs for Tat, IB or IBN. IB is an NF-B-binding protein that sequesters NF-B in the cytoplasm until IB becomes phosphorylated by an upstream signalling kinase and is degraded by the ubiquitin-proteasome pathway, thus releasing active NF-B to the nucleus. IBN is an N-terminally truncated mutant of IB that lacks two serine residues that are phosphorylated upon activation of the NF-B pathway and are required for degradation via the ubiquitin-proteasome pathway (Ghoda et al., 1997). Whereas IB had no effect on Tat-dependent BKVE transcription, co-expression of IBN with Tat abolished the Tat-dependent BKVE transcriptional response (Fig. 2a). Similarly, site-directed mutagenesis of the BKVE-B motif (BKVE-B-CAT) resulted in loss of Tat responsiveness (Fig. 2b, lanes 3 and 4) compared with the wild-type promoter (Fig. 2b, lanes 1 and 2).
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B-dependent. Furthermore, since Tat does not bind DNA, we sought to determine whether Tat induced the binding of NF-B to its cognate site. To this end, we employed EMSA using Tat protein and the B DNA motif as a probe. The biological activity of the recombinant GST–Tat fusion protein used in this experiment was confirmed as follows. Transient assays were performed by treating HL3T1 cells, a HeLa-cell derivative that contains multiple integrated copies of the CAT gene under control of the HIV-1 LTR, with GST or GST–Tat. As shown in Fig. 3(a), treatment of HL3T1 cells with GST–Tat, but not GST, caused a dramatic induction of CAT activity, demonstrating that the GST–Tat preparation was biologically active. Next, nuclear extracts prepared from HeLa cells treated with GST (Fig. 3b, lanes 4–8) or GST–Tat (Fig. 3b, lanes 9–13) were incubated with a double-stranded 32P-labelled oligonucleotide probe containing the BKVE B motif. The presence of the p50 and/or p65 subunits of NF-B in the protein–DNA complexes was determined with the use of anti-p50 (Fig. 3b, lanes 7 and 12) or anti-p65 (Fig. 3b, lanes 8 and 13) antibodies. Nuclear extract from PMA-treated HeLa cells was included as a positive control for NF-B binding activity (Fig. 3b, lane 3). In contrast to extracts from untreated and GST-treated cells (Fig. 3b, lanes 2 and 4–8, respectively), GST–Tat induced the binding of a band that co-migrated with extract from PMA-treated cells (Fig. 3b, compare lane 9 with lane 3). This band disappeared with p65-specific antibody (Fig. 3b, lane 13), but not with control sera (Fig. 3b, lanes 10 and 11) or with p50-specific antibody (lane 12), demonstrating the presence of p65 in the complex.
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B and the transcriptional activity was determined (Fig. 4a). In agreement with the EMSA results, overexpressed p65, but not p50, dramatically induced BKVE promoter activity. Furthermore, co-expression of p65 and p50 gave results similar to p65 alone. Next, we used siRNA transiently to knock down the expression of the p65 subunit of NF-B and repeated the transfection experiment with BKVE reporter alone or in combination with overexpressed Tat. Transient siRNA-dependent knock-down of p65 in HeLa cells significantly reduced Tat-dependent induction of BKVE promoter activity, indicating the importance of endogenous p65 in Tat stimulation of BKVE (Fig. 4b). The efficacy of siRNA for the suppression of p65 protein level was tested by Western blot analysis (Fig. 4c). Non-specific siRNA did not suppress the level of p65 protein, nor did it affect the rate of BKVE transcription significantly.
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; Rounseville & Kumar, 1992). Next, we sought to determine the importance of this sequence, which we designated BKVE-TAR, for Tat transactivation of the BKVE promoter. Hence, we deleted the BKVE-TAR pentanucleotide by site-directed mutagenesis to give the construct BKVE-TAR and tested for responsiveness to Tat. Vero cells were transfected with wild-type BKVE or the BKVE-TAR construct alone or in combination with the Tat expression construct and the transcriptional activities were compared (Fig. 5a). Whereas Tat activated the wild-type BKVE reporter, mutation of the BKVE-TAR site extinguished Tat responsiveness, demonstrating the requirement of the TAR-like site for Tat transactivation of the BKVE promoter. We also performed an RNA EMSA using a 32P-labelled RNA oligonucleotide containing the pentanucleotide BKVE-TAR plus flanking sequences. Recombinant GST–Tat was cleaved from GST beads with thrombin (Fig. 5c). Incubation of cleaved Tat with the BKVE-TAR probe resulted in a retarded band (Fig. 5b), demonstrating that Tat binds to BKVE-TAR. Hence, we concluded that Tat also requires the BKV TAR sequence for transactivation of the BKVE promoter.
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B and TAR) for the effect of PMA stimulation, which activates NF-B, and the relative effect of expression of p65 or Tat (Fig. 7). PMA stimulated the wild-type BKVE promoter 3.2-fold (Fig. 7, compare lanes 1 and 2). The basal levels of activity of both the B and TAR mutant promoters were lower than the wild-type BKVE promoter by 0.28-fold and 0.43-fold, respectively (Fig. 7, compare lanes 1, 5 and 9). The activity of these promoters in the presence of PMA was also much reduced (Fig. 7, compare lanes 2, 6 and 10) and no statistically significant difference was seen for the B promoter in the presence and absence of PMA (Fig. 7, compare lanes 5 and 6). Both of the mutant promoters were unresponsive to Tat (Fig. 7, compare lane 5 with lane 8 and lane 9 with lane 12), as was also shown in Figs 2 and 5. The TAR but not the B promoter was responsive to p65 expression (compare lane 9 with lane 11 and lane 5 with lane 7).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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; Ferguson & Subramani, 1994; Markowitz & Dynan, 1988; Moens & Rekvig, 2001). Functional analysis by site-directed mutagenesis of the BKVE B sequence demonstrated that this site was required for activation by p65 and by Tat. Furthermore, co-expression of the NF-B inhibitor IBN and the use of siRNA directed against p65 demonstrated that NF-B activation was required for Tat transactivation of the BKVE promoter. An EMSA with an oligonucleotide probe containing the BKVE B sequence showed that Tat induced the binding of the p65 subunit of NF-B to the BKVE promoter. Finally, we identified and characterized an HIV-1 TAR-like sequence within the BKVE 5' UTR that was also required for transactivation of the BKVE promoter by Tat.
In addition to the HIV-1 LTR, Tat has also been shown to activate several cellular promoters (e.g. TNF-; Darbinian et al., 2001) and viral promoters (e.g. JCV; Tada et al., 1990). Activation of BKV by Tat through a TAR-like motif is not without precedent. It has been shown that Tat activates IL-6 gene expression through interaction of Tat with a TAR-like sequence located in the 5' UTR of the IL-6 RNA transcript (Ambrosino et al., 1997). Hence, there is a precedent for the regulation by Tat of the TAR-like sequence within the 5' UTR of BKVE transcripts.
Furthermore, it is well documented that, in the absence of the TAR region, Tat can activate transcription of the HIV-1 LTR, as well as several heterologous promoters, through its association with NF-B (Biswas et al., 1995; Demarchi et al., 1996). For example, Tat directs E-selectin expression in human umbilical vein endothelial cells by inducing the binding of NF-B to a B motif in the E-selectin promoter (Cota-Gomez et al., 2002). In this study, we have demonstrated likewise the requirement for a B motif for initiation of transcription from the BKVE promoter by Tat.
In vivo, there are several modes of action by which Tat potentially could activate the BKVE promoter and cause BKV reactivation. First, biologically active Tat is secreted by HIV-1-infected cells and could affect neighbouring cells by transcellular means or by binding to cell-surface receptors (reviewed by Peruzzi, 2006). Alternatively, if an HIV-1-infected cell becomes superinfected with BKV or vice versa, newly synthesized Tat could activate BKVE transcription, as has been shown to occur for transactivation of Human herpesvirus 5 (human cytomegalovirus) (Ho et al., 1991; Skolnik et al., 1988). From the dual requirement for NF-B activation and BKV TAR binding in the present study, we can propose a model in which Tat first initiates transcription by inducing the binding of NF-B p65 to the BKVE promoter. Subsequently, Tat may bind to the 5' UTR of BKVE transcripts and enhance transcriptional activation through an HIV-1 TAR-like mechanism. Furthermore, when we replaced the TAR of the HIV-1 LTR with the 5' UTR from the BKVE promoter, the BKV TAR sequence failed to rescue Tat responsiveness of the TAR-less HIV-1 LTR (Fig. 6). Therefore, the possibility of only a TAR-like mechanism for transcriptional activation of the BKVE promoter in response to Tat is probably an oversimplification. It may be that BKV TAR-bound Tat must also interact either directly or indirectly with promoter-bound NF-B p65 to activate BKVE transcription.
As regards the mechanism of NF-B activation by Tat, this has been the subject of intensive investigation in this laboratory and others. It has become clear that this activation can occur by both direct and indirect mechanisms. Direct interaction with NF-B is involved in TAR-independent activation of the HIV-1 LTR in cells from the CNS (Taylor et al., 1995) and may involve a third protein named NFBP (Sweet et al., 2005). As regards indirect interaction, it has been demonstrated previously, by ourselves and others, that Tat induces the expression of TNF- (Buonaguro et al., 1992; Darbinian et al., 2001; Rautonen et al., 1994; Sawaya et al., 1998), which can activate the NF-B pathway (reviewed by Chen & Goeddel, 2002). Thus, there exist direct and indirect mechanisms for the induction of NF-B by Tat. As we report here for the BKV NCCR, it has also been reported that NF-B and Tat act synergistically to increase transcription from the HIV-1 LTR (Nabel & Baltimore, 1987; West et al., 2001).
In conclusion, the data presented in this communication provide evidence for HIV-1 action on the BKVE promoter that complements clinical data in the literature that indicate an emerging role for BKV in AIDS pathology and point to the importance of a role for HIV-1 Tat in BKV reactivation in AIDS patients.
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ACKNOWLEDGEMENTS |
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Received 29 September 2005;
accepted 16 February 2006.
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