Tat-RNase H and its use in HIV gene therapy

I. Introduction

AIDS is caused by the human immunodeficiency virus type-1, HIV-1, the genome of which consists of two “+” strand RNAs. Destruction of HIV RNA molecules within the cell or within virion could prove to be a successful strategy in inhibiting HIV replication. The therapeutic potential of a number of strategies based on the ability of ribozymes and ribonucleases to cleave RNA/DNA molecules is currently being investigated.

Three RNase-based strategies may be used to inactivate HIV RNA (Singwi and Joshi, 2000). In the first strategy, “cytotoxic” RNases may be employed to specifically destroy cells that become infected by HIV (Singwi and Joshi, 2000). In the second strategy, “co-localized” RNases may be designed to be packaged within the progeny virus and cleave the virion RNA (Singwi and Joshi, 2000). This would inactivate the progeny virus by cleaving the virion RNA. The Staphylococcal nuclease (SN) fused with the HIV-1 virion protein R (Vpr) or HIV-2 virion protein X (Vpx) has been shown to allow its packaging within the HIV virion (Wu et al, 1995); however, this nuclease was inactivated by the HIV protease. In a third strategy, others and we have developed targeted RNases that can cleave specific RNA sequences (Melekhovets and Joshi, 1996; Singwi et al, 1999). The probability of escape mutants interfering with the function of targeted RNases is significantly lower since it would require mutations in both the viral protein from which the binding moiety of the RNase is derived and the RNA sequence it binds to.

Two targeted RNases were designed and constructed in our laboratory by fusing an RNase molecule with HIV RNA binding proteins, a Tat-RNase H (Melekhovets and Joshi, 1996) and a Tev-RNase T1 (Singwi et al, 1999). The Tat-RNase H protein consists of the TAR (trans-activation response) element-binding domain of the HIV-1 Tat and the RNase H domain of the HIV-1 reverse transcriptase (RT) (Melekhovets and Joshi, 1996). The Tat portion of the fusion protein spans the first 72 amino acids of the Tat protein and it is essential and sufficient for the transport of the protein to the nucleus and specific binding to the TAR element present in all HIV-1 RNA molecules (Hoffmann et al, 1997). The RNase portion of the HIV-1 RT specifically degrades the RNA moiety within RNA/DNA hybrid in vivo (Skalka and Goff, 1993; Telesnitsky and Goff, 1997) and to a lesser degree within RNA/RNA hybrid in vitro (Ben-Artzi et al, 1992; Gotte et al, 1995). Although the two domains (i.e., the TAR binding domain of the Tat protein and the RNase H domain of HIV-1 RT) when not fused, were shown to be inactive, the chimeric Tat-RNase H protein was demonstrated to specifically recognize and cleave TAR RNA in vitro (Melekhovets and Joshi, 1996). The Tev-RNase T1 protein consists of the RNA binding domain of HIV-1 Tev protein and the RNase domain of the Aspergillus oryzae RNase T1 protein. In vitro, the Tev-RNase T1 protein was shown to exhibit poor specificity, however, when expressed in a CD4⁺ human T lymphoid (MT4) cell line or human peripheral blood T-lymphocytes (PBLs) no cytotoxicity was observed (Singwi et al, 1999). In addition, HIV-1 replication was inhibited both in transduced MT4 cells and PBLs (Singwi et al, 1999).

The Tat-RNase H was previously shown to specifically recognize and cleave HIV-1 TAR RNA in vitro in RNA/RNA hybrid (Melekhovets and Joshi, 1996). In this paper, we demonstrate that Tat-RNase H-mediated cleavage of TAR RNA in vitro in RNA/DNA hybrid occurs much more efficiently. However, this activity was shown to be non-specific as both TAR RNA and mutant TAR RNA/DNA hybrids were efficiently cleaved. A retroviral vector was then engineered for the delivery and expression of tat-RNase H gene in the human CD4⁺ T lymphoid cells. However, Tat-RNase H failed to inhibit HIV-1 replication in transduced MT4 cells and PBLs. The possible explanations for the inability of the Tat-RNase H to cleave HIV-RNA in vivo are discussed.

II. Results

A. Tat-RNase H-mediated cleavage in vitro of TAR RNA and mutant TAR RNA in RNA/RNA or RNA/DNA hybrid

We have previously designed and expressed a fusion Tat-RNase H protein (Melekhovets and Joshi, 1996). Tat-RNase H produced in E. coli was shown to specifically recognize and cleave RNAs containing the HIV-1 TAR element. Tat-RNase H could potentially cleave HIV-1 RNA in RNA/RNA or RNA/DNA hybrid. The RNA cleaving activity of Tat-RNase H in RNA/RNA hybrid had been previously demonstrated (Melekhovets and Joshi, 1996). The RNA cleaving activity of Tat-RNase H in RNA/DNA hybrid was studied as follows. A 75 nt-long TAR/mutant TAR RNA was transcribed and 5’ end-labeled in vitro. This RNA contained the 59 nt-long stem loop structure forming the TAR/mutant TAR element at its 5’ end and a 16 nt-long single stranded RNA at its 3’ end. This 75 nt-long TAR/mutant TAR RNA and a 18 nt-long oligodeoxynucleotide complementary to the single stranded RNA were used to provide Tat-RNase H with a substrate containing TAR/mutant TAR element at the 5’ end and RNA/DNA hybrid at the 3’ end (Figure 1A). As the oligodeoxynucleotide is not designed to hybridize to the nucleotides forming the TAR element, the TAR stem-loop structure will form. Thus, while testing RNA cleavage in RNA/DNA hybrid, the TAR stem loop structure at the 5’ end would allow Tat-RNase H/TAR element interaction as well as cleavage in an RNA/RNA hybrid. Cleavage in RNA/RNA hybrid was previously shown to yield 37, 46-49, 56-58 nt-long fragments (Melekhovets and Joshi, 1996). RNA cleavage in RNA/DNA hybrid should yield 57-75 nt-long fragments. If the cleavage was complete, only the shortest 57 nt-long fragment would be detected (Figure 1A); alternatively, as the two nucleotides at the 3’ end of the oligodeoxynucleotide are complementary to the nts 58 and 59 forming the TAR stem structure, a 59 nt-long fragment may be detected if the formation of the TAR structure was to prevent these two nucleotides from hybridizing to the oligodeoxynucleotides.

Cleavage products were analyzed on a 12-15% polyacrylamide gel. As shown in Figure 1B, no TAR RNA (lane 1) or TAR RNA/DNA (lane 2) cleavage was observed in control samples that were incubated in the absence of Tat-RNase H. Tat-RNase H-mediated cleavage of TAR RNA is shown in lane 3. The expected 37, 46-49 and 46-48 nt-long cleavage products were observed. Tat-RNase H-mediated cleavage of TAR RNA/DNA hybrid is shown in lane 4. Almost 99% of the TAR RNA is cleaved in RNA/DNA hybrid; both 57 and 59 nt-long products were observed. Bands corresponding to RNA cleavage in the RNA/RNA hybrid are also observed (lane 4); however, the intensity of these bands is very week compared to those obtained from cleavage in RNA/DNA hybrid. Thus, the efficiency of Tat-RNase H-mediated cleavage of TAR RNA in RNA/DNA hybrid is much higher than in RNA/RNA hybrid.

To determine cleavage specificity of Tat-RNase H in cleaving TAR RNA in RNA/DNA hybrid, TAR-RNase H was used to cleave both TAR and mutant TAR RNA/DNA hybrids (Figure 1C). Cleavage was performed for 5, 10 and 15 min. RNA was effectively cleaved within both TAR RNA/DNA (lanes 1-3) and mutant TAR RNA/DNA (lanes 4-6) hybrids. Mutant TAR RNA/DNA hybrid cleavage by Tat-RNase H indicates that Tat-RNase H can

the Tat-RNase H fusion protein does not inhibit HIV-1 replication in transduced MT4 cells and PBLs.

III. Discussion

A number of functions have been attributed to the RNase H domain of the HIV-1 RT (Hostomsky et al, 1992, 1994). RNase H activity is required for strand transfer and strand displacement both of which are essential functions for the viral replication (Pop, 1996; Gabbara et al, 1999). RNase H shows a strong affinity for RNA/DNA hybrids in vivo (Skalka and Goff, 1993; Telesnitsky and Goff, 1997) and to a lesser degree for RNA/RNA hybrids in vitro (Ben-Artzi et al, 1992; Gotte et al, 1995). The HIV-1 RNase H has been shown to exhibit both endonuclease and exonuclease activities. The exonucleolytic activity on RNA/DNA complex is also referred to as "directional processing" (Zhan et al, 1994). It has been proposed that the dual function of RNase H might be the result of two distinct conformations (Zhan et al, 1994; Cirino et al, 1995). The two conformations are regulated by the presence and relative amounts of divalent ions (Davis et al, 1991; Cirino et al, 1995). HIV-1 RNase H binds two divalent ions, namely Mn²⁺ or Mg²⁺. The presence of Mn²⁺ has been implicated in the exonucleolytic activity of the enzyme in RNA/DNA and RNA/RNA hybrids, while Mg²⁺ is required for both the exonucleolytic and endonucleotic activities (Ben-Artzi et al, 1993). The intimate association of the RNase H function with viral replication and its ability to recognize and cleave both RNA/RNA and RNA/DNA hybrids, renders it an attractive candidate for anti-HIV-1 gene therapy.

We have previously reported the design and expression of the fusion gene tat-RNase H, composed of the sequence encoding a portion of the Tat protein of HIV-1 and of the sequence encoding the RNase H domain of the HIV-1 RT (Melekhovets and Joshi, 1996). In vitro studies using Tat-RNase H fusion protein expressed in E. coli and an HIV-1 TAR RNA had previously shown that the fusion protein specifically recognizes the TAR element and cleaves RNA in RNA/RNA hybrid (Melekhovets and Joshi, 1996). Tat-RNase H could cleave RNA in RNA/RNA and also in RNA/DNA hybrid (Ben-Artzi et al, 1992; Skalka and Goff, 1993; Gotte et al, 1995; Telesnitsky and Goff, 1997). The RNA cleaving activity of Tat-RNase H in RNA/DNA hybrid was therefore studied. The efficiency of Tat-RNase H-mediated cleavage of TAR RNA in RNA/DNA hybrid was found to be much higher than in RNA/RNA hybrid. However, the RNA cleaving activity of Tat-RNase H in RNA/DNA hybrid was non-specific and did not require Tat-RNase H/TAR element interaction as both TAR and mutant TAR RNAs were efficiently cleaved in RNA/DNA hybrid. How the mutant TAR RNA/DNA hybrid is recognized by Tat-RNase H was not investigated.

Tat-RNase H-mediated cleavage of HIV RNAs, which all contain the TAR element at their 5’ end, should result in inhibition of HIV-1 replication. Thus, a retroviral vector MoTN-TH was designed to allow constitutive expression of tat-RNase H gene under the control of the SV 40 promoter. Note that constitutive expression is the only effective way to express Tat-RNase H. Tat-inducible expression would require Tat-RNase H mRNA to contain the TAR element and therefore would be suicidal for Tat-RNase H’s own production. Rev-inducible expression would make it too late for Tat-RNase-H to inhibit virus replication.

Tat-RNase H mediated inhibition of HIV-1 replication was tested in both transduced human CD4⁺ T-lymphoid cell line (MT4) and in PBLs. Amphotropic MoTN and MoTN-TH vector particles were used to transduce MT4 cells and PBLs. PCR and RT-PCR analyses confirmed the presence and expression of MoTN and MoTN-TH vectors. Also the viability of MoTN or MoTN-TH transduced packaging cell lines, MT4 cells and PBLs was similar, suggesting lack of cytotoxicity. This result indicates that the RNA/DNA cleaving activity of Tat-RNase H is not predominant in vivo. As this activity is not dependent on Tat-RNase H/TAR element interaction, if present it was expected to cause cytotoxicity.

However, Tat-RNase H failed to inhibit HIV-1 replication in both MT4 transductants challenged with a laboratory strain of HIV-1 and PBL transductants challenged with a clinical isolate of HIV-1. As virus production in MoTN-TH transduced cells was not delayed as compared to cells transduced with the MoTN vector, it seems unlikely, that the lack of inhibition of HIV-1 replication by the Tat-RNase H protein would be due to escape virus production. These results indicate that, the Tat-RNase H fusion protein cannot cleave HIV-1 RNA in MT4 cells or PBLs.

There are three stages in the virus life cycle that could be affected by the Tat-RNase H activity: during reverse transcription in the cytoplasm, during transcription in the nucleus, or post-transcription in the nucleus or in the cytoplasm. However, in order for the Tat-RNase H to cleave HIV-1 RNA in RNA/RNA or RNA/DNA hybrid during reverse transcription, it would have to enter the partially uncoated virion which is rather unlikely. Next, Tat-RNase H could cleave RNA in RNA/RNA or RNA/DNA hybrid during transcription in the nucleus. Note that Tat-RNase H contains the nuclear localization signal of Tat and therefore it should have been localized in the nucleus (Endo et al, 1989; Hoffmann et al, 1997). Lastly, HIV RNA in RNA/RNA hybrid could have been cleaved post-transcription in the nucleus or in the cytoplasm. However, as Tat is predominantly localized in the nucleus, chances of Tat-RNase H localization and cleaving HIV RNA in the nucleus are much greater than in the cytoplasm. Failure of the Tat-RNase H to inhibit HIV-1 replication, by virtue of destroying HIV-1 RNA, could be attributed to a number of reasons discussed below.

As Tat-RNase H-mediated cleavage of RNA in RNA/DNA hybrid is non-specific in vitro and does not require Tat/TAR interaction, this activity could not have resulted in specific inhibition of HIV replication. If present, this activity would have caused cytotoxicity, which was not observed. The RNA cleaving activity of Tat-RNase H in RNA/RNA hybrid observed in vitro (Melekhovets and Joshi, 1996) is the one that was expected to specifically inhibit virus replication. Thus, it seems that this activity was poorly exhibited in vivo.

Tat-RNase H was designed to contain the nuclear localization signal of Tat for transport of the fusion protein to the nucleus to be localized in the nucleus. This should have enabled the Tat-RNase H to recognize and cleave nascent HIV-1 transcripts. However, the last few amino acids present in the C-terminus of the Tat protein have not been included in the Tat-RNase H protein. Although the significance of this sequence is not clear, some evidence exists to implicate it in the nuclear localization of the protein (Hoffmann et al, 1997). Therefore, it is possible that inadequate localization of the Tat-RNase H protein in the nucleus might have resulted in poor inhibition of HIV-1 replication. Alternatively, it is also possible that Tat-RNase H does enter the nucleus but that TAR RNA recognition and/or cleavage by the fusion protein is inhibited in vivo. This could be due to steric interference caused by other TAR RNA binding proteins, or those typically interacting with the Tat protein (Kato et al, 1992; Kaczmarski and Khan, 1993). High levels of viral Tat protein could also out-compete the Tat-RNase H protein for TAR RNA binding, rendering the HIV-1 RNA inaccessible to the Tat-RNase H activity.

A poor Tat-RNase H-mediated cleavage of TAR RNA in RNA/RNA hybrid in vivo is consistent with the fact that RNase H activity of RT/RNase H during viral reverse transcription only shows RNase activity within RNA/DNA hybrids and not within RNA/RNA hybrids. Our in vitro studies have shown that the Tat-RNase H protein can cleave TAR RNA in an RNA/RNA or RNA/DNA hybrid in the presence of Mn²⁺ but not in the presence of Mg²⁺. It is possible that the physiological concentrations of Mn²⁺ in MT4 cells or PBLs are sub-optimal for RNA cleaving activity of Tat-RNase H. As this activity (RNA cleavage in RNA/RNA hybrid) would have been detrimental for virus evolution, it seems that the reason why this targeted RNase failed to inhibit HIV-1 replication is because of the choice of the RNase used. Indeed, Tev-RNase T1 was shown to inhibit HIV-1 replication in both MT4 cells and PBLs (Singwi et al, 1999).

IV. Materials and Methods

A. In vitro cleavage of TAR RNA and mutant TAR RNA in RNA/RNA and RNA/DNA hybrids by Tat-RNase H

Tat-RNase H was produced and purified from E. coli (Melekhovets and Joshi, 1996). TAR RNA and mutant TAR RNA (75 nt-long) were transcribed in vitro and 5’ end-labeled as described previously (Melekhovets and Joshi, 1996). These RNAs (30,000 cpm) were then subjected to Tat-RNase H cleavage in the presence of 10 mM Mn²⁺ as described previously (Melekhovets and Joshi, 1996). Control samples lacked Tat-RNase H. In order to study the RNA/DNA cleaving activity of Tat-RNase H, an oligodeoxynucleotide (5’-TTG-AGG-CTT-AAG-CAG-TGG-3’) complementary to the last 18 nucleotides of the TAR/mutant TAR RNA was added. All cleavage reactions were performed for 1 hour at 37^oC, unless specified otherwise. The cleaved and uncleaved RNA was then ethanol precipitated and analyzed by electrophoresis on a 12-15% polyacrylamide gel.

B. MoTN-TH vector construction

The tat-RNase H gene was constructed as described previously (Melekhovets and Joshi, 1996). The SV 40 promoter and the tat-RNase H gene were then subcloned in a retroviral vector using a two step PCR strategy. In the first PCR, the Tat-RNase H-5’ primer (5’-CGG-AAG-ATC-TAA-TAC-GAC-TCA-CTA-T-3’) and Tat-RNase H-3’ primer (5’-GCG-CAT-CGA-TCT-ATA-GTA-CTT-TCC-TGA-TTC-C-3’) were used to amplify the tat-RNase H gene from the pET-TH plasmid. This PCR product and the SV-5’ primer (5’-CGG-AAG-ATC-TAA-TAC-GAC-TCA-CTA-T-3’) were then used in a second PCR reaction with the pB1-SVR6842 plasmid DNA, to amplify the SV40 promoter and the tat-RNase H gene. The resulting PCR product contained a Bgl II restriction site, the SV40 promoter, the tat-RNase H gene and a Cla I restriction site. This cassette was digested with Bgl II and Cla I and cloned at the BamH I and Cla I sites of the MoTN vector. Clones containing the SV40 promoter and the tat-RNase H gene in the correct orientation were confirmed using restriction enzyme and PCR analyses.

C. Transduction of MT4 cells and PBLs with MoTN and MoTN-TH

Amphotropic MoTN and MoTN-TH vector particles were produced as described previously (Joshi et al, 1990). Either MoTN or MoTN-TH vector particles were used to transduce the human CD4⁺ T lymphoid (MT4) cell line (Pauwels et al, 1987; Larder et al, 1989) as described previously (Liem et al, 1993). Stable MT4 transductants containing either the MoTN or MoTN-TH vector were selected for resistance to the antibiotic G418 over a period of three weeks.

Human peripheral blood mononuclear cells were isolated using Ficoll-Hypaque gradient centrifugation. Following a phosphate buffered saline (PBS) wash, 1 x 10⁶ cells/ml were cultured for a day at 37^oC in RPMI 1640 medium that contained 10% fetal bovine serum, 20 units/ml recombinant human interleukin (IL)-2 (Boehringer Mannheim) and 5 mg/ml phytohemagglutinin (Sigma). Cells in suspension were collected and cultured for two more days. T cell-specific monoclonal antibodies were used to determine T lymphocytes by flow cytometry. Approximately 1 x 10⁶ PBLs were transduced with MoTN or MoTN-TH vector particles as described previously (Singwi et al, 1999) in the presence of 16 mg/ml polybrene and 20 units/ml IL-2. The cells were then collected by centrifugation at 200 x g for 1 hour at 32^oC and cultured for 16 hours at 32^oC. Prior to transduction, the cells were incubated at 37^oC for 6 hours in medium containing IL-2. Three rounds of transduction were carried out. Following the final round, transduced cells were selected for four days in medium containing G418 (500 mg/ml).

D. Detection of tat-RNase H DNA and RNA in transduced cells

Genomic DNA isolation from cells transduced with MoTN and MoTN-TH vector was carried out as described earlier (Sambrook et al, 1989). PCR analysis was performed as described earlier (Ramezani and Joshi, 1996) using the Tat-RNase H-5' and 3' primers. Total cellular RNA was extracted from MoTN and MoTN-TH vector transduced cells as described elsewhere (Chomezynski and Sacchi, 1987). Reverse transcription was performed using the Tat-RNase H-3’ primer followed by PCR using the Tat-RNase H-5’ and 3’ primers as described previously (Ramezani and Joshi, 1996). PCR and RT-PCR products were analyzed by electrophoresis on a 1.5% agarose gel.

E. HIV-1 susceptibility of MT4 and PBL transductants expressing Tat-RNase H

The pools of actively dividing stable MT4 transductants lacking or expressing Tat-RNase H (2 x 10⁶ cells) were each infected with the HIV-1 strain NL4-3 (500 ng p24 equivalent) for 6 hours at 37^oC as described previously (Ramezani and Joshi, 1996). Transduced PBLs (5 x 10⁶ cells) were infected with 1.5 ng (p24 equivalent) of a clinical isolate of HIV-1 for 16 hours at 37^oC as described previously (Singwi et al, 1999). Half of the culture supernatants was collected every third day and replaced with fresh medium. The amount of HIV-1 p24 antigen released in the cell culture supernatant was measured by enzyme linked immunosorbent assay (ELISA, Abbott) as described by the manufacturer.

Acknowledgements

This work was supported by a grant from the Canadian Institutes of Health Research. The following reagents were obtained through the AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, NIH: HIV-1 strain NL4-3 from Dr. R.C. Gallo, pNL4-3 from Dr. A. Adachi; MT4 cell line from Dr. D. Richman.

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