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Functional characteristics of a reverse transcriptase encoded by an endogenous retrovirus from Drosophila melanogaster
ARTICLE IN PRESS Insect Biochemistry and Molecular Biology Insect Biochemistry and Molecular Biology 35 (2005) 323–331 www.elsevier.com/locate/ibmb
Functional characteristics of a reverse transcriptase encoded by an endogenous retrovirus from Drosophila melanogaster F. Arnauda, E. Peyretailladeb, B. Dastuguea, C. Vaurya, a
b
INSERM U384, Faculty de Medecine, 28 Place Henri Dunant, 63000 Clermont-Ferrand, France De´partement Ge´nie Biologique, I.U.T. de Clermont-Ferrand, 100, rue de l’Egalite´, 15013 Aurillac Cedex
Received 17 November 2004; received in revised form 17 December 2004; accepted 27 December 2004
Abstract ZAM is an LTR-retrotransposon from Drosophila melanogaster that belongs to the genus errantivirus, viruses similar in structure and replication cycle to vertebrate retroviruses. A key component to its lifecycle is its reverse transcriptase which copies singlestranded genomic RNA into DNA. Here, we provide a detailed characterization of the enzymatic activities of the reverse transcriptase encoded by ZAM. When expressed in vitro, the reverse transcriptase domain associated with the RNase H domain encoded by the ZAM pol gene forms homodimers and displays an efficient RNA-dependent DNA-polymerase activity. It requires either Mg2+ or Mn2+ divalent cations, and works in basic pH, with a peak at around pH9. The so-called [RT-RH] polypeptide displays an optimal activity at 22 1C, a property that makes it well-adapted to the temperature of its host. This study contributes to our understanding of the general structures and functions of retroviral reverse transcriptases, a necessary process in the search for novel inhibitors. r 2005 Elsevier Ltd. All rights reserved. Keywords: Reverse transcriptase; Endogenous retrovirus; Errantivirus; Drosophila melanogaster; ZAM
1. Introduction LTR-retrotransposons are mobile genetic entities that replicate via reverse transcription of a genomic singlestrand RNA into an unintegrated double-strand linear DNA. This reaction is catalysed by a reverse transcriptase encoded by their pol gene. Sequence comparisons from their reverse transcriptase domains have split LTR-retrotransposons into two main groups: Ty1-copia and Ty3-gyspy. The two families are structurally distinguished by the order of the domains encoded by their pol gene: protease–reverse-transcriptase–integrase (PR–RT–IN) for the Ty3-gypsy group, and PR–IN–RT for the Ty1-copia group. Nevertheless, it should be noted that certain exceptions exist, including a group of Corresponding author. Tel.: +33 4 73 60 80 24; fax: +33 4 73 27 61 32. E-mail address: [email protected] (C. Vaury).
0965-1748/$ - see front matter r 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.ibmb.2004.12.008
deuterostome Ty3/gypsy-like retrotransposons with Ty1/copia-like pol-domain orders (Goodwin and Poulter, 2002). Although many steps in the lifecycle of these LTRretrotransposons, especially those which are readily amenable to genetic analysis, have been extensively studied, there have been few biochemical studies on the enzymes that support these events. Studies performed on retroviruses to which the Ty3-gypsy group is closely related have reported that reverse transcriptase is the product of a Gag-Pol polyprotein precursor which is subsequently cleaved by the Pol-encoded protease to yield the active form of the enzyme. Nevertheless, the subunit composition and organization of various retroviral reverse transcriptases are markedly different. As an example taken from studies performed on mammalian retroviruses, the murine leukaemia virus reverse transcriptase (MuLV RT) is functionally active—at least in solution—in a monomeric form and contains both DNA polymerase and RNase H domains in a single
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polypeptide (Roth et al., 1985). In contrast, Lentivirus reverse transcriptases such as human immunodeficiency virus (HIV-1 and HIV-2), feline immunodeficiency virus and equine infectious anaemia virus are only active as heterodimers. For HIV-1, two polypeptide chains, designated p66 and p51, are necessary (di Marzo Veronese et al., 1986; Fan et al., 1996; North et al., 1990; Rubinek et al., 1994). The p51 subunit is generated via endoproteolytic cleavage of the p66 subunit (di Marzo Veronese et al., 1986). The larger subunit (p66) contains both polymerase and RNase H activity, while the smaller subunit (p51) lacks the RNase H activity (Amacker and Hubscher, 1998; Kohlstaedt et al., 1992). There are few data available on the mechanisms of action of reverse transcriptases from LTR-retrotransposons, excepted those obtained in the course of studies performed on the yeast Ty1 and Ty3 reverse transcriptases (Cristofari et al., 1999; Lener et al., 2002; Wilhelm et al., 2000). We therefore aimed to elucidate the general characteristics of a reverse transcriptase encoded by an LTR-retrotransposon, ZAM, from Drosophila melanogaster (Leblanc et al., 1997). ZAM is very similar to vertebrate retroviruses in its structure and replication cycle. Its three open reading frames (ORFs), analogous in position and coding potential to the retroviral gag, pol and env genes, are flanked by two long terminal repeats (LTRs). Given these structural and functional similarities with mammalian retroviruses, especially the presence of an envelope gene, such elements have been called errantiviruses or insect endogenous retroviruses. Due to the structural and functional similarities of errantiviruses and retroviruses, their study has common and complementary objectives. One objective of the present study is to contribute to the search for novel inhibitors of reverse transcriptases. ZAM reverse transcriptase displays an YxDD box, a motif known to be the catalytic centre of reverse transcriptases and highly conserved between all the retroviruses. Together with retroviruses, ZAM also displays specific amino acids within the RNase H domain (Leblanc et al., 1997). In the present study, we prepared recombinant ZAM polypeptides potentially encoded by its pol gene, and analysed both the nucleoprotein complexes and enzymatic properties mediating their RNA-dependent DNApolymerase activity. In addition, we compared many of the biochemical features of ZAM reverse transcriptase with the equivalent features of the most extensively studied HIV-1 reverse transcriptase (Hizi et al., 1991).
DNA. The following oligonucleotides were designed: a 50 primer ZAM3267 (50 -GGATCCCA ATATTCTTATACACT-30 ), and a 30 primer ZAM4636 (50 GGATCCTTCGAAGTCTG GGTGTATG-30 ) or ZAM5350 (50 -GGATCCTA CGATTTCGTGGAGAAG-30 ). The additional BamH1 restriction site used for cloning is underlined. Primer pairs ZAM3267/ ZAM4636 and ZAM3267/ZAM5350 were used to amplify the [RT] and [RT-RH] sequences, respectively (see Fig. 1). The purified fragments were digested with BamH1 and ligated to the expression plasmid pGEX-5XT1 digested with BamH1, thus generating pGEX-ZAM[RT] and pGEX-ZAM-[RT-RH] vectors.
2. Materials and methods
The RNA-dependent DNA polymerase (RDDP) activity of the different polypeptides was assayed by monitoring the rates of poly(rA)n-oligo(dT)12–18directed incorporation of 32Pd[TTP]. RDDP activity was determined in a mixture with a final volume of 20 ml containing 50 mM Tris-HCL (pH7), 10 mM
2.1. Constructs Amplification of the reverse transcriptase-encoding regions of the ZAM pol gene was performed on genomic
2.2. In vitro expression of the fusion proteins Expression of both GST-RT and GST-RT-RH fusion proteins in Escherichia coli BL21 was induced with 0.1 mM IPTG (isopropyl-b-D-thiogalactopyranoside), and cells were grown at 37 1C for 4 h. The two bacterial pellets were resuspended in a lysis buffer. The cells were broken by sonication and both supernatants were incubated independently with 500 ml of glutathione (GSH)-agarose beads (Sigma) for 1 h. In parallel, a PCR product corresponding to ZAM-[RT-RH] was cloned in pGEMT easy vector (Promega) and the corresponding 35S-methioninelabelled polypeptide was produced in vitro using the Promega TNTs coupled reticulocyte lysate system. In addition, oligonucleotides were designed to produce the well-characterized HIV reverse transcriptase subunits in vitro, i.e. p51 (RT) and p66 (RT-RH); these are presented in Fig. 1. 2.3. In vitro pull-down assays Twenty microlitres of the supernatants containing the GST-RT and GST-RT-RH fusion proteins and crosslinked on agarose beads were incubated for 4 h at room temperature with 10 ml of the 35S-methionine-labelled ZAM-[RT-RH] protein. After three washes, the beads were loaded onto an SDS-PAGE gel and stained by coomassie, followed by an autoradiography to detect the 35S-methionine-labelled ZAM-[RT-RH] potentially retained. A GST protein fixed on beads was also used as a negative control. 2.4. Reverse transcriptase assays
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HHCC domain
HIV-1 pol:
RVP
RVT_1
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ZAM pol:
RVP
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rve
----- ---------
integrase
---------------- [RT]-------------------- [RT-RH] Nucleotides: 2795 3267
4636
5351
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Fig. 1. Comparative analysis of protein Pfam domains of the Pol polyprotein of ZAM LTR-retrotransposon (TrEMBL Accession number: O46115) and HIV-1 retrovirus (Swissprot Accession number P03366). This analysis allows to detect four Pfam homologous domains (boxes): Retroviral aspartyl protease (RVP), Reverse transcriptase (RVT_1), RNase (RNase H) and Integrase core domain (rve). Using these results with the zinc finger HHCC domain located at the NH2-terminal end of both integrase proteins and the well-characterized N- and C-terminal extremities of each protein of the HIV-1 Pol polyprotein: PR (protease), RT p51, RT p66 and integrase, we designed oligonucleotides (bold arrows) to, respectively, amplify RT and RT-RH genes of ZAM and HIV-1 (i.e. p51, p66). Nucleotide numbers according to ZAM and HIV-1 sequence are indicated below.
MgCl2, 1 mM dithiothreitol, 20 mM 32Pd[TTP], 0.5 mg of poly(rA)n-oligo(dT)1218, 0.05% (vol/vol) Triton X-100 and 2 mL of enzymes synthesized in a rabbit reticulocyte lysate (T7 TNT; Promega). The activities of ZAM-[RT], ZAM-[RT-RH] and a mixture of both were tested and compared to HIV-p51, HIV-p66 and the heterodimer HIVp51/p66. For the HIV heterodimer, two types of experiments were performed: one at 22 1C (temperature of flies) and one at 37 1C (body temperature of humans). Reaction buffers used to evaluate pH requirement contained 50 mM Tris-HCL at a pH ranging from 5 to 9. Reaction buffers used to evaluate magnesium requirement contained 50 mM Tris-HCL with a magnesium concentration ranging from 0 to 20 mM. Reverse transcriptase reactions were performed at room temperature (22 1C) for 1 h. Moreover, the manganese requirement was investigated with a Mn2+ concentration ranging from 0 to 10 mM in 50 mM of Tris-HCl pH7 buffer. Reactions evaluating temperature requirements contained 50 mM Tris-HCL buffer (pH7.5) and 10 mM MgCl2. Reactions were performed for 1 h at a temperature ranging from 4 to 42 1C. Samples were then heated at 70 1C for 15 min to inactivate the enzyme. Radioactivity was measured by scintillation counting before and after several washes to remove unspecific and
unincorporated radioactivity. The percentage of the remaining 32Pd[TTP] was determined for all reverse transcriptase reactions.
3. Results 3.1. ZAM-[RT-RH] forms dimers in vitro In order to analyse the properties of the ZAM reverse transcriptase, we accurately determined all the domains in the Pol polyprotein of ZAM in comparison with the HIV-1 Pol polyprotein by Pfam analyses (Bateman et al., 2004). The sensitivity of detection using the Pfam HMM database was increased by selecting the E-value cut-off (value: 3.0). This analysis allowed to characterize the protease (RVP), reverse transcriptase (RVT_1), RNase H (RNase H), and integrase (rve) domains (Fig. 1). Since the protease domain corresponds to the complete protease sequence, we were able to determine the N-terminal end of the RT domain and therefore define the oligonucleotide of its 50 end. Moreover, the N-terminal end of the HIV RNase H domain corresponds to the cleavage site between p51 and p66 subunits. This allowed us to determine the N-terminal end of the RNase H domain of ZAM predicted by Pfam analysis, and thereby design an oligonucleotide. The
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determination of the putative C-terminal end of ZAM[RT-RH] was based on the zinc finger HHCC domain of ZAM integrase. Based on this analysis, the functional domains in the polyprotein encoded by the pol gene of ZAM were delimited as follows: Protease domain 2795–3266; RT domain 3267–4636; RNase H domain 4637–5350; and Integrase domain 5351–6448 (Accession number AJ000387). Next, we aimed to determine whether the functional reverse transcriptase of ZAM acts as a homodimeric and/or heterodimeric enzyme. A system for ZAM reverse transcriptase expression in E. coli was developed. Retroviral reverse transcriptases have been shown to possess their RDDP function associated with their RNase H activity within a single polypeptide. Thus, a protein encompassing the reverse transcriptase and the RNase H domains from nucleotides 3267–5350 according to ZAM sequence was expressed in E. coli, and is referred to as GST-ZAM-[RT-RH] product (Fig. 2, lane 3, upper panel). Two main bands instead of one can be observed on the gel. This can be easily explained by the fact that chimeric proteins bearing the GST polypeptide
1
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66 45
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Fig. 2. Reverse transcriptase of ZAM forms homodimers and heterodimers in vitro. Glutatione-S-Transferase (GST) (lane 2), GST-ZAM-[RT-RH] (lane 3) and GST-ZAM-[RT] (lane 4) crosslinked on beads were incubated with an in vitro transcribed and translated peptide corresponding to 35S-methionine-labelled ZAM[RT-RH]. All reactions were analysed on SDS-PAGE electrophoresis followed by coomassie staining (upper panel) and an autoradiography to detect the 35S-labelled protein (lower panel). The GST alone was unable to retain the ZAM-[RT-RH] peptide (lane 2, lower panel), whereas GST-ZAM-[RT-RH] and GST-ZAM-[RT] fusion proteins (lanes 3 and 4, lower panel, respectively) retained 35S-labelled ZAM[RT-RH]. The input used for each reaction (10 ml) of in vitro translated and 35S-labelled ZAM-[RT-RH] peptide is shown in lane 1 with a coomassie staining (upper panel) followed by an autoradiography (lower panel). The molecular masses of proteins are indicated in kilodalton on the right.
are vulnerable to proteolytic cleavage. Indeed, such a cleavage is described as frequently occurring near the junction of the GST and the fused polypeptide. This proteolytic activity generates an approximately 30 kDa GST domain and separates the ZAM domain [RT-RH] from 97 to 66 kDa, a protein encompassing the GST domain was expressed in E. coli, and is referred to as the GST product of 30 kDa (Fig. 2, lane 2, upper panel). In parallel, a 35S-methionine-labelled ZAM-[RT-RH] was translated in vitro with the Promega TNTs coupled reticulocyte lysate system. GST pull-down experiments were then performed with these two series of proteins, i.e. ZAM-[RT-RH] fused to GST, and 35S-methionine-labelled ZAM-[RTRH]. Results showed that the fusion protein GSTZAM-[RT-RH] is able to retain the 35S-labelled ZAM[RT-RH] protein (Fig. 2, lane 3, lower panel) whereas the GST-protein alone is not (Fig. 2, lane 2, lower panel). This indicates that ZAM reverse transcriptase is able to dimerize as a homodimer [RT-RH] in vitro. The HIV-1 reverse transcriptase is composed of two subunits p51 and p66, the first resulting from the processing of the RNase H domain from the p66 fulllength polypeptide by the viral protease (di Marzo Veronese et al., 1986). These reverse transcriptases are able to form both homodimers p66/p66 and heterodimers p51/p66. In order to determine whether ZAM reverse transcriptase could also form heterodimers with a truncated form of the full-length protein, primers were designed to amplify the reverse transcriptase domain without the RNase H domain from nucleotide 3267 to 4636 (Fig. 2, lane 4, upper panel; and Materials and Methods). This truncated ZAM-[RT] subunit was produced as a GST-fusion protein, fixed on beads and used in GST pull-down experiments as described previously. Again, two main bands were detected on the gel resulting from the proteolytic cleavage of the GST polypeptide (Fig. 2, lane 4). However, since both products contain the full-length RT domain under the test, artefactual responses due to the presence or absence of GST are very unlikely to occur. GST pull-down experiments with 35S-methionine-labelled ZAM-[RTRH] showed that the GST-fusion protein ZAM-[RT] was able to interact with the 35S-labelled ZAM-[RT-RH] protein (Fig. 2, lane 4, lower panel). This experiment indicates that heterodimeric polypeptides bearing the reverse transcriptase domain of ZAM can to be formed in vitro, such as HIV or FIV. 3.2. ZAM-RT displays a high RNA-dependent DNApolymerase activity as RT-RH/RT-RH dimers To investigate the enzymatic RNA-dependent DNA-polymerase (RDDP) activity of this reverse transcriptase, we performed experiments with a polyr(A)n-oligo(dt)1218 used as a substrate (see Materials
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and Methods). Both ZAM-[RT-RH] and ZAM-[RT] proteins were produced in vitro using the Promega TNTs coupled reticulocyte lysate system, and then 35Smethionine-labelled (Fig. 3A, lanes 1 and 2). The ability of both ZAM-[RT-RH] and ZAM-[RT] to support a strand displacement and 32Pd[TTP] incorporation was assessed. Fig. 3B provides a graphic representation showing that ZAM-[RT] displays no RDDP activity (Fig. 3B lane1), whereas ZAM-[RT-RH] demonstrates 27% 32Pd[TTP] incorporation (Fig. 3B, lane2). When both subunits were put together, a significant decrease of reverse transcriptase activity from 27% to 15% was observed (Fig. 3B, lane3). This decrease of activity could easily be explained if inactive heterodimers ZAM-[RTRH]/[RT] formed and thus led to a lower amount of active ZAM-[RT-RH]/[RT-RH] homodimers.
kDa
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51
%OFTTP P32 REMAINING
35 30 25 20 15 10 5 0 1 (B)
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This result was compared with data obtained from similar experiments testing the RNA-dependent DNApolymerase activity of HIV-1. This function is known to act better when heterodimeric HIV1-[RT-RH/RT] peptides are formed (Hottiger and Hubscher, 1996). Both subunits p51 and p66 corresponding to the HIV1-[RT] peptide and HIV1-[RT-RH] peptides, respectively, were produced in vitro (Fig. 3A, lanes 3 and 4). Tests of activity presented in Fig. 3B (lanes 4, 5 and 6) were performed at 22 1C, a temperature that does not allow a comparison between the efficiency of the reverse transcriptases of ZAM and HIV1, but only allows a test of the influence of one or both polypeptides p51 and p66 on the polymerase activity. Results confirmed that HIV-p51 alone had no activity whereas HIV-p66 alone had a weak activity (Fig. 3, lanes 4 and 5). When both of them were put together, the RNA-dependent DNApolymerase activity was 2.5 times higher, indicating that the presence of both p51 and p66 is necessary for a maximal reverse transcriptase activity (Fig. 3B, lane 6). We verified that the RT activity of all these experiments was not brought by the free TNT mix independently of the synthesized reverse transcriptases. Enzymatic activity was ensured with the same amount of free TNT mix but no parasitic reverse transcriptase activity was detected (Fig. 3B, lane 7). These results indicate that ZAM reverse transcriptase demonstrates a maximal activity in its full-length form. Its lower activity when the [RT] is added to the reaction can be interpreted as a partial inhibition by an inactive heterodimer [RT-RH/RT] formation. This data should be taken together with the fact that no proteolytic processing of the full-length peptide potentially resulting from the cleavage of the RNase H domain by the ZAMencoded viral protease could be detected through in vitro assays (unpublished data).
7
3.3. Biochemical properties of ZAM [RT-RNase H] ZAM
HIV
Fig. 3. Functional characterization of ZAM reverse transcriptase. (A) ZAM-[RT], ZAM-[RT-RH], HIV p51 and HIV p66 sequences were cloned in pGEMT easy vector (Promega), and the corresponding 35Smethionine-labelled polypeptides were produced in vitro using the Promega TNTs coupled reticulocyte lysate system. Lane 1, ZAM[RT]; lane 2, ZAM-[RT-RH]; lane 3, HIV p51; lane 4, HIV p66. The proteins were analysed by SDS-PAGE electrophoresis followed by autoradiography. The molecular masses of protein standards are indicated in kilodalton on the left. (B) The RNA-dependent DNApolymerase activity of reverse transcriptases was determined as described in the Materials and Methods section. The activities of ZAM-[RT], ZAM-[RT-RH], and ZAM-[RT-RH/RT] (lanes 1–3) were assayed and compared to HIV-p51, HIV-p66 and the heterodimer HIV-p51/p66 (lanes 4–6, respectively). Each reaction was incubated at room temperature and the percentage of remaining 32Pd[TTP] was calculated to determine enzyme activity. A negative control was performed with fresh TNTs coupled reticulocyte lysate mix to ensure the absence of parasite RNA-dependent DNA-polymerase activity (lane 7). Each experiment was performed in triplicate, and standard deviations were calculated.
To go further in the characterization of ZAM reverse transcriptase, its activity was investigated in relation to three different parameters: divalent cations, pH, and temperature. 3.3.1. Divalent cations It has been previously demonstrated that reverse transcriptase is dependent on the presence of divalent cations, most commonly magnesium, but MLV RT displays optimal activity in the presence of manganese (Verma, 1975). The requirement of divalent cations Mg2+ and Mn2+ for ZAM reverse transcriptase was therefore investigated. First, RNA-dependent DNApolymerase activity was performed with an Mg2+ concentration ranging from 0 to 20 mM. Results presented in Fig. 4A (left) show that in the absence of divalent cations (0 mM), ZAM reverse transcriptase is
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completely inactive. The presence of 1 mM of Mg2+ was enough to promote an efficient DNA-polymerase activity that increased at 2 mM of Mg2+, and remained comparable at Mg2+ concentrations of up to 10 mM. Higher concentrations such as 20 mM of Mg2+ gave rise to a weaker reverse transcriptase activity. A similar experiment was performed with a buffer containing Mn2+ cations ranging from 0 to 10 mM (Fig. 4A, right). We observed that the RNA-dependent DNApolymerase activity of ZAM [RT] was higher at weak Mn2+ cation concentrations, reaching a maximum at 1 and 2 mM, and then decreasing at 5 and 10 mM. These experiments demonstrate the requirement of divalent cations for ZAM reverse transcriptase activity with a maximal activity at 10 mM Mg2+, or at only 2 mM Mn2+. Furthermore, the highest RT activity was observed with Mn2+ divalent cation and in a narrower window of concentration (1–2 mM for Mn2+ compared to 2–10 mM for Mg2+).
3.3.2. pH Results presented in Fig. 4B show the effect of pH on ZAM-[RT-RH] RNA-dependent DNA-polymerase activity. ZAM-[RT-RH] exhibits a low activity at acidic pH (3–5). This activity increased from pH 3 to 7, reaching an optimum at pH9 and then decreasing at pH10.5. Thus, ZAM-[RT-RH] works better at basic pH than at acidic pH with an optimum activity at pH9.
3.3.3. Temperature We aimed to determine the extent to which processivity could be influenced by temperature. Several assays were performed at reaction temperatures varying from 0 to 45 1C (Fig. 4C). ZAM-[RT-RH] showed a high reverse transcriptase activity at 22 1C, which corresponds to the temperature of its host Drosophila melanogaster. This property was compared with that of the wellstudied heterodimeric reverse transcriptase of HIV-1 that also shows a three-fold increase of activity at a specific temperature, i.e. 37 1C, the temperature of its host (Fig. 4C).
4. Discussion The last decade has seen a major effort devoted to the research of the reverse transcriptases of HIV-1 and HIV2, the viruses responsible for AIDS, because most of the drugs approved to date for the treatment of this disease are inhibitors of the viral reverse transcriptases. More recently, with the appearance of drug-resistant HIV variants, the molecular, structural and catalytic properties of other retroviral reverse transcriptases have become the focus of numerous recent studies aiming to gain a fuller understanding of the general structures and functions of these enzymatic activities, and then to develop novel potent and specific reverse transcriptase inhibitors. However, very few studies to date have addressed the catalytic activities of reverse transcriptases encoded by closely related retroviruses called errantiviruses and found in insect genomes, despite the fact that they display considerable sequence homology with reverse transcriptases encoded by their mammalian counterparts (Wilhelm and Wilhelm, 2001; Xiong and Eickbush, 1988). In order to explore the properties of a reverse transcriptase encoded by an errantivirus from Drosophila melanogaster, we expressed an active form of a recombinant ZAM reverse transcriptase, and analysed its biochemical characteristics. Most lentiviral reverse transcriptases studied to date exhibit a dimeric structure of asymmetrically organized subunits. However, data obtained with reverse transcriptases purified from viruses such as MLV or MMTV support the possibility of a monomeric reverse transcriptase organization (Taube et al., 1998; Verma, 1975). In the present report, we show that the reverse transcriptase and the RNase H domains encoded by ZAM are both necessary in a single polypeptide for a DNA-polymerase activity. If a C-terminal deletion affecting the RNase H domain is introduced, the amount of reverse transcription is abolished. This transcription is partially recovered when the two peptides ZAM-[RT-RH] and ZAM-[RT] are put together for the test. When ZAM-[RT-RH] is the only peptide used in the reaction, the transcription is then maximal. This result suggests that ZAM-[RT] peptides act as dominant negative components counteracting the
Fig. 4. Cation, pH and temperature requirements of the RNA-dependent DNA-polymerase activity of ZAM reverse transcriptase. The RNAdependent DNA-polymerase activity of ZAM-[RT-RH] was determined by calculating the percentage of remaining 32Pd[TTP]. (A) Reactions were performed in a Tris-HCl buffer (pH7) and incubated for 1 h at room temperature (22 1C) with a concentration ranging from 0 to 20 mM of Mg2+ or 0 to 10 mM of Mn2+. The optimum concentrations of reverse transcriptase activity in the presence of Mg2+ and Mn2+ are indicated by arrows. (B) The RNA-dependent DNA-polymerase reactions of ZAM-[RT-RH] were performed in a Tris-HCl buffer (10 mM of Mg2+) with a pH ranging from 3 to 10.5, and incubated for 1 h at room temperature (22 1C). (C) The RNA-dependent DNA-polymerase reactions of ZAM-[RT-RH] were performed at 10 mM Mg2+ concentration in a Tris-HCl buffer (pH7), and incubated at the following temperatures: 0, 22, 37 and 45 1C, as indicated below the graph. Furthermore, HIV-p51/p66 heterodimer was assayed in the same buffer and reactions were performed at Drosophila temperature (22 1C) and human body temperature (37 1C). Each experiment was performed in triplicate, and standard deviations were calculated. A negative control was performed for each experiment with fresh TNTs coupled reticulocyte lysate mix to ensure the absence of parasite RNA-dependent DNA-polymerase activity within the medium (data not shown).
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full activity of ZAM-[RT-RH] peptides. It also indicates that ZAM-[RT-RH] and ZAM-[RT] can potentially interact through their RT domain and provides evidence for an active form of the ZAM reverse transcriptase as ZAM-[RT-RH/RT-RH] homodimers.
Similarly to all other retroviral reverse transcriptases, ZAM-[RT-RH] needs divalent cations for its activity. It has been shown that most retroviral reverse transcriptases have a marked preference for Mg2+ over Mn2+. Here, we show that ZAM-[RT-RH] displays a good
Mg2+
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efficiency in the presence of Mg2+, although the highest efficiency was detected with Mn2+ in a very narrow window of low concentration (1–2 mM). For HIV, Mg2+ has been found to have an effect on dimerization with an increased amount of dimers and activity related to an increased MgCl2 concentration (Cabodevilla et al., 2001). From this angle, the effect of Mg2+ and Mn2+ might be indirect and play a role in the association of ZAM-[RT-RH] rather than acting directly on its reverse transcriptase activity. ZAM reverse transcriptase was efficient at basic pH with a major activity detected at pH9, which was slightly higher than the optimum pH of 7.5 required for the DNA-polymerase activity of HIV-1 RT or MMTV RT (Hizi et al., 1991; Taube et al., 1998). Lastly, we analysed the temperature dependence of ZAM-[RT-RH]. Our data showed that ZAM reverse transcriptase activity was highly temperature-sensitive, with a maximal incorporation of labelled desoxynucleotides observed at its host temperature 22 1C. An increase in temperature severely affected the amount of reverse transcribed RNA, with a very low amount of reverse transcription observed at 37 1C, almost absent at 45 1C. Host-temperature-dependent reverse transcriptase regulation has been previously demonstrated for other insect reverse transcriptases (Ivanov et al., 1991; Lescault et al., 1989.; Pyatkov et al., 2004), as well as for HIV reverse transcriptase. For this latter, Huang et al. (1998) demonstrated that a temperature lower than 37 1C gives rise to a decrease in HIV reverse transcriptase activity, a decrease attributed to a reduced dimerization process (Cabodevilla et al., 2001). For ZAM reverse transcriptase, a similar mechanism could certainly be considered, with a decrease of dimerization at 37 1C rather than 22 1C. These data indicate that ZAM reverse transcriptase is well-adapted to the temperature of its host, and although expressed in vitro the polypeptide displays an optimal temperature sensitivity corresponding to the conditions necessary for a replication process occurring in vivo. In vivo, the reverse transcription of viral genomic RNA occurs inside the viral capsid. The influence of all the tested parameters in this study was investigated on ZAM reverse transcriptase in vitro, and these experiments did not take into consideration the micro-environment yielded by the core structure of viral particles. However, there is still no reconstituted system recapitulating events in vivo, our results argue for an active homodimeric form of ZAM-[RT-RH] in vivo, its need for divalent cations, and its specific temperature dependence.
Acknowledgements We thank the members of the laboratory for their help, comments and encouragement. This work was
supported by grants from the INSERM (U384) and the CNRS (GDR2157), and by a project grant from the Association pour la Recherche contre le Cancer (ARC 3441) to C.V. F.A. received a grant from the Ministe`re de l’Enseignement Supe´rieur et de la Recherche (MESR), and the Fondation Recherche Me´dicale (FRM).
References Amacker, M., Hubscher, U., 1998. Chimeric HIV-1 and feline immunodeficiency virus reverse transcriptases: critical role of the p51 subunit in the structural integrity of heterodimeric lentiviral DNA polymerases. J. Mol. Biol. 278, 757–765. Bateman, A., Coin, L., Durbin, R., Finn, R.D., Hollich, V., GriffithsJones, S., Khanna, A., Marshall, M., Moxon, S., Sonnhammer, E.L., Studholme, D.J., Yeats, C., Eddy, S.R., 2004. The Pfam protein families database. Nucleic Acids Res. 32 (Database issue), D138–D141. Cabodevilla, J.F., Odriozola, L., Santiago, E., Martinez-Irujo, J.J., 2001. Factors affecting the dimerization of the p66 form of HIV-1 reverse transcriptase. Eur. J. Biochem. 268, 1163–1172. Cristofari, G., Gabus, C., Ficheux, D., Bona, M., Le Grice, S.F., Darlix, J.L., 1999. Characterization of active reverse transcriptase and nucleoprotein complexes of the yeast retrotransposon Ty3 in vitro. J. Biol. Chem. 274, 36643–36648. di Marzo Veronese, F., Copeland, T.D., DeVico, A.L., Rahman, R., Oroszlan, S., Gallo, R.C., Sarngadharan, M.G., 1986. Characterization of highly immunogenic p66/p51 as the reverse transcriptase of HTLV-III/LAV. Science 231, 1289–1291. Fan, N., Rank, K.B., Poppe, S.M., Tarpley, W.G., Sharma, S.K., 1996. Characterization of the p68/p58 heterodimer of human immunodeficiency virus type 2 reverse transcriptase. Biochemistry 35, 1911–1917. Goodwin, T.J., Poulter, R.T., 2002. A group of deuterostome Ty3/ gypsy-like retrotransposons with Ty1/ copia-like pol-domain orders. Mol. Genet. Genomics 267, 481–491 (Epub 2002 May 2023). Hizi, A., Tal, R., Shaharabany, M., Loya, S., 1991. Catalytic properties of the reverse transcriptases of human immunodeficiency viruses type 1 and type 2. J. Biol. Chem. 266, 6230–6239. Hottiger, M., Hubscher, U., 1996. Human immunodeficiency virus type 1 reverse transcriptase. Biol. Chem. Hoppe Seyler 377, 97–120. Huang, M., Zensen, R., Cho, M., Martin, M.A., 1998. Construction and characterization of a temperature-sensitive human immunodeficiency virus type 1 reverse transcriptase mutant. J. Virol. 72, 2047–2054. Ivanov, V.A., Melnikov, A.A., Siunov, A.V., Fodor II, Ilyin, Y.V., 1991. Authentic reverse transcriptase is coded by jockey, a mobile Drosophila element related to mammalian LINEs. Embo J. 10, 2489–2495. Kohlstaedt, L.A., Wang, J., Friedman, J.M., Rice, P.A., Steitz, T.A., 1992. Crystal structure at 3.5 A resolution of HIV-1 reverse transcriptase complexed with an inhibitor. Science 256, 1783–1790. Leblanc, P., Desset, S., Dastugue, B., Vaury, C., 1997. Invertebrate retroviruses: ZAM a new candidate in D.melanogaster. Embo J. 16, 7521–7531. Lener, D., Budihas, S.R., Le Grice, S.F., 2002. Mutating conserved residues in the ribonuclease H domain of Ty3 reverse transcriptase affects specialized cleavage events. J. Biol. Chem. 277, 26486–26495 (Epub 22002 May 26486). Lescault, A., Becker, J.L., Barre-Sinoussi, F., Chermann, J.C., BestBelpomme, M., Ono, K., 1989. Characterization of a reverse
ARTICLE IN PRESS F. Arnaud et al. / Insect Biochemistry and Molecular Biology 35 (2005) 323–331 transcriptase activity associated with retrovirus-like particles in a Drosophila cell line. Cell Mol. Biol. 35, 163–171. North, T.W., Cronn, R.C., Remington, K.M., Tandberg, R.T., Judd, R.C., 1990. Characterization of reverse transcriptase from feline immunodeficiency virus. J. Biol. Chem. 265, 5121–5128. Pyatkov, K.I., Arkhipova, I.R., Malkova, N.V., Finnegan, D.J., Evgen’ev, M.B., 2004. Reverse transcriptase and endonuclease activities encoded by Penelope-like retroelements. Proc. Natl. Acad. Sci. U. S. A. 101, 14719–14724 (Epub 12004 Oct 14701). Roth, M.J., Tanese, N., Goff, S.P., 1985. Purification and characterization of murine retroviral reverse transcriptase expressed in Escherichia coli. J. Biol. Chem. 260, 9326–9335. Rubinek, T., Loya, S., Shaharabany, M., Hughes, S.H., Clark, P.K., Hizi, A., 1994. The catalytic properties of the reverse transcriptase of the lentivirus equine infectious anemia virus. Eur. J. Biochem. 219, 977–983. Taube, R., Loya, S., Avidan, O., Perach, M., Hizi, A., 1998. Reverse transcriptase of mouse mammary tumour virus: expression in
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Functional characteristics of a reverse transcriptase encoded by an endogenous retrovirus from Drosophila melanogaster F. Arnauda, E. Peyretailladeb, B. Dastuguea, C. Vaurya, a
b
INSERM U384, Faculty de Medecine, 28 Place Henri Dunant, 63000 Clermont-Ferrand, France De´partement Ge´nie Biologique, I.U.T. de Clermont-Ferrand, 100, rue de l’Egalite´, 15013 Aurillac Cedex
Received 17 November 2004; received in revised form 17 December 2004; accepted 27 December 2004
Abstract ZAM is an LTR-retrotransposon from Drosophila melanogaster that belongs to the genus errantivirus, viruses similar in structure and replication cycle to vertebrate retroviruses. A key component to its lifecycle is its reverse transcriptase which copies singlestranded genomic RNA into DNA. Here, we provide a detailed characterization of the enzymatic activities of the reverse transcriptase encoded by ZAM. When expressed in vitro, the reverse transcriptase domain associated with the RNase H domain encoded by the ZAM pol gene forms homodimers and displays an efficient RNA-dependent DNA-polymerase activity. It requires either Mg2+ or Mn2+ divalent cations, and works in basic pH, with a peak at around pH9. The so-called [RT-RH] polypeptide displays an optimal activity at 22 1C, a property that makes it well-adapted to the temperature of its host. This study contributes to our understanding of the general structures and functions of retroviral reverse transcriptases, a necessary process in the search for novel inhibitors. r 2005 Elsevier Ltd. All rights reserved. Keywords: Reverse transcriptase; Endogenous retrovirus; Errantivirus; Drosophila melanogaster; ZAM
1. Introduction LTR-retrotransposons are mobile genetic entities that replicate via reverse transcription of a genomic singlestrand RNA into an unintegrated double-strand linear DNA. This reaction is catalysed by a reverse transcriptase encoded by their pol gene. Sequence comparisons from their reverse transcriptase domains have split LTR-retrotransposons into two main groups: Ty1-copia and Ty3-gyspy. The two families are structurally distinguished by the order of the domains encoded by their pol gene: protease–reverse-transcriptase–integrase (PR–RT–IN) for the Ty3-gypsy group, and PR–IN–RT for the Ty1-copia group. Nevertheless, it should be noted that certain exceptions exist, including a group of Corresponding author. Tel.: +33 4 73 60 80 24; fax: +33 4 73 27 61 32. E-mail address: [email protected] (C. Vaury).
0965-1748/$ - see front matter r 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.ibmb.2004.12.008
deuterostome Ty3/gypsy-like retrotransposons with Ty1/copia-like pol-domain orders (Goodwin and Poulter, 2002). Although many steps in the lifecycle of these LTRretrotransposons, especially those which are readily amenable to genetic analysis, have been extensively studied, there have been few biochemical studies on the enzymes that support these events. Studies performed on retroviruses to which the Ty3-gypsy group is closely related have reported that reverse transcriptase is the product of a Gag-Pol polyprotein precursor which is subsequently cleaved by the Pol-encoded protease to yield the active form of the enzyme. Nevertheless, the subunit composition and organization of various retroviral reverse transcriptases are markedly different. As an example taken from studies performed on mammalian retroviruses, the murine leukaemia virus reverse transcriptase (MuLV RT) is functionally active—at least in solution—in a monomeric form and contains both DNA polymerase and RNase H domains in a single
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polypeptide (Roth et al., 1985). In contrast, Lentivirus reverse transcriptases such as human immunodeficiency virus (HIV-1 and HIV-2), feline immunodeficiency virus and equine infectious anaemia virus are only active as heterodimers. For HIV-1, two polypeptide chains, designated p66 and p51, are necessary (di Marzo Veronese et al., 1986; Fan et al., 1996; North et al., 1990; Rubinek et al., 1994). The p51 subunit is generated via endoproteolytic cleavage of the p66 subunit (di Marzo Veronese et al., 1986). The larger subunit (p66) contains both polymerase and RNase H activity, while the smaller subunit (p51) lacks the RNase H activity (Amacker and Hubscher, 1998; Kohlstaedt et al., 1992). There are few data available on the mechanisms of action of reverse transcriptases from LTR-retrotransposons, excepted those obtained in the course of studies performed on the yeast Ty1 and Ty3 reverse transcriptases (Cristofari et al., 1999; Lener et al., 2002; Wilhelm et al., 2000). We therefore aimed to elucidate the general characteristics of a reverse transcriptase encoded by an LTR-retrotransposon, ZAM, from Drosophila melanogaster (Leblanc et al., 1997). ZAM is very similar to vertebrate retroviruses in its structure and replication cycle. Its three open reading frames (ORFs), analogous in position and coding potential to the retroviral gag, pol and env genes, are flanked by two long terminal repeats (LTRs). Given these structural and functional similarities with mammalian retroviruses, especially the presence of an envelope gene, such elements have been called errantiviruses or insect endogenous retroviruses. Due to the structural and functional similarities of errantiviruses and retroviruses, their study has common and complementary objectives. One objective of the present study is to contribute to the search for novel inhibitors of reverse transcriptases. ZAM reverse transcriptase displays an YxDD box, a motif known to be the catalytic centre of reverse transcriptases and highly conserved between all the retroviruses. Together with retroviruses, ZAM also displays specific amino acids within the RNase H domain (Leblanc et al., 1997). In the present study, we prepared recombinant ZAM polypeptides potentially encoded by its pol gene, and analysed both the nucleoprotein complexes and enzymatic properties mediating their RNA-dependent DNApolymerase activity. In addition, we compared many of the biochemical features of ZAM reverse transcriptase with the equivalent features of the most extensively studied HIV-1 reverse transcriptase (Hizi et al., 1991).
DNA. The following oligonucleotides were designed: a 50 primer ZAM3267 (50 -GGATCCCA ATATTCTTATACACT-30 ), and a 30 primer ZAM4636 (50 GGATCCTTCGAAGTCTG GGTGTATG-30 ) or ZAM5350 (50 -GGATCCTA CGATTTCGTGGAGAAG-30 ). The additional BamH1 restriction site used for cloning is underlined. Primer pairs ZAM3267/ ZAM4636 and ZAM3267/ZAM5350 were used to amplify the [RT] and [RT-RH] sequences, respectively (see Fig. 1). The purified fragments were digested with BamH1 and ligated to the expression plasmid pGEX-5XT1 digested with BamH1, thus generating pGEX-ZAM[RT] and pGEX-ZAM-[RT-RH] vectors.
2. Materials and methods
The RNA-dependent DNA polymerase (RDDP) activity of the different polypeptides was assayed by monitoring the rates of poly(rA)n-oligo(dT)12–18directed incorporation of 32Pd[TTP]. RDDP activity was determined in a mixture with a final volume of 20 ml containing 50 mM Tris-HCL (pH7), 10 mM
2.1. Constructs Amplification of the reverse transcriptase-encoding regions of the ZAM pol gene was performed on genomic
2.2. In vitro expression of the fusion proteins Expression of both GST-RT and GST-RT-RH fusion proteins in Escherichia coli BL21 was induced with 0.1 mM IPTG (isopropyl-b-D-thiogalactopyranoside), and cells were grown at 37 1C for 4 h. The two bacterial pellets were resuspended in a lysis buffer. The cells were broken by sonication and both supernatants were incubated independently with 500 ml of glutathione (GSH)-agarose beads (Sigma) for 1 h. In parallel, a PCR product corresponding to ZAM-[RT-RH] was cloned in pGEMT easy vector (Promega) and the corresponding 35S-methioninelabelled polypeptide was produced in vitro using the Promega TNTs coupled reticulocyte lysate system. In addition, oligonucleotides were designed to produce the well-characterized HIV reverse transcriptase subunits in vitro, i.e. p51 (RT) and p66 (RT-RH); these are presented in Fig. 1. 2.3. In vitro pull-down assays Twenty microlitres of the supernatants containing the GST-RT and GST-RT-RH fusion proteins and crosslinked on agarose beads were incubated for 4 h at room temperature with 10 ml of the 35S-methionine-labelled ZAM-[RT-RH] protein. After three washes, the beads were loaded onto an SDS-PAGE gel and stained by coomassie, followed by an autoradiography to detect the 35S-methionine-labelled ZAM-[RT-RH] potentially retained. A GST protein fixed on beads was also used as a negative control. 2.4. Reverse transcriptase assays
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325
HHCC domain
HIV-1 pol:
RVP
RVT_1
RNaseH
rve
RT p51
RT
RT p66 Nucleotides:
1799
2065
integrase 3415
3775
4639
HHCC domain
ZAM pol:
RVP
RVT_1
RNaseH
rve
----- ---------
integrase
---------------- [RT]-------------------- [RT-RH] Nucleotides: 2795 3267
4636
5351
6448
Fig. 1. Comparative analysis of protein Pfam domains of the Pol polyprotein of ZAM LTR-retrotransposon (TrEMBL Accession number: O46115) and HIV-1 retrovirus (Swissprot Accession number P03366). This analysis allows to detect four Pfam homologous domains (boxes): Retroviral aspartyl protease (RVP), Reverse transcriptase (RVT_1), RNase (RNase H) and Integrase core domain (rve). Using these results with the zinc finger HHCC domain located at the NH2-terminal end of both integrase proteins and the well-characterized N- and C-terminal extremities of each protein of the HIV-1 Pol polyprotein: PR (protease), RT p51, RT p66 and integrase, we designed oligonucleotides (bold arrows) to, respectively, amplify RT and RT-RH genes of ZAM and HIV-1 (i.e. p51, p66). Nucleotide numbers according to ZAM and HIV-1 sequence are indicated below.
MgCl2, 1 mM dithiothreitol, 20 mM 32Pd[TTP], 0.5 mg of poly(rA)n-oligo(dT)1218, 0.05% (vol/vol) Triton X-100 and 2 mL of enzymes synthesized in a rabbit reticulocyte lysate (T7 TNT; Promega). The activities of ZAM-[RT], ZAM-[RT-RH] and a mixture of both were tested and compared to HIV-p51, HIV-p66 and the heterodimer HIVp51/p66. For the HIV heterodimer, two types of experiments were performed: one at 22 1C (temperature of flies) and one at 37 1C (body temperature of humans). Reaction buffers used to evaluate pH requirement contained 50 mM Tris-HCL at a pH ranging from 5 to 9. Reaction buffers used to evaluate magnesium requirement contained 50 mM Tris-HCL with a magnesium concentration ranging from 0 to 20 mM. Reverse transcriptase reactions were performed at room temperature (22 1C) for 1 h. Moreover, the manganese requirement was investigated with a Mn2+ concentration ranging from 0 to 10 mM in 50 mM of Tris-HCl pH7 buffer. Reactions evaluating temperature requirements contained 50 mM Tris-HCL buffer (pH7.5) and 10 mM MgCl2. Reactions were performed for 1 h at a temperature ranging from 4 to 42 1C. Samples were then heated at 70 1C for 15 min to inactivate the enzyme. Radioactivity was measured by scintillation counting before and after several washes to remove unspecific and
unincorporated radioactivity. The percentage of the remaining 32Pd[TTP] was determined for all reverse transcriptase reactions.
3. Results 3.1. ZAM-[RT-RH] forms dimers in vitro In order to analyse the properties of the ZAM reverse transcriptase, we accurately determined all the domains in the Pol polyprotein of ZAM in comparison with the HIV-1 Pol polyprotein by Pfam analyses (Bateman et al., 2004). The sensitivity of detection using the Pfam HMM database was increased by selecting the E-value cut-off (value: 3.0). This analysis allowed to characterize the protease (RVP), reverse transcriptase (RVT_1), RNase H (RNase H), and integrase (rve) domains (Fig. 1). Since the protease domain corresponds to the complete protease sequence, we were able to determine the N-terminal end of the RT domain and therefore define the oligonucleotide of its 50 end. Moreover, the N-terminal end of the HIV RNase H domain corresponds to the cleavage site between p51 and p66 subunits. This allowed us to determine the N-terminal end of the RNase H domain of ZAM predicted by Pfam analysis, and thereby design an oligonucleotide. The
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determination of the putative C-terminal end of ZAM[RT-RH] was based on the zinc finger HHCC domain of ZAM integrase. Based on this analysis, the functional domains in the polyprotein encoded by the pol gene of ZAM were delimited as follows: Protease domain 2795–3266; RT domain 3267–4636; RNase H domain 4637–5350; and Integrase domain 5351–6448 (Accession number AJ000387). Next, we aimed to determine whether the functional reverse transcriptase of ZAM acts as a homodimeric and/or heterodimeric enzyme. A system for ZAM reverse transcriptase expression in E. coli was developed. Retroviral reverse transcriptases have been shown to possess their RDDP function associated with their RNase H activity within a single polypeptide. Thus, a protein encompassing the reverse transcriptase and the RNase H domains from nucleotides 3267–5350 according to ZAM sequence was expressed in E. coli, and is referred to as GST-ZAM-[RT-RH] product (Fig. 2, lane 3, upper panel). Two main bands instead of one can be observed on the gel. This can be easily explained by the fact that chimeric proteins bearing the GST polypeptide
1
2
3
4
kDa 97
66 45
30
35S
ZAM-[RT-RH]
66
Fig. 2. Reverse transcriptase of ZAM forms homodimers and heterodimers in vitro. Glutatione-S-Transferase (GST) (lane 2), GST-ZAM-[RT-RH] (lane 3) and GST-ZAM-[RT] (lane 4) crosslinked on beads were incubated with an in vitro transcribed and translated peptide corresponding to 35S-methionine-labelled ZAM[RT-RH]. All reactions were analysed on SDS-PAGE electrophoresis followed by coomassie staining (upper panel) and an autoradiography to detect the 35S-labelled protein (lower panel). The GST alone was unable to retain the ZAM-[RT-RH] peptide (lane 2, lower panel), whereas GST-ZAM-[RT-RH] and GST-ZAM-[RT] fusion proteins (lanes 3 and 4, lower panel, respectively) retained 35S-labelled ZAM[RT-RH]. The input used for each reaction (10 ml) of in vitro translated and 35S-labelled ZAM-[RT-RH] peptide is shown in lane 1 with a coomassie staining (upper panel) followed by an autoradiography (lower panel). The molecular masses of proteins are indicated in kilodalton on the right.
are vulnerable to proteolytic cleavage. Indeed, such a cleavage is described as frequently occurring near the junction of the GST and the fused polypeptide. This proteolytic activity generates an approximately 30 kDa GST domain and separates the ZAM domain [RT-RH] from 97 to 66 kDa, a protein encompassing the GST domain was expressed in E. coli, and is referred to as the GST product of 30 kDa (Fig. 2, lane 2, upper panel). In parallel, a 35S-methionine-labelled ZAM-[RT-RH] was translated in vitro with the Promega TNTs coupled reticulocyte lysate system. GST pull-down experiments were then performed with these two series of proteins, i.e. ZAM-[RT-RH] fused to GST, and 35S-methionine-labelled ZAM-[RTRH]. Results showed that the fusion protein GSTZAM-[RT-RH] is able to retain the 35S-labelled ZAM[RT-RH] protein (Fig. 2, lane 3, lower panel) whereas the GST-protein alone is not (Fig. 2, lane 2, lower panel). This indicates that ZAM reverse transcriptase is able to dimerize as a homodimer [RT-RH] in vitro. The HIV-1 reverse transcriptase is composed of two subunits p51 and p66, the first resulting from the processing of the RNase H domain from the p66 fulllength polypeptide by the viral protease (di Marzo Veronese et al., 1986). These reverse transcriptases are able to form both homodimers p66/p66 and heterodimers p51/p66. In order to determine whether ZAM reverse transcriptase could also form heterodimers with a truncated form of the full-length protein, primers were designed to amplify the reverse transcriptase domain without the RNase H domain from nucleotide 3267 to 4636 (Fig. 2, lane 4, upper panel; and Materials and Methods). This truncated ZAM-[RT] subunit was produced as a GST-fusion protein, fixed on beads and used in GST pull-down experiments as described previously. Again, two main bands were detected on the gel resulting from the proteolytic cleavage of the GST polypeptide (Fig. 2, lane 4). However, since both products contain the full-length RT domain under the test, artefactual responses due to the presence or absence of GST are very unlikely to occur. GST pull-down experiments with 35S-methionine-labelled ZAM-[RTRH] showed that the GST-fusion protein ZAM-[RT] was able to interact with the 35S-labelled ZAM-[RT-RH] protein (Fig. 2, lane 4, lower panel). This experiment indicates that heterodimeric polypeptides bearing the reverse transcriptase domain of ZAM can to be formed in vitro, such as HIV or FIV. 3.2. ZAM-RT displays a high RNA-dependent DNApolymerase activity as RT-RH/RT-RH dimers To investigate the enzymatic RNA-dependent DNA-polymerase (RDDP) activity of this reverse transcriptase, we performed experiments with a polyr(A)n-oligo(dt)1218 used as a substrate (see Materials
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and Methods). Both ZAM-[RT-RH] and ZAM-[RT] proteins were produced in vitro using the Promega TNTs coupled reticulocyte lysate system, and then 35Smethionine-labelled (Fig. 3A, lanes 1 and 2). The ability of both ZAM-[RT-RH] and ZAM-[RT] to support a strand displacement and 32Pd[TTP] incorporation was assessed. Fig. 3B provides a graphic representation showing that ZAM-[RT] displays no RDDP activity (Fig. 3B lane1), whereas ZAM-[RT-RH] demonstrates 27% 32Pd[TTP] incorporation (Fig. 3B, lane2). When both subunits were put together, a significant decrease of reverse transcriptase activity from 27% to 15% was observed (Fig. 3B, lane3). This decrease of activity could easily be explained if inactive heterodimers ZAM-[RTRH]/[RT] formed and thus led to a lower amount of active ZAM-[RT-RH]/[RT-RH] homodimers.
kDa
1
2
3
4
80 66 (A)
51
%OFTTP P32 REMAINING
35 30 25 20 15 10 5 0 1 (B)
2
3
4
5
6
327
This result was compared with data obtained from similar experiments testing the RNA-dependent DNApolymerase activity of HIV-1. This function is known to act better when heterodimeric HIV1-[RT-RH/RT] peptides are formed (Hottiger and Hubscher, 1996). Both subunits p51 and p66 corresponding to the HIV1-[RT] peptide and HIV1-[RT-RH] peptides, respectively, were produced in vitro (Fig. 3A, lanes 3 and 4). Tests of activity presented in Fig. 3B (lanes 4, 5 and 6) were performed at 22 1C, a temperature that does not allow a comparison between the efficiency of the reverse transcriptases of ZAM and HIV1, but only allows a test of the influence of one or both polypeptides p51 and p66 on the polymerase activity. Results confirmed that HIV-p51 alone had no activity whereas HIV-p66 alone had a weak activity (Fig. 3, lanes 4 and 5). When both of them were put together, the RNA-dependent DNApolymerase activity was 2.5 times higher, indicating that the presence of both p51 and p66 is necessary for a maximal reverse transcriptase activity (Fig. 3B, lane 6). We verified that the RT activity of all these experiments was not brought by the free TNT mix independently of the synthesized reverse transcriptases. Enzymatic activity was ensured with the same amount of free TNT mix but no parasitic reverse transcriptase activity was detected (Fig. 3B, lane 7). These results indicate that ZAM reverse transcriptase demonstrates a maximal activity in its full-length form. Its lower activity when the [RT] is added to the reaction can be interpreted as a partial inhibition by an inactive heterodimer [RT-RH/RT] formation. This data should be taken together with the fact that no proteolytic processing of the full-length peptide potentially resulting from the cleavage of the RNase H domain by the ZAMencoded viral protease could be detected through in vitro assays (unpublished data).
7
3.3. Biochemical properties of ZAM [RT-RNase H] ZAM
HIV
Fig. 3. Functional characterization of ZAM reverse transcriptase. (A) ZAM-[RT], ZAM-[RT-RH], HIV p51 and HIV p66 sequences were cloned in pGEMT easy vector (Promega), and the corresponding 35Smethionine-labelled polypeptides were produced in vitro using the Promega TNTs coupled reticulocyte lysate system. Lane 1, ZAM[RT]; lane 2, ZAM-[RT-RH]; lane 3, HIV p51; lane 4, HIV p66. The proteins were analysed by SDS-PAGE electrophoresis followed by autoradiography. The molecular masses of protein standards are indicated in kilodalton on the left. (B) The RNA-dependent DNApolymerase activity of reverse transcriptases was determined as described in the Materials and Methods section. The activities of ZAM-[RT], ZAM-[RT-RH], and ZAM-[RT-RH/RT] (lanes 1–3) were assayed and compared to HIV-p51, HIV-p66 and the heterodimer HIV-p51/p66 (lanes 4–6, respectively). Each reaction was incubated at room temperature and the percentage of remaining 32Pd[TTP] was calculated to determine enzyme activity. A negative control was performed with fresh TNTs coupled reticulocyte lysate mix to ensure the absence of parasite RNA-dependent DNA-polymerase activity (lane 7). Each experiment was performed in triplicate, and standard deviations were calculated.
To go further in the characterization of ZAM reverse transcriptase, its activity was investigated in relation to three different parameters: divalent cations, pH, and temperature. 3.3.1. Divalent cations It has been previously demonstrated that reverse transcriptase is dependent on the presence of divalent cations, most commonly magnesium, but MLV RT displays optimal activity in the presence of manganese (Verma, 1975). The requirement of divalent cations Mg2+ and Mn2+ for ZAM reverse transcriptase was therefore investigated. First, RNA-dependent DNApolymerase activity was performed with an Mg2+ concentration ranging from 0 to 20 mM. Results presented in Fig. 4A (left) show that in the absence of divalent cations (0 mM), ZAM reverse transcriptase is
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completely inactive. The presence of 1 mM of Mg2+ was enough to promote an efficient DNA-polymerase activity that increased at 2 mM of Mg2+, and remained comparable at Mg2+ concentrations of up to 10 mM. Higher concentrations such as 20 mM of Mg2+ gave rise to a weaker reverse transcriptase activity. A similar experiment was performed with a buffer containing Mn2+ cations ranging from 0 to 10 mM (Fig. 4A, right). We observed that the RNA-dependent DNApolymerase activity of ZAM [RT] was higher at weak Mn2+ cation concentrations, reaching a maximum at 1 and 2 mM, and then decreasing at 5 and 10 mM. These experiments demonstrate the requirement of divalent cations for ZAM reverse transcriptase activity with a maximal activity at 10 mM Mg2+, or at only 2 mM Mn2+. Furthermore, the highest RT activity was observed with Mn2+ divalent cation and in a narrower window of concentration (1–2 mM for Mn2+ compared to 2–10 mM for Mg2+).
3.3.2. pH Results presented in Fig. 4B show the effect of pH on ZAM-[RT-RH] RNA-dependent DNA-polymerase activity. ZAM-[RT-RH] exhibits a low activity at acidic pH (3–5). This activity increased from pH 3 to 7, reaching an optimum at pH9 and then decreasing at pH10.5. Thus, ZAM-[RT-RH] works better at basic pH than at acidic pH with an optimum activity at pH9.
3.3.3. Temperature We aimed to determine the extent to which processivity could be influenced by temperature. Several assays were performed at reaction temperatures varying from 0 to 45 1C (Fig. 4C). ZAM-[RT-RH] showed a high reverse transcriptase activity at 22 1C, which corresponds to the temperature of its host Drosophila melanogaster. This property was compared with that of the wellstudied heterodimeric reverse transcriptase of HIV-1 that also shows a three-fold increase of activity at a specific temperature, i.e. 37 1C, the temperature of its host (Fig. 4C).
4. Discussion The last decade has seen a major effort devoted to the research of the reverse transcriptases of HIV-1 and HIV2, the viruses responsible for AIDS, because most of the drugs approved to date for the treatment of this disease are inhibitors of the viral reverse transcriptases. More recently, with the appearance of drug-resistant HIV variants, the molecular, structural and catalytic properties of other retroviral reverse transcriptases have become the focus of numerous recent studies aiming to gain a fuller understanding of the general structures and functions of these enzymatic activities, and then to develop novel potent and specific reverse transcriptase inhibitors. However, very few studies to date have addressed the catalytic activities of reverse transcriptases encoded by closely related retroviruses called errantiviruses and found in insect genomes, despite the fact that they display considerable sequence homology with reverse transcriptases encoded by their mammalian counterparts (Wilhelm and Wilhelm, 2001; Xiong and Eickbush, 1988). In order to explore the properties of a reverse transcriptase encoded by an errantivirus from Drosophila melanogaster, we expressed an active form of a recombinant ZAM reverse transcriptase, and analysed its biochemical characteristics. Most lentiviral reverse transcriptases studied to date exhibit a dimeric structure of asymmetrically organized subunits. However, data obtained with reverse transcriptases purified from viruses such as MLV or MMTV support the possibility of a monomeric reverse transcriptase organization (Taube et al., 1998; Verma, 1975). In the present report, we show that the reverse transcriptase and the RNase H domains encoded by ZAM are both necessary in a single polypeptide for a DNA-polymerase activity. If a C-terminal deletion affecting the RNase H domain is introduced, the amount of reverse transcription is abolished. This transcription is partially recovered when the two peptides ZAM-[RT-RH] and ZAM-[RT] are put together for the test. When ZAM-[RT-RH] is the only peptide used in the reaction, the transcription is then maximal. This result suggests that ZAM-[RT] peptides act as dominant negative components counteracting the
Fig. 4. Cation, pH and temperature requirements of the RNA-dependent DNA-polymerase activity of ZAM reverse transcriptase. The RNAdependent DNA-polymerase activity of ZAM-[RT-RH] was determined by calculating the percentage of remaining 32Pd[TTP]. (A) Reactions were performed in a Tris-HCl buffer (pH7) and incubated for 1 h at room temperature (22 1C) with a concentration ranging from 0 to 20 mM of Mg2+ or 0 to 10 mM of Mn2+. The optimum concentrations of reverse transcriptase activity in the presence of Mg2+ and Mn2+ are indicated by arrows. (B) The RNA-dependent DNA-polymerase reactions of ZAM-[RT-RH] were performed in a Tris-HCl buffer (10 mM of Mg2+) with a pH ranging from 3 to 10.5, and incubated for 1 h at room temperature (22 1C). (C) The RNA-dependent DNA-polymerase reactions of ZAM-[RT-RH] were performed at 10 mM Mg2+ concentration in a Tris-HCl buffer (pH7), and incubated at the following temperatures: 0, 22, 37 and 45 1C, as indicated below the graph. Furthermore, HIV-p51/p66 heterodimer was assayed in the same buffer and reactions were performed at Drosophila temperature (22 1C) and human body temperature (37 1C). Each experiment was performed in triplicate, and standard deviations were calculated. A negative control was performed for each experiment with fresh TNTs coupled reticulocyte lysate mix to ensure the absence of parasite RNA-dependent DNA-polymerase activity within the medium (data not shown).
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full activity of ZAM-[RT-RH] peptides. It also indicates that ZAM-[RT-RH] and ZAM-[RT] can potentially interact through their RT domain and provides evidence for an active form of the ZAM reverse transcriptase as ZAM-[RT-RH/RT-RH] homodimers.
Similarly to all other retroviral reverse transcriptases, ZAM-[RT-RH] needs divalent cations for its activity. It has been shown that most retroviral reverse transcriptases have a marked preference for Mg2+ over Mn2+. Here, we show that ZAM-[RT-RH] displays a good
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efficiency in the presence of Mg2+, although the highest efficiency was detected with Mn2+ in a very narrow window of low concentration (1–2 mM). For HIV, Mg2+ has been found to have an effect on dimerization with an increased amount of dimers and activity related to an increased MgCl2 concentration (Cabodevilla et al., 2001). From this angle, the effect of Mg2+ and Mn2+ might be indirect and play a role in the association of ZAM-[RT-RH] rather than acting directly on its reverse transcriptase activity. ZAM reverse transcriptase was efficient at basic pH with a major activity detected at pH9, which was slightly higher than the optimum pH of 7.5 required for the DNA-polymerase activity of HIV-1 RT or MMTV RT (Hizi et al., 1991; Taube et al., 1998). Lastly, we analysed the temperature dependence of ZAM-[RT-RH]. Our data showed that ZAM reverse transcriptase activity was highly temperature-sensitive, with a maximal incorporation of labelled desoxynucleotides observed at its host temperature 22 1C. An increase in temperature severely affected the amount of reverse transcribed RNA, with a very low amount of reverse transcription observed at 37 1C, almost absent at 45 1C. Host-temperature-dependent reverse transcriptase regulation has been previously demonstrated for other insect reverse transcriptases (Ivanov et al., 1991; Lescault et al., 1989.; Pyatkov et al., 2004), as well as for HIV reverse transcriptase. For this latter, Huang et al. (1998) demonstrated that a temperature lower than 37 1C gives rise to a decrease in HIV reverse transcriptase activity, a decrease attributed to a reduced dimerization process (Cabodevilla et al., 2001). For ZAM reverse transcriptase, a similar mechanism could certainly be considered, with a decrease of dimerization at 37 1C rather than 22 1C. These data indicate that ZAM reverse transcriptase is well-adapted to the temperature of its host, and although expressed in vitro the polypeptide displays an optimal temperature sensitivity corresponding to the conditions necessary for a replication process occurring in vivo. In vivo, the reverse transcription of viral genomic RNA occurs inside the viral capsid. The influence of all the tested parameters in this study was investigated on ZAM reverse transcriptase in vitro, and these experiments did not take into consideration the micro-environment yielded by the core structure of viral particles. However, there is still no reconstituted system recapitulating events in vivo, our results argue for an active homodimeric form of ZAM-[RT-RH] in vivo, its need for divalent cations, and its specific temperature dependence.
Acknowledgements We thank the members of the laboratory for their help, comments and encouragement. This work was
supported by grants from the INSERM (U384) and the CNRS (GDR2157), and by a project grant from the Association pour la Recherche contre le Cancer (ARC 3441) to C.V. F.A. received a grant from the Ministe`re de l’Enseignement Supe´rieur et de la Recherche (MESR), and the Fondation Recherche Me´dicale (FRM).
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