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Functional differences between the human LINE retrotransposon and retroviral reverse transcriptases for invivo mRNA reverse transcription

1997; Springer Nature; Volume: 16; Issue: 21 Linguagem: Inglês

10.1093/emboj/16.21.6590

1460-2075

O. Dhellin,

Advanced biosensing and bioanalysis techniques

Article1 November 1997free access Functional differences between the human LINE retrotransposon and retroviral reverse transcriptases for in vivo mRNA reverse transcription Olivier Dhellin Olivier Dhellin Unité de Physicochimie et Pharmacologie des Macromolécules Biologiques, CNRS URA147, Institut Gustave Roussy, 39 rue Camille Desmoulins, 94805 Villejuif, Cedex, France Search for more papers by this author Joël Maestre Joël Maestre Unité de Physicochimie et Pharmacologie des Macromolécules Biologiques, CNRS URA147, Institut Gustave Roussy, 39 rue Camille Desmoulins, 94805 Villejuif, Cedex, France Search for more papers by this author Thierry Heidmann Corresponding Author Thierry Heidmann Unité de Physicochimie et Pharmacologie des Macromolécules Biologiques, CNRS URA147, Institut Gustave Roussy, 39 rue Camille Desmoulins, 94805 Villejuif, Cedex, France Search for more papers by this author Olivier Dhellin Olivier Dhellin Unité de Physicochimie et Pharmacologie des Macromolécules Biologiques, CNRS URA147, Institut Gustave Roussy, 39 rue Camille Desmoulins, 94805 Villejuif, Cedex, France Search for more papers by this author Joël Maestre Joël Maestre Unité de Physicochimie et Pharmacologie des Macromolécules Biologiques, CNRS URA147, Institut Gustave Roussy, 39 rue Camille Desmoulins, 94805 Villejuif, Cedex, France Search for more papers by this author Thierry Heidmann Corresponding Author Thierry Heidmann Unité de Physicochimie et Pharmacologie des Macromolécules Biologiques, CNRS URA147, Institut Gustave Roussy, 39 rue Camille Desmoulins, 94805 Villejuif, Cedex, France Search for more papers by this author Author Information Olivier Dhellin1, Joël Maestre1 and Thierry Heidmann 1 1Unité de Physicochimie et Pharmacologie des Macromolécules Biologiques, CNRS URA147, Institut Gustave Roussy, 39 rue Camille Desmoulins, 94805 Villejuif, Cedex, France *Corresponding author. E-mail: [email protected] The EMBO Journal (1997)16:6590-6602https://doi.org/10.1093/emboj/16.21.6590 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info We have analysed the reverse transcriptase (RT) activity of the human LINE retrotransposon and that of two retroviruses, using an in vivo assay within mammalian (murine and human) cells. The assay relies on transfection of the cells with expression vectors for the RT of the corresponding elements and PCR analysis of the DNA extracted 2–4 days post-transfection using primers bracketing the intronic domains of co-transfected reporter genes or of cellular genes. This assay revealed high levels of reverse-transcribed cDNA molecules, with the intron spliced out, with expression vectors for the LINE. Generation of cDNA molecules requires LINE ORF2, whereas ORF1 is dispensable. Deletion derivatives within the 3.8 kb LINE ORF2 allowed further delineation of the RT domain: >0.7 kb at the 5′-end of the LINE ORF2 is dispensable for reverse transcription, consistent with this domain being an endonuclease-like domain, as well as 1 kb at the 3′-end, a putative RNase H domain. Conversely, the RT of the two retroviruses tested, Moloney murine leukemia virus and human immunodeficiency virus, failed to produce similar reverse transcripts. These experiments demonstrate a specific and high efficiency reverse transcription activity for the LINE RT, which applies to RNA with no sequence specificity, including those from cellular genes, and which might therefore be responsible for the endogenous activity that we previously detected within mammalian cells through the formation of pseudogene-like structures. Introduction Mammalian genomes contain two major types of reverse transcriptase (RT)-encoding elements: the retroviral-like long terminal repeat (LTR) retrotransposons and the non-LTR (or LINE) retrotransposons (reviewed in Gabriel and Boeke, 1993; Eickbush, 1994). These two classes of elements transpose in a replicative manner, via reverse transcription mediated by the transposon-encoded RT of a genomic RNA transcript from the element (Boeke et al., 1985; Heidmann and Heidmann, 1991; Jensen and Heidmann, 1991; Pélisson et al., 1991; Moran et al., 1996). Phylogenetic examination of the RT-encoding domains from both types of elements reveal strong similarities, with seven highly conserved boxes (Xiong and Eickbush, 1990; McClure, 1993) which are also found in other RT-containing sequences, including prokaryotic retrons (Inouye et al., 1989), mitochondrial plasmids (Nargang et al., 1984; Kuiper and Lambowitz, 1988) and group II introns (Michel and Lang, 1985; Lambowitz and Belfort, 1993). In the LINE elements (reviewed in Martin, 1991a; Eickbush, 1992) the RT domain is part of a large ORF. A sequence has been identified in its 5′-region, by database analysis, as most probably encoding an endonuclease (Barzilay and Hickson, 1995; Martin et al., 1995, 1996). This prediction was recently confirmed experimentally for the human LINE (L1) element (Feng et al., 1996). At the 3′-end a domain with homology to RNase H can be detected, as is also observed in retroviral RTs (McClure, 1991), as well as a zinc finger motif (Fanning and Singer, 1987; Schwarz-Sommer et al., 1987). Phylogenetic analyses have shown that the LINE RTs are distantly related to those of retroviruses and retroviral-like elements, but functional studies have not yet revealed any significant difference between these two classes of polymerases. However, there exist fundamental differences between the mechanisms involved in transposition of the two types of elements. LTR elements make reverse transcripts of their genomic RNA within retroviral-like particles, initiation of reverse transcription being mediated by a three-component association between a specific viral sequence (the primer binding site, PBS), a complementary tRNA and the RT (reviewed in Coffin, 1996). The extrachromosomal proviral DNA copies thus generated then integrate into the genome via an integrase-directed process. Conversely, LINE most probably undergo both processes simultaneously, through in situ reverse transcription of their genomic RNA intermediate, initiated at the level of a 3′-OH from a nick within the target genomic DNA. The nick is generated by a transposon-encoded endonuclease, as demonstrated for the R2Bm element (Luan et al., 1993) and strongly suggested for the human LINE (Feng et al., 1996). Such differences in the overall transposition process could be due to the nature of the structural intermediates for reverse transcription and replication. Retroviral-like elements are associated with particles of known content and organization (reviewed in Coffin, 1996), whereas LINE elements are associated with ribonucleoprotein particulate intermediates of still poorly defined structure (Martin, 1991b; Hohjoh and Singer, 1996). In fact, another possibility could be that they more directly result from intrinsic differences between the encoded RTs themselves. In this respect, previous assays in which the RT domain of the yeast Ty1 LTR retrotransposon had been replaced by the human LINE RT-containing ORF (Mathias et al., 1991; Dombroski et al., 1994; Teng et al., 1996) have resulted in transposed elements with unusual structures (see Discussion), suggesting that the two RTs are not exchangeable. To investigate this issue we have developed an in vivo assay for reverse transcription, whereby the human LINE RT is not forced into a retroviral-like particle but is assayed in a biologically more relevant situation within homologous human cells (as well as in murine or feline cells). Using this assay we show that a RT domain can be delineated within the functional human L1 element (Dombroski et al., 1991; Moran et al., 1996), which shows a high efficiency RT activity allowing in vivo reverse transcription of RNAs with no sequence specificity, a property which is not shared by the retroviral RTs (from the murine MoMLV and human HIV retroviruses) that we have similarly tested. Hence, LINE and retroviral RTs have distinct reverse transcription capacities and the 'wide spectrum' RT activity of the former is likely to be responsible for the endogenous activity that we previously revealed within mammalian cells through de novo formation of pseudogene-like structures (Tchénio et al., 1993; Maestre et al., 1995). Results Rationale of the assay The rationale of the assay (Figure 1) relies on the use of expression vectors for the human LINE ORFs and for retroviral RTs and on the capacity of these expression vectors to make a cDNA copy of an intron-containing reporter gene in mammalian cells in culture. In a standard experiment cells (human NTera2D1 or 293 cells, murine 3TDM1 cells or feline G355.5 cells) were co-transfected with both the expression vector and the reporter gene. Two to 4 days post-transfection the transfected cell DNA was extracted and PCR carried out to test for occurrence of cDNA copies of transcripts from the reporter gene, which can be unambigously identified as a result of splicing out of the intron (Figure 2). Figure 1.Structure of retroviral and LINE elements and rationale of the RT assay. (A) Structure of the MoMLV provirus with the GAG, POL and ENV ORFs and of the human LINE retrotransposon with ORF1 and ORF2. The boundaries of the cleavage products within the retroviral ORFs are indicated with dotted lines, with the matrix (Ma), capsid (Ca) and nucleocapsid (Nc) proteins within Gag and the protease (Pr), reverse transcriptase (RT) and integrase (In) within Pol. Transcription start sites are indicated by arrows. RT-containing expression vectors derived from both elements are schematized in Figures 4 and 5. Reporter genes for the RT assay contain an intron (from the previously devised neoRT indicator gene; Heidmann et al., 1988; Heidmann and Heidmann, 1991), the CMV promoter and the SV40 polyadenylation signal and are schematized in Figure 3. Spliced transcripts, if reverse transcribed by the products of the RT-containing expression vectors, should result in intronless cDNA molecules that can be identified upon PCR analysis using primers bracketing the splice junction (see also Figure 2). (B) Experimental procedure for the in vivo RT assay. Mammalian cells (of human, murine or feline origin; see text) are co-transfected with both intron-containing reporter genes and RT-containing expression vectors and DNA is extracted 2–4 days post-transfection for PCR analysis as indicated in (A) and in Figure 2. Download figure Download PowerPoint Figure 2.In vivo RT activity of LINE elements. The complementation assay in Figure 1 was carried out in human NTera2D1 cells using an intron-containing reporter gene (Figure 3, a) and the expression vector for the full-length LINE element (Figure 4, c). (A) Structure of the expected cDNA molecules and results. (Top) Primers bracketing the intronic domain of the reporter gene (primers neo1 and tk1, neo2 and tk2, d2 and tk2) are indicated, with the length of the PCR fragments expected upon splicing out of the intron; for reverse transcripts of the reporter gene the neo1–tk1 fragment obtained after PCR amplification (387 bp) should yield two fragments (of 193 and 194 bp) after SacI restriction (SacI generated at the splice junction; see Heidmann et al., 1988). (Bottom) PCR fragments were analysed by electrophoresis of 10 μl of the PCR reactions in 1.5% agarose gels and ethidium bromide staining: lane a, PCR products (restricted or not by SacI) with both the intron-containing reporter gene and the LINE expression vector; lane b, reporter gene alone; lane –, control PCR without DNA; M, size marker. (B) Assay for double-strand DNA synthesis in the RT assay. (Top) Double-stranded DNA should be restricted by the indicated enzymes (XbaI and BglII). PCR amplification as in (A) using primers neo1 and tk1 should then result in a 387 bp amplified fragment in the case of single-strand cDNA synthesis and no amplified fragment in the case of double-strand synthesis, whereas control amplification using primers d2 and tk2 (both on the same side of the cut DNA) should yield a 180 bp fragment even after restriction. (Bottom) PCR fragments obtained with the primers indicated for each panel, with cellular DNA either uncut (nc) or cut (c) with XbaI and BglII prior to amplification. Lane –, control PCR without DNA. (C) Assay for second-strand DNA synthesis. Primer extension specific for the putative DNA plus strand was first achieved by a 45 cycle 'linear' PCR amplification with the tk1 primer, resulting in multiple single-strand copies as schematized. PCR amplification with two primers (neo2 and tk2) was then allowed to proceed for a limited number of cycles (23 cycles). PCR results obtained with or without preliminary primer extension are presented for transfection experiments as in (A), with both the expression vector and the reporter gene (a) or with the reporter gene alone (b). Controls for RNAs (for instance within putative RNA–DNA hybrids) as possible templates were performed by prior treatment of the extracted nucleic acids with RNase A (after denaturation at 95°C [+RNase A(1)] or with NaOH [RNase A(2)]) or upon treatment with RNase H. The expected 277 bp PCR fragments are indicated and their identity was ascertained upon restriction with SacI (not shown). Download figure Download PowerPoint Figure 3.Absence of effect of LINE sequences in the reporter gene for in vivo reverse transcription. Co-transfection experiments were as in Figure 2, with the CMVLINE expression vector (Figure 4, c) and the indicated LINE sequence-containing (or not containing) reporter genes (see reporter gene structures on the left); LINE sequences [including for (a) the 5′ untranslated domain, ORF1, part of ORF2 and the 3′ untranslated domain] are represented in grey and the intron-containing neoRT cassette with open boxes; the size of the expected PCR fragments before and after SacI restriction (−s, +s) are indicated. Download figure Download PowerPoint A first series of expression vectors were constructed containing either the entire LINE element (i.e. both ORFs and the 5′- and 3′-untranslated domains) or only the LINE ORFs under control of the potent immediate early cytomegalovirus (CMV) promoter. Since it has been shown that LINE ORF2 is expressed within the bicistronic LINE at a level at least 100-fold lower than ORF1 (McMillan and Singer, 1993), we also constructed expression vectors harbouring only ORF2 (see vector structures in Figure 4). Similarly, several reporter genes were constructed harbouring the previously described intron-containing indicator gene for retrotransposition (see for example Heidmann et al., 1988; Heidmann and Heidmann, 1991). In a first series of reporter genes the indicator gene was inserted into a LINE element, with a small deletion to limit the overall size of the transcript, to take into account the possibility that some sequences might be essential in cis for reverse transcription. Other reporter genes were also constructed in which LINE sequences were deleted, encompassing domains 5′ and/or 3′ of the indicator gene (see reporter gene structures in Figure 3). Figure 4.LINE sequences required in trans for in vivo reverse transcription. (A) Co-transfection experiments were as in Figure 2, with the CMVLINEneoRT reporter gene and the LINE-derived expression vectors whose structures are indicated on the left, lanes a–d. Lane –, control PCR without DNA. (B) Experimental conditions as in (A) with the CMVORF2 expression vector and CMVLINEneoRT (lane 1), CMVneoRT (lane 2) or no reporter gene (lane 3). (C) Quantitation of in vivo RT activity in homologous and heterologous cells. Human (NTera2D1) or murine (3TDM1) cells were co-transfected with the CMVneoRT reporter gene and the LINE CMVORF2 expression vector and DNA was extracted 2 days post-transfection. Aliquots of 1.5 μg cellular DNA and serial dilutions (by 10-fold steps) were used for PCR amplifications as in Figure 2.The indicated amount of pre-spliced plasmid copies (diluted into genomic DNA from non-transfected cells) were amplified in parallel as a reference. Lane –, control PCR without DNA. Download figure Download PowerPoint Figure 5.Assay for in vivo reverse transcription using retroviral elements. (A) Co-transfection assays were as in Figure 2, with the CMVneoRT reporter gene and the indicated expression vectors (structure on the left): lane a, LINE ORF2; lane b, MoMLV GAG–POL; lane c, MoMLV RT; lane d, HIV RT (using the CMV p66 and SV p51 expression vectors for the full-length and RNase H-truncated subunits constituting the dimeric HIV RT). PCR and controls were as in Figure 2. (B) Assay for quantitation of the relative efficiency of the LINE RT versus the MoMLV RT for in vivo reverse transcription. Co-transfection was as in (A) with CMVORF2 (lane a) or MoMLV CMVRT (lane c). Cellular DNAs (1 μg) were PCR amplified either directly or after serial dilutions (10-fold steps, the total amount of DNA before amplification being maintained constant by complementation with cellular DNA from untransfected cells). Lane –, control PCR without DNA. Download figure Download PowerPoint Evidence for in vivo reverse transcription by LINE-containing vectors As illustrated in Figure 2A, co-transfection of cells with both a marked LINE element under control of the CMV promoter and an expression vector for the full-length LINE element resulted in cDNA copies that could be easily detected upon PCR amplification of the transfected cell DNA. PCR amplification of the DNA extracted 4 days post-transfection using primers bracketing the intronic domain of the indicator gene generated a 387 bp band, consistent with splicing out of the intron, which further contained the SacI site expected to be generated at the splice junction (Heidmann et al., 1988; Figure 2A). This band was not observed in the absence of the expression vector for the LINE element (Figure 2A) and no fragment could be detected after a second nested PCR amplification, which further increases PCR sensitivity at least 10-fold (data not shown). Amplification of larger fragments using primers located distantly from the reporter intron (namely at the 5′-end of the neo gene and at the polyadenylation signal at the 3′-end of the reporter gene) were also positive, although with a reduced intensity, again only in the presence of the expression vector for the LINE element (data not shown). The nature of the cDNA molecules revealed by the PCR assay was further investigated to determine whether they corresponded to single- or double-stranded DNA (both would result in production of the 387 bp PCR fragment) and, more precisely, to determine whether the DNA plus strand was generated during reverse transcription. In a first series of experiments the extracted cellular DNA was first treated with a combination of restriction enzymes (XbaI and BglII) which do not cut single-stranded DNA and should disrupt the DNA region to be amplified (see scheme in Figure 2B). PCR amplification was then carried out as previously described. As illustrated in Figure 2B, under these conditions no PCR product was generated. As an internal positive control PCR amplification was also carried out before and after enzymatic digestion but using a pair of oligonucleotide primers located on the same side of the XbaI and BglII restriction sites, which should produce a PCR fragment of a distinct size (see Figure 2B, bottom): in that case, as expected, amplification was observed with both cut and uncut DNA. Altogether, these data suggest that a major fraction of the LINE-induced cDNA molecules are double-stranded (among which RNA–DNA hybrids cannot be excluded, but see below). In a second series of experiments (see scheme in Figure 2C), primer extension of the putative plus strand DNA was first performed by repeated cycles of PCR using a single primer (45 cycles) and occurrence of extended DNA molecules was then tested by standard PCR amplification using two primers, with a reduced number of cycles (23 cycles). As illustrated in the figure, PCR amplification under these conditions resulted in the expected fragment only upon preliminary primer extension, thus again strongly suggesting synthesis of plus strand DNA in the reverse transcription assay. As expected, amplification was only observed in the presence of the LINE expression vector and was insensitive to treatment of the extracted nucleic acids, prior to primer extension, by RNase A (after denaturation) or RNase H, which degrade RNAs and RNA strands within putative RNA–DNA hybrids (Figure 2C). To measure the efficacy of in vivo reverse transcription mediated by the LINE expression vectors the amount of reverse transcript was estimated by semi-quantitative PCR analysis, using a previously described method (Ségal-Bendirdjian and Heidmann, 1991). Basically, PCR was allowed to proceed for a number of cycles adjusted so as to be in the range of linearity between the amount of DNA to be amplified and the amount of PCR products generated, as quantitated after gel electrophoresis and ethidium bromide staining (i.e. during the exponential phase of PCR). DNA plasmid molecules, diluted into genomic DNA from non-transfected cells, were amplified in parallel as standards (Figure 4C). Accordingly, the number of cDNA molecules could be systematically determined and was found to be in the range 104–105/μg DNA, which corresponds to 0.01–0.1 molecules/cell, depending on the LINE expression vector tested (see below). The reverse transcripts found in relatively large amounts are most probably not integrated into the genome, but rather correspond to extrachromosomal cDNA molecules, as previously observed using rat LINE reporters (Ségal-Bendirdjian and Heidmann, 1991). This conclusion is consistent with the fact that despite the large number of cDNA copies generated in these transiently transfected cells and taking advantage of the indicator neo gene (see for example Heidmann et al., 1988; Heidmann and Heidmann, 1991) contained in the reporter plasmids, no G418-resistant clones could be isolated (unpublished results). LINE sequences required in trans or in cis for in vivo reverse transcription The demonstration that a full-length LINE expression vector can generate double-stranded reverse transcripts of an intron-containing, LINE-derived reporter gene and the simple in vivo assay described above allow an investigation of both the LINE-coding sequences in the expression vector which are required in trans for this process and the LINE sequences in the reporter gene which might be required in cis. A series of deletion derivatives of the initial LINE-derived reporter gene were therefore constructed (Figure 3) and assayed as described above to test for the possible role in reverse transcription of LINE sequences in cis. As illustrated in Figure 3, similar levels of reverse transcript were obtained using reporter genes with complete deletion of the 5′- and/or 3′-domains of the LINE elements (including the LINE 3′-untranslated domain), ending in the minimal CMVneoRT reporter gene completely devoid of LINE sequences (Figure 3, lane d): clearly, no LINE sequence is specifically required in cis for reverse transcription (see also Figure 6 and the related section). Figure 6.LINE-mediated reverse transcription of mRNA from cellular genes. Cells (293) were transfected with expression vectors for the LINE ORF2, the MoMLV RT or no vector, either alone (B and C) or together with an expression vector for genomic TNFβ (A). Reverse transcripts of mRNAs from the TNFβ (A), E1A (B) or c-myc (C) genes were assayed by PCR using the pairs of primers indicated in the figure for the corresponding genes. Introns are indicated together with the expected size of the PCR products. For the TNFβ gene the second intron has a splicing efficiency close to unity, whereas the third intron is not fully processed (Neel et al., 1995). For the E1A gene two alternative splices have been described (Stephens and Harlow, 1987). Download figure Download PowerPoint Deletion derivatives of the initial LINE expression vector were then constructed and assayed to delineate the role of the LINE ORFs in trans for reverse transcription. The assays were performed either with the minimal CMVneoRT reporter gene mentioned above or with the LINE-containing reporter, in which case a deletion within ORF1 was introduced to prevent expression of this ORF from the reporter gene itself (see Materials and methods). As illustrated in Figure 4A for the LINE reporter gene and in Figure 4B for the CMVneoRT reporter, ORF1 is dispensable for reverse transcription activity of the LINE expression vector. Reverse transcription efficiency was even higher (up to 10-fold) with the CMVORF2 vector than with CMVLINE, as expected, since ORF2 expression in the former vector does not require translational re-initiation. An alternative interpretation involving a possible negative effect of ORF1 expression was ruled out by an experiment using a LINE expression vector with an in-phase deletion within ORF1, which gave the same result as the full-length LINE vector (not shown). Finally, a vector with a deletion within ORF2 (CMVORF2*, Figure 4), assayed as a control, actually lacked reverse transcription activity in the assay. The LINE ORF2 is therefore necessary and sufficient to generate reverse transcripts in vivo. The number of cDNA molecules synthesized under these conditions was determined as indicated in the previous section and was found to be close to 105/μg DNA, i.e. close to 0.1 molecules/cell, taking into account the transfection efficiency measured by X-gal staining of cells transfected with a lacZ-containing plasmid. Figure 4C also demonstrates, interestingly, that similar levels of reverse transcript were obtained in murine and human cells (as well as in feline cells; data not shown), strongly suggesting that the reverse transcription activity mediated by the LINE ORF2 is not dependent on species-specific factors, but is rather a property of the protein per se. In vivo reverse transcription of the reporter gene cannot be induced by a provirus or by retroviral RTs Since the LINE-mediated in vivo reverse transcription activity revealed in the present assay does not require specific sequences in cis, we tested whether similar results would be obtained with retroviral RTs. We previously demonstrated that a cloned MoMLV provirus was competent for both viral particle formation and intracellular transposition in murine as well as human cells (Heidmann et al., 1988; Tchénio and Heidmann, 1991, 1992) and this proviral construct was tested. As illustrated in Figure 5A (lane b) for murine cells (similar results were obtained for human cells; not shown), this element was negative in the in vivo reverse transcription assay, whatever the reporter gene used. We checked that the transfected plasmid was 'functional' as a provirus by measuring RT activity in the viral particles released in the supernatant of transfected cells, using a classical in vitro RT assay with synthetic templates and primers (Table I). Since particle formation could impair reverse transcription from a non-viral RNA template, we then derived an expression vector for the retroviral RT, as described in Jean-Jean et al. (1989). Rather unexpectedly, this retroviral RT (Figure 5A, lane c) was similarly unable to generate cDNA copies of transcripts from the reporter gene (whatever the reporter gene and the cells tested) and these remained undetectable even after a second nested PCR amplification. Quantitation of the PCR assay using 10-fold serial dilutions of the DNA from the transfected cells demonstrated an at least 1000-fold lower in vivo reverse transcription activity, if any, of the MoMLV RT as compared with the LINE ORF2 (Figure 5B). In that case also we could demonstrate that the MoMLV RT was actually produced in the transient transfection assay. This was tested upon protein extraction from the transfected cells and assaying in vitro RT activity as described above, using synthetic templates and primers: as indicated in Table II, the MoMLV RT was active and its activity was even higher than that of the LINE RT (extracted under identical conditions), as determined under two assay conditions for divalent cations (Mg2+ or Mn2+). The inefficiency of the MoMLV RT to achieve reverse transcription of the reporter gene was also observed with another retroviral RT. Indeed, using expression vectors encoding the two subunits of the HIV RT described in Ansari-Lari and Gibbs (1994), no spliced reverse transcripts could be detected by the in vivo assay in either murine (Figure 5A, lane d) or human cells (not shown); in that case the in vitro RT activity of the HIV RT was not assayed, but its functionality has been previously documented (Ansari-Lari and Gibbs, 1994). Table 1. Assay for the MoMLV GAG–POL expression vector Cells GAG–POL transfected Untransfected Ψ2 Control RT activity in cell supernatanta 9200 ± 1000 410 ± 70 7500 ± 600 330 ± 30 a RT activity in supernatants from the transfected (and untransfected) cells was assayed in vitro as described in Materials and methods in 0.6 mM MnCl2 (c.p.m./30 min reaction). Supernatant from recombinant virus-producing Ψ2 cells was taken as a reference and control refers to the assay without supernatant. Values are the means of at least three experiments. Table 2. RT activity in cellular extracts from cells transfected with expression vectors for the MoMLV RT, LINE ORF2 and deletion deriva