Department of Microbiology and Immunology and Markey Cancer Center, University of Kentucky, Combs Research Bldg Room 206, 800 Rose Street, KY 40536-0096, Lexington, USA1
Author for correspondence: Jiayou Zhang. Fax +1 606 257 8940. e-mail jzhan1{at}pop.uky.edu
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Abstract |
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Introduction |
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Retroviral recombination plays an important role in retroviral carcinogenesis and in the AIDS epidemic (Coffin et al., 1997 ). Most retroviral recombinations occur during minus-strand DNA synthesis (Zhang et al., 2000
). Two conflicting in vitro observations suggest that retroviral recombinations are temperature dependent. Ouhammouch & Brody (1992)
demonstrated that during in vitro cDNA synthesis, the avian myeloblastosis virus (AMV) reverse transcriptase could switch from one template to another in a temperature-dependent manner. Chimeric cDNA molecules were generated with an increasing efficiency from lower temperature to higher temperature. This result suggested that retroviral recombination rates should increase as temperature increases. However, Shimomaye & Salvato (1989)
and Brooks et al. (1995)
found that raising the reaction temperature was the simplest way to overcome template secondary structure to prevent premature termination of cDNA synthesis. Their results suggested that the recombination rate should decrease as temperature increases.
In this report, we have established an in vivo system to demonstrate that the rate of retroviral recombination decreases as the temperature of reverse transcription is increased from 31 to 43 °C.
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Methods |
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Vector constructions.
All recombinant techniques were carried out by conventional procedures (Sambrook et al., 1989 ). All vector sequences are available upon request.
(i) Introduction of restriction enzyme sites into the gfp gene.
The gfp gene was from the jellyfish Aequorea victoria (pEGFP; Clontech) (Chalfie et al., 1994 ). Introduction of frame-shift mutations has been described previously (Zhang et al., 2000
). Briefly, the gfp gene was mutated by PCR to create a BstBI site at position 624 and the mutated gfp was designated gfp-BstBI. To introduce a frame-shift mutation into the gfp gene, the gfp-BstBI gene sequence was digested with BstBI, followed by repair with Klenow fragment. BstBI digestion created two DNA ends that contained a two-base overhang. When the overhangs were repaired by the Klenow fragment two blunt ends were created. Ligation of these two blunt ends with T4 ligase created a 2 bp insertion, which shifted the gfp open reading frame by two (+2).
The gfp gene was also mutated so that it contained a NcoI site at position 169. This resulting gfp gene was designated gfp-NcoI. Gfp-NcoI was digested with NcoI followed by repair with Klenow fragment and ligation. NcoI digestion created two DNA ends that contained a four-base overhang. As a result, this gfp open reading frame was shifted by one (+1) (Zhang et al., 2000 ).
(ii) Construction of pJZ481.
pJZ481 (Fig. 1A) is an MLV vector which contains two mutated gfp genes and a neomycin resistance gene (neo) (Li & Zhang, 2000
). The pJZ481 construct, from 5' to 3', was assembled as follows. The 5·4 kb NdeIBamHI fragment (from positions 3810 to 1630), which contained the neo gene and the two MLV long terminal repeats (LTRs), was isolated from pLN (Miller & Rosman, 1989
). The 0·7 kb BamHIBglII (from positions 1631 to 2150) fragment of gfp-BstBI- was derived from pEGFP and the frame-shift mutation was located at the 3' end of the gfp gene. The 0·7 kb BglIINotI (from positions 2151 to 3190) fragment of gfp-NcoI-1 was also derived from pEGFP and the frame-shift mutation was located at the 5' end of the gfp gene. The 0·6 kb NotINdeI fragment (from positions 3191 to 3809) was isolated from pCITE-1 (Novagen) and contained the internal ribosome entry segment (IRES) sequence.
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Introduction of JZ481 and JZ442+3' Hyg into helper cell line PG13.
Plasmid DNAs of pJZ481 and pJZ442+3' Hyg were transfected into an MLV amphotropic helper cell line PA317 (Miller & Buttimore, 1986 ). The supernatant media containing the viruses were collected and designated STEP 1 virus. The STEP 1 virus was used to infect the MLV xenotropic helper cell line PG13 (Miller et al., 1991
). The viruses released from the infected PG13 cells were unable to infect NIH 3T3 derivatives including PG13 (Miller et al., 1991
). This procedure ensured that the infection of D17 target cells with viruses collected from PG13 cells represented only a single round of infection. Infected PG13 cells were selected for Neor and Hygr for JZ481 and JZ442+3' Hyg respectively. Visible colonies appeared after 10 days of selection. The cells of well-separated clear colonies were isolated and designated STEP 2 cells for JZ481. Green colonies were isolated as JZ442+3' Hyg STEP 2 cells. Viruses harvested from STEP 2 cells were designated STEP 2 viruses.
Infection of target D17 cells.
STEP 2 viruses were incubated with D17 cells for 5 min at 37 °C. The cells were washed with TD three times to remove viruses that did not attach to the cells. Fresh medium was added and the cells were incubated at 24, 31, 37 and 43 °C. Nine hours after infection cells were transferred to 37 °C. Medium with hygromycin for JZ442+3' Hyg or medium with G418 for JZ481 was added 24 h after infection. Visible colonies were observed 1012 days after infection and designated STEP 3 cells.
Cells, transfection and infection.
The processing of D17 cells (ATCC CRL-8468), PA317 helper cells (ATCC CRL-9078), PG13 helper cells (ATCC CRL-10686), DNA transfections, virus harvesting and virus infections were as previously described (Zhang & Temin, 1993 ).
Fluorescence microscopy.
A fluorescence inverted microscope (Zeiss Axiovert 25) with a mercury arc lamp (100 W) and a fluorescence filter set (CZ909) consisting of a 470/40 nm exciter, a 515 nm emitter, and a 500 nm beam splitter was used to detect green fluorescent protein in living cells.
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Results |
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The second vector encoded a functional gfp gene and a hyg gene (Fig. 1B). From 5' to 3', JZ442+3' Hyg carried a drug resistance gene, hyg, an IRES sequence, a functional gfp and a 290 bp sequence of the 3' hyg gene, so that the gfp gene was flanked by two identical 290 bp sequences. (Zhang & Sapp, 1999
). Recombination between the two identical 290 bp sequences resulted in deletion of the gfp gene. Cells containing a recombinant JZ442+3' Hyg were clear under the microscope, while cells containing a parental-type JZ442+3' Hyg were green.
Retroviral recombinations are temperature dependent
JZ481 and JZ442+3' Hyg were introduced into the helper cell line PG13 by infection, as described in Methods, to avoid a high frequency of deletion during transfection (Zhang & Sapp, 1999 ). The viruses released from each PG13 clone, which contained JZ481 or JZ442+3' Hyg provirus, were used to infect D17 cells. Five min after viruses were added, the infected cells were incubated at 24, 31, 37 or 43 °C for 9 h to allow completion of reverse transcription. Then the cells were transferred back to 37 °C to allow integration and late stage infection to occur in a natural condition and to avoid further damage of the cells at higher or lower temperature. Twenty-four hours post-infection, the cells were selected for neomycin resistance (Neor) for JZ481 [or for hygromycin resistance (Hygr) for JZ442+3' Hyg]. Visible colonies were observed 1012 days after infection. Because D17 cells do not contain viral gagpol and env gene products to support retrovirus replication, no progeny viruses were released from these cells (Zhang & Temin, 1993
). Therefore, each drug resistant colony represented a single round of viral infection. Neor and Hygr colonies were analysed by fluorescence microscopy. For D17 cells infected with JZ481, green cells represented recombinants carrying a functional gfp gene (Fig. 1A
), whereas clear cells contained parental mutated gfp genes. The rate of recombination was determined by the ratio of the number of green colonies to the number of total Neor colonies (green plus clear colonies) (Table 1
and Fig. 2
). For D17 cells infected with JZ442+3' Hyg (Fig. 1B
), the rate of recombination was determined by the ratio of the number of clear colonies over the number of total Hygr colonies (Table 2
and Fig. 2
). As indicated in Fig. 2
, the recombination rate decreased as the temperature of reverse transcription increased from 31 to 43 °C. A slight decrease in the recombination rate at 24 °C was observed.
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To determine the nature of the recombinants of JZ442+3' Hyg, DNAs from clear and green colonies were digested with EcoRV and hybridized with a hyg probe. EcoRV digested within the two LTRs of both parental and recombinant proviruses (Fig. 1B). The parental proviruses from green colonies produced a 4·2 kb fragment and the recombinant proviruses from clear colonies produced a 2·5 kb fragment (Zhang & Sapp, 1999
). The Southern analysis indicated that the phenotype of most clear colonies resulted from deletion between the two identical 290 bp sequences of 3' hyg.
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Discussion |
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Retroviral RNA molecules usually form numerous secondary structures (Leis et al., 1993 ; Coffin et al., 1997
). One possible explanation for such an observation is that temperature change induces alternation in the secondary structure of RNA molecules. At higher temperature, the looser conformation of RNA molecules tends to form, which may facilitate the processing of reverse transcription. Since most retroviral recombinations occur during minus-strand DNA synthesis (Zhang et al., 2000
), at low temperature these tightly folded structures may hinder reverse transcription proceeding along the RNA template, which increases its chance of dissociating from the template (Mikkelsen et al., 2000
; Pathak & Temin, 1992
). In vitro studies have shown that pausing of retroviral reverse transcriptase enhances strand transfer (Wu et al., 1995
, 1996
; Kim et al., 1997
). The rate of recombination did not decrease much when the temperature increased from 31 to 43 °C in this system, probably because the secondary structures within the gfp gene or the hyg gene sequences were not strong. A more dramatic change of rates is expected if recombination occurs between two sequences with tighter secondary structures. In addition, the temperature range in this report was limited due to tissue culture conditions, which were different from in vitro experiments (Shimomaye & Salvato, 1989
; Brooks et al., 1995
).
Retroviral recombination may involve three steps: (1) pausing of reverse transcription; (2) the reverse transcription growing point leaving the original template; and (3) the growing point landing on the target/alternate template. In addition to affecting RNA secondary structures and the pausing of reverse transcriptase (step 1), higher temperature may also promote the dissociation of reverse transcriptase from its template (step 2), which increases the change of template switches. However, our in vivo results suggest that step 1 (pausing) is more crucial than step 2 (leaving) in retroviral recombination when reverse transcriptions are between 31 and 43 °C.
Another possibility is that decreasing temperature reduced elongation rates of reverse transcriptase, which also results in the pausing of reverse transcription. The pausing increased the chance of template switching, thereby increased the rate of recombination at lower temperature. It is also possible that decreasing temperature might have an effect upon the core particle structure, or it might have a direct effect upon reverse transcriptase as well as other unforeseen effect(s), which in turn affects viral recombinations.
Our data for 24 °C showed a slight decrease in the recombination rate compared to that for 31 °C. Two factors might have contributed to this observation. First, at temperatures lower than 31 °C RNA molecules might undergo little change as temperature altered, or secondly, the frequency of dissociation of the reverse transcriptase from its template was reduced.
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Acknowledgments |
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This research was supported by Public Health Service research grants CA70407 from the National Institutes of Health.
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References |
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Received 6 December 2000;
accepted 6 February 2001.
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