Host RNA polymerase II makes minimal contributions to retroviral frame-shift mutations

Jiayou Zhang

Department of Microbiology, Immunology and Molecular Genetics and Markey Cancer Center, University of Kentucky, 206 Combs Research Bldg, 800 Rose Street, Lexington, KY 40536-0096, USA

Correspondence
Jiayou Zhang
jzhan1{at}uky.edu


   ABSTRACT
Top
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
The rate of mutation during retrovirus replication is high. Mutations can occur during transcription of the viral genomic RNA from the integrated provirus or during reverse transcription from viral RNA to form viral DNA or during replication of the proviral DNA as the host cell is dividing. Therefore, three polymerases may all contribute to retroviral evolution: host RNA polymerase II, viral reverse transcriptases and host DNA polymerases, respectively. Since the rate of mutation for host DNA polymerase is very low, mutations are more likely to be caused by the host RNA polymerase II and/or the viral reverse transcriptase. A system was established to detect the frequency of frame-shift mutations caused by cellular RNA polymerase II, as well as the rate of retroviral mutation during a single cycle of replication in vivo. In this study, it was determined that RNA polymerase II contributes less than 3 % to frame-shift mutations that occur during retrovirus replication. Therefore, the majority of frame-shift mutations detected within the viral genome are the result of errors during reverse transcription.


   INTRODUCTION
Top
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Retroviral virions carry RNA molecules as their genetic material. After entering cells, the RNA is reverse transcribed into DNA, which is then integrated into host chromosomal DNA to become a provirus. Viral RNA is transcribed by the host transcription machinery and is packaged into progeny virions, which are released from host cells. Therefore, retrovirus replication cycles can be divided into two discrete stages: the early stage from parental viral RNA to proviral DNA and the late stage from proviral DNA to progeny viral RNA. The early stage involves reverse transcription of viral RNA by the retrovirus-encoded RNA-dependent DNA polymerase, whereas the late stage requires RNA polymerase II, a DNA-dependent RNA polymerase, which is encoded by the host cell. Any errors introduced by either of these polymerases will result in mutations within the progeny viruses. Current experimental protocols have been unable to distinguish between a mutation made during early or late stages. In most in vitro experimental systems studying the error rate of human immunodeficiency virus (HIV) reverse transcriptase, the RNA templates were obtained by transcription from a bacteriophage T7 promoter (Boyer et al., 1992). Such experimental procedures do not distinguish between errors made by T7 RNA polymerase and those made by the reverse transcriptase. Furthermore, current studies of mutation in vivo also cannot distinguish between the two processes. In such previous studies, after one round of replication, the mutations were identified and mutation rates calculated. The mutations were assumed to have resulted from the viral reverse transcriptase.

While the contribution of host RNA polymerase II to retroviral recombination and mutation has long been speculated (Preston & Dougherty, 1996), RNA polymerase II is assumed to be a high-fidelity polymerase because it has been found to have 3' to 5' repair activity (Thomas et al., 1998; Gnatt et al., 2001). In addition, the mutation rates for different retroviruses vary significantly. For example, mutation rates of HIV are much higher than those of spleen necrosis virus, Moloney murine leukaemia virus (MoMLV) and human T cell leukaemia virus (Preston et al., 1988; Mansky & Temin, 1995; Mansky, 2000), suggesting that reverse transcriptase is most likely to be a low-fidelity polymerase and therefore responsible for the mutations.

Studies suggest that the error frequency of prokaryotic RNA polymerase is between 10–4 and 10–5 (Blank et al., 1986; Erie et al., 1993; Libby & Gallant, 1991), while that of wheat-germ RNA polymerase II in vitro is less than 1x10–3 (de Mercoyrol et al., 1992). However, the fidelity of mammalian RNA polymerase II is still unknown. Errors caused by RNA polymerase II have been described in Alzheimer's patients as well as in patients with Down's syndrome (van Leeuwen et al., 1998). Moreover, specific RNA polymerase II errors were found within an adenine homopolymeric run in patients with severe haemophilia A within the factor VIII gene (Young et al., 1997), as well as within the apolipoprotein B gene (Linton et al., 1992). In damaged rat liver cells, the frequency of error in transcription of p53 was also reported to be increased (Ba et al., 2000).

When mutations occurring within the two LTRs of retroviral proviruses were analysed, it was suggested that about half of the mutations in both LTRs could have been caused by either the RNA polymerase II and/or the reverse transcriptase (Kim et al., 1996; O'Neil et al., 2002). However, the other half of the mutations, which were found only in one LTR, could only have been introduced by the reverse transcriptase during plus-strand DNA synthesis. This fact suggested that the reverse transcriptase most likely contributes 50 % or more of the mutations in retroviral evolution (Kim et al., 1996; O'Neil et al., 2002). However, no work has been done to compare directly the mutation rate for RNA polymerase II versus the mutation rate for reverse transcriptase.

One of the most common mutations that occurs during retrovirus replication is a frame-shift mutation. Frame-shift mutations occur frequently within homo-oligomeric runs. In this report, a system was established to differentiate between the errors made by the two polymerases. A colour reporter gene encoding the green fluorescent protein (GFP) with poly-oligomeric runs at the 5' end of its open reading frame (ORF) was inserted into a vector. If the insertion was in frame, the colour reporter gene would be functional so that cells that encode this gene would emit a green light. On the other hand, if the insertion was out of frame, for example, if an extra A was inserted, the colour reporter gene would be non-functional. We assumed that the rate of frame-shift mutation caused by polymerase II was low, so that the intensity of green light from the in-frame gfp gene thus represented the value of 100 % of functional transcripts within cells. By comparing the intensity of the green light from the in-frame gfp product with that of an out-of-frame gfp gene, the backward frame-shift mutation frequency of host RNA polymerase II and the ribosome could be estimated. The backward mutation frequency was thus the ratio of intensity of green light from cells with an out-of-frame gfp gene to the fluorescence emitted from cells with an in-frame gfp gene. When a run of 10 adenosine (A) nucleotides was inserted, this frequency was only about 0·3 %, while the rate of mutation during a single cycle of retrovirus replication within the same 10 A stretch was about 11 %. This evidence suggested that the reverse transcriptase contributes to more than 97 % [(11–0·3)/11] of retroviral frame-shift mutations.


   METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Vector constructions
All recombinant techniques were carried out according to conventional procedures (Sambrook et al., 1989). All vector sequences are available upon request.

Construction of a gfp gene with homo-oligomeric runs to encode a protein that emits green light.
The gfp gene was isolated from the jellyfish Aequorea victoria. Plasmid pEGFP-N1 (Clontech) contained a cytomegalovirus (CMV) promoter, a restriction site polylinker including a BglII site, the gfp gene (Chalfie et al., 1994) and the SV40 polyadenylation sequence.

Homo-oligomeric runs were inserted into the 5' end of the gfp gene after its ATG start codon. The construction of the vectors is described in Fig. 1. To insert homo-oligomeric runs at the 5' end of the gfp gene, a gfp gene was created with a silent mutation at the 5' end so that this segment (nt 19–24 or GAGCTC) could be digested with SacI. The resulting vector was designated pJZ506. A linker was then made by hybridization of two artificially synthesized oligonucleotides. For example, for the insertion of 15 As, the first oligonucleotide was 5'-GATCTACCATGGTGAAAAAAAAAAAAAAAGAGCT-3' and the second oligonucleotide was 5'-CTTTTTTTTTTTTTTTCACCATGGTA-3'. The 5' end of the annealed DNA was identical to the sticky end that was formed by digestion with BglII and the 3' end was identical to the sticky end that was formed by digestion with SacI. This linker contained homo-oligomeric runs of 15 As and was ligated into the pJZ506 vector, which had been digested with BglII and SacI. The resulting vector was designated p15A-GFP. Different numbers of As were inserted as described above. All insertions of oligomeric linkers were confirmed by sequence analysis.



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Fig. 1. Detection of slippage within oligomeric runs. Vectors were constructed based on pEGFP-N1, which contains the CMV promoter, the gfp gene and the SV40 polyadenylation sequence. A linker containing an oligomeric run was inserted at the 5' end of the gfp ORF after its start codon. Vector structure and light and fluorescence microscopy of CHO-K1 cells transfected with the vector are shown for vectors containing 15 (a), 1 (b) and 16 (c) As.

 
Construction of retroviral vectors containing the gfp gene with homo-oligomeric runs.
Retroviral vectors were constructed based on MoMLV (Sapp et al., 1999) and contained a gfp gene as described above, an internal ribosome entry site (IRES) and the rfp (red fluorescent protein, RFP) gene. The rfp gene was derived from pDsRed1-N1 (Clontech). The vector JZ508, which has been described previously, contains a gfp gene, an IRES and a hyg gene (Li & Zhang, 2002). The hyg gene of JZ508 was replaced by the rfp gene and the gfp gene of JZ508 was replaced by a gfp gene with homo-oligomeric runs, as described above.

Cells, transfection and infection.
PG13 is an NIH3T3-derived MoMLV-based helper cell line that supports the propagation of MoMLV-based vector virus (Miller et al., 1991). D17 is a dog osteosarcoma cell line obtained from ATCC (CRL-6248). Transfection, infection and the maintenance of cells have been described previously (Zhang & Temin, 1993).

Fluorescence microscopy.
A fluorescence inverted microscope (Zeiss, Axiovert 25) with a mercury arc lamp (100 W) and a fluorescent filter set (CZ909) consisting of a 470/40 nm exciter, a 515 nm emitter and a 500 nm beamsplitter were used to detect GFP in living cells.

Flow cytometric analysis.
This was conducted using a FACSCalibar (Becton Dickinson). A total of 10 000 events was acquired using the SL2-H histogram with the marker set at position 305 on the left. The FL-1 emission channel was used to monitor the intensity of fluorescence of the GFP.


   RESULTS
Top
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Detection of slippage within oligomeric runs
To detect errors made by RNA polymerase II, in-frame or out-of-frame oligomeric A runs were added to the 5' end of the ORF of the gfp gene, after the ATG start codon. The mutated gfp genes were expressed by a CMV promoter and had the SV40 polyadenylation signal (Fig. 1). Plasmid DNA encoding the mutated gfp gene was then used for transfection into CHO-K1 cells (ATCC CCL-61). The cells were examined by fluorescence microscopy 2 days after transfection. When 15 As were added to the gfp gene, resulting in the addition of five lysines to the end of the GFP, cells that were transfected with the 15A–gfp expression vector emitted a very bright green fluorescence (Fig. 1a). However, when only one A was added to the gfp gene, no fluorescence was detected because this resulted in the reading frame of the gfp gene being out of frame (Fig. 1b). When cells were transfected with the 16A–gfp expression vector, insertion of 16 As should also cause the gfp gene to be out of frame. Thus, if no frame-shift mutation occurred during transcription or translation, every protein translated from the RNA should be non-functional. However, the cells did emit a dim green fluorescence (Fig. 1c), suggesting that this long A run allowed the formation of a small proportion of functional GFP.

Frequency of slippage at an adenosine oligomeric run increases with length of the run
The experiment described above could not accurately determine the fluorescence of each vector, since the number of transfected cells could not be determined. To determine quantitatively the proportion of functional GFP, a gfp gene with an additional 1, 4, 7, 10, 16 or 22 As was cloned into a retroviral vector (Fig. 2a). This retroviral vector was derived from MoMLV and contained, from 5' to 3', the 5' LTR, the packaging signal ({Psi}), the gfp gene with various oligomeric A runs, the IRES, the rfp gene and the 3' LTR (Fig. 2a). Each vector thus encoded two colour reporter genes, the gfp gene with additional As and the rfp gene. The resulting vectors were designated 1A-, 4A-, 7A-, 10A-, 16A- and 22A-GFP+RFP. The gfp gene was expressed via the traditional scanning model from the 5' end of the transcript, while the rfp gene was expressed via the IRES. Plasmid DNAs were transfected into a retroviral helper cell line, PG13 (Miller et al., 1991), derived from murine NIH3T3 cells. The virus (xenotropic) released from PG13 cells was packaged by envelope proteins derived from gibbon ape leukaemia virus, which is unable to infect murine cells including PG13 cells (Miller et al., 1991; Zhang & Temin, 1993). Transfected PG13 cells were analysed by flow cytometry 3 days after transfection. Red fluorescent cells represented cells transfected with the retroviral vector containing the rfp gene. Red cells were selected by flow cytometry, after which the green fluorescence of these cells was analysed (Fig. 3 and Fig. 4). The vector JZ517 encoding the rfp gene but not the gfp gene was used as a negative control. Untransfected PG13 cells and cells transfected with JZ517 did not emit green fluorescence. The relative fluorescence of each gfp gene with different out-of-frame insertions is shown in Fig. 4(a). Cells transfected with 1A-, 4A- and 7A-GFP+RFP did not emit detectable green fluorescence (Fig. 4a), while cells transfected with 10A-GFP+RFP emitted a discernible amount of green light. Cells transfected with 16A-GFP+RFP emitted the brightest green fluorescence, while cells transfected with 22A-GFP+RFP emitted less green fluorescence than 16A-GFP+RFP. It was assumed initially that the longer the oligomeric run, the more frequently slippage would occur. However, it was possible that the GFP with seven N-terminal lysines (21 As) emitted a less green fluorescence than the GFP with five N-terminal lysines (15 As). As a result, although more slippage error may have occurred within the 22 A oligomeric run, the individual functional protein emitted less fluorescence, resulting in a weaker green fluorescence overall.



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Fig. 2. (a) Structure of a retroviral vector encoding a gfp gene with an oligomeric run, the rfp gene and an IRES between the two genes. (b) Single cycle of retrovirus replication. Retroviral vectors were transfected into the helper cell line PG13. The viral structural proteins gag–pol and env were expressed in the PG13 cells. Transfected PG13 cells were analysed by flow cytometry for errors made by the host RNA polymerase II within the oligomeric run. Virus released from the helper cells was used to infect the target cells, D17. Infected cells were also analysed by flow cytometry for errors made by the viral reverse transcriptase.

 


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Fig. 3. Flow cytometric analysis of PG13 cells transfected with retroviral vectors. Transfected cells were selected based on red fluorescence, which represented cells infected with virus encoding RFP. Red cells were then analysed based on their green fluorescence. Three histograms are superimposed representing the vectors 1A-, 16A- and 15A-GFP+RFP. Each analysis is one of the three replicate experiments shown in Fig. 4.

 


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Fig. 4. Green fluorescence intensity of retroviral vectors in PG13 cells. The control vector (Cntl) was JZ517, which does not encode a gfp gene and thus represents background fluorescence. Other vectors were constructed with the gfp gene containing between 1 and 22 As added to the 5' ORF of the gfp gene. Cells were transfected with a vector containing an out-of-frame (a) or in-frame (b) insertion. The value for each vector is the mean of three replicate experiments.

 
To determine the relative effect of the number of N-terminal lysine residues on the fluorescence of GFP, vectors encoding in-frame gfp transcripts with 9, 15 and 21 As and the rfp gene were also constructed and analysed (Fig. 4b). The fluorescence of GFP with an additional three lysines (9 As) was 78 % (2191/2823) that of the protein without any insertion at the 5' end of the ORF. When the number of additional lysines was five (15 As) and seven (21 As), the fluorescence of GFP was 33 % (927/2823) and 9 % (250/2823), respectively. These results indicated that the more lysines there were at the N terminus of GFP, the less green fluorescence the protein emitted. Functional GFP from templates of the 10A-, 16A- and 22A-GFP+RFP vectors would result from the deletion of an A to make the gfp gene in frame. Therefore, when the fluorescence of the 10A-, 16A- and 22A-GFP+RFP vectors was normalized against the fluorescence of the 9A-, 15A- and 21A-GFP+RFP vectors, the frequency of the frame-shift mutation in 10A-GFP+RFP was 0·3 % [(16·81–10·90)/2191, or the difference in fluorescence intensity for 10A and JZ517 divided by the fluorescence intensity for 9A], in 16A-GFP+RFP was 3 % [(38·34–10·90)/927] and in 22A-GFP+RFP was 8 % [(30·92–10·90)/250]. As a result, we concluded that the frequency of errors increased when the length of the oligomeric run increased. The decrease in fluorescence resulted from a frame-shift by the host RNA polymerase II during transcription and/or frame-shift by the host ribosome during translation. Although we could not distinguish between these two frame-shift mechanisms, we could still conclude that the frame-shift mutation rate of RNA polymerase II must be less than 0·3 % for 10A-GFP+RFP and less than 3 % for 16A-GFP+RFP.

RNA polymerase II alone contributes less than 3–13 % of frame-shift mutations that occur during retrovirus replication
To determine the contribution of RNA polymerase II to the frame-shift mutations that occur during retrovirus replication, the virus released from the transfected PG13 helper cells, which were originally transfected with the retroviral vectors as described above, was used to infect D17 cells (Fig. 2b). Since D17 cells do not encode any viral proteins, any infection that occurs represents a single round of replication. The beginning of the replication cycle could be defined as the parental provirus in the helper cell line, PG13 (Fig. 2b), while the end of the replication cycle could be defined as the formation of progeny proviruses in the target D17 cells. When D17 cells were infected with vector GFP+RFP containing an rfp gene, the infected cells emitted a red fluorescence. Cells without red fluorescence represented uninfected cells. The red infected cells were further separated into two groups. The first group of red cells had a dim green fluorescence, indicating that most had an out-of-frame gfp gene. The second group of infected cells emitted a bright green fluorescence, which represented the fact that the proviruses encoded an in-frame gfp gene. The ratio of the number of bright green cells to the number of total red cells gave the rate of the frame-shift mutation within the gfp gene that resulted in a functional GFP. The total number of red cells and the number of bright green cells was determined by flow cytometry. The rate of frame-shift mutation was 11 % for 10A-GFP+RFP and 23 % for 16A-GFP+RFP. As described above, the frequency of error by the RNA polymerase II was 0·3 % and 3 % for 10A- and 16A-GFP+RFP, respectively, suggesting that 3 % (0·3/11) to 13 % (3/23) of the retroviral frame-shift mutations were the result of errors made by the host RNA polymerase II. Fig. 4 shows that the shorter runs resulted in a lower frequency of RNA polymerase II and ribosome frame-shifts. Therefore, the contribution of RNA polymerase II to retroviral frame-shift mutations was less than 3 %.


   DISCUSSION
Top
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Previous studies have demonstrated that reverse transcriptase contributes to 50 % or more of retroviral mutations (Kim et al., 1996; O'Neil et al., 2002). Our report is the first to compare mutations made by the reverse transcriptase with mutations made by the host RNA polymerase II. During a single cycle of retrovirus replication within a 16 A oligomeric run, at most 13 % of frame-shift mutations could be the result of errors made by the host RNA polymerase II. Since the contribution of RNA polymerase II to retroviral frame-shift mutations was less than 3 % when the oligomeric runs were 10 As and most retroviral genomes do not contain oligomeric runs that are as long as 10 As, then the contribution of RNA polymerase II to frame-shift mutations in wild-type retroviruses is probably less than 3 %. Thus, we estimated that more than 97 % of frame-shift mutations were caused by the reverse transcriptase.

The difference between 3 % for 10 As and 13 % for 16 As probably resulted from an underestimate of the total rate of frame-shift mutation during virus replication when the oligomeric run was 16 As. If the rate of the frame-shift mutation by reverse transcriptase was extremely high, the highest rate detected by this system should be 33 % because all three of the reading frames would be equally distributed. However, the total rate of mutation for 16A was determined to be 23 %, probably because many proviral DNAs contained double frame-shift mutations, resulting in a dim green fluorescence. Therefore, although there were two percentages, 3 % for 10A and 13 % for 16A, the estimate of 3 % is more likely to be accurate for the contribution that the host RNA polymerase II makes to retroviral frame-shift mutation.

The frequency of frame-shift mutation for a 10 A oligomeric run by RNA polymerase II was 0·3 % while the total rate of frame-shift mutation was 11 % for this sequence. It is not difficult to find an oligomeric run within retroviral sequences. For example, one strain of HIV-1 (AF033819) contains a run of 12 As (with a G in the middle of the run). Comparison of homologous sequences in GenBank indicates that this region is much more variable than its flanking sequence. This suggests that frame-shift mutations may play an important role in HIV evolution. In addition, the rate of frame-shift mutations was determined during a single cycle of replication in this report; however, countless cycles of replication occur in patients with HIV over the many years that it takes for this disease to run its course.

The first limitation of this study was that we could not test mutations other than frame-shift mutations made by RNA polymerase II since the frequencies of other mutations such as point mutations by the polymerase is low, so they could not be detected utilizing the assay described in this report. Further work needs to be done to detect other kinds of mutations. The second limitation of this study was that the frequency of errors we detected was actually the sum of errors made by the RNA polymerase II during transcription and the slippage errors made by the ribosome during translation. If the ribosome also demonstrates slippage within an oligomeric run, our conclusion that more than 97 % of frame-shift mutations are made by reverse transcription should still stand.


   ACKNOWLEDGEMENTS
 
We thank W. Bargmann, R. Hoppe and C. Sapp for helpful comments on the manuscript. We thank J. Strange and C. Bauman for flow cytometric analysis and M. Russ for sequencing analysis. This research was supported by Public Health Service research grant CA70407.


   REFERENCES
Top
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
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Received 2 March 2004; accepted 7 May 2004.



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