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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 104 and 105 (Blank et al., 1986; Erie et al., 1993
; Libby & Gallant, 1991
), while that of wheat-germ RNA polymerase II in vitro is less than 1x103 (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 % [(110·3)/11] of retroviral frame-shift mutations.
![]() |
METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 1924 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.
|
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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 (
), 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.
|
|
|
RNA polymerase II alone contributes less than 313 % 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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Blank, A., Gallant, J. A., Burgess, R. R. & Loeb, L. A. (1986). An RNA polymerase mutant with reduced accuracy of chain elongation. Biochemistry 25, 59205928.[Medline]
Boyer, J. C., Bebenek, K. & Kunkel, T. A. (1992). Unequal human immunodeficiency virus type 1 reverse transcriptase error rates with RNA and DNA templates. Proc Natl Acad Sci U S A 89, 69196923.[Abstract]
Chalfie, M., Tu, Y., Euskirchen, G., Ward, W. W. & Prasher, D. C. (1994). Green fluorescent protein as a marker for gene expression. Science 263, 802805.[Medline]
de Mercoyrol, L., Corda, Y., Job, C. & Job, D. (1992). Accuracy of wheat-germ RNA polymerase II. General enzymatic properties and effect of template conformational transition from right-handed B-DNA to left-handed Z-DNA. Eur J Biochem 206, 4958.[Abstract]
Erie, D. A., Hajiseyedjavadi, O., Young, M. C. & von Hippel, P. H. (1993). Multiple RNA polymerase conformations and GreA: control of the fidelity of transcription. Science 262, 867873.[Medline]
Gnatt, A. L., Cramer, P., Fu, J., Bushnell, D. A. & Kornberg, R. D. (2001). Structural basis of transcription: an RNA polymerase II elongation complex at 3·3 A resolution. Science 292, 18761882.
Kim, T., Mudry, R. A., Jr, Rexrode, C. A., II & Pathak, V. K. (1996). Retroviral mutation rates and A-to-G hypermutations during different stages of retroviral replication. J Virol 70, 75947602.[Abstract]
Li, T. & Zhang, J. (2002). Intramolecular recombinations of Moloney murine leukemia virus occur during minus-strand DNA synthesis. J Virol 76, 96149623.
Libby, R. T. & Gallant, J. A. (1991). The role of RNA polymerase in transcriptional fidelity. Mol Microbiol 5, 9991004.[Medline]
Linton, M. F., Pierotti, V. & Young, S. G. (1992). Reading-frame restoration with an apolipoprotein B gene frameshift mutation. Proc Natl Acad Sci U S A 89, 1143111435.[Abstract]
Mansky, L. M. (2000). In vivo analysis of human T-cell leukemia virus type 1 reverse transcription accuracy. J Virol 74, 95259531.
Mansky, L. M. & Temin, H. M. (1995). Lower in vivo mutation rate of human immunodeficiency virus type 1 than that predicted from the fidelity of purified reverse transcriptase. J Virol 69, 50875094.[Abstract]
Miller, A. D., Garcia, J. V., von Suhr, N., Lynch, C. M., Wilson, C. & Eiden, M. V. (1991). Construction and properties of retrovirus packaging cells based on gibbon ape leukemia virus. J Virol 65, 22202224.[Medline]
O'Neil, P. K., Sun, G., Yu, H., Ron, Y., Dougherty, J. P. & Preston, B. D. (2002). Mutational analysis of HIV-1 long terminal repeats to explore the relative contribution of reverse transcriptase and RNA polymerase II to viral mutagenesis. J Biol Chem 277, 3805338061.
Preston, B. D. & Dougherty, J. P. (1996). Mechanisms of retroviral mutation. Trends Microbiol 4, 1621.[CrossRef][Medline]
Preston, B. D., Poiesz, B. J. & Loeb, L. A. (1988). Fidelity of HIV-1 reverse transcriptase. Science 242, 11681171.[Medline]
Sambrook, J., Fritsch, E. F. & Maniatis, T. (1989). Molecular Cloning: a Laboratory Manual, 2nd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
Sapp, C. M., Li, T. & Zhang, J. (1999). Systematic comparison of a color reporter gene and drug resistance genes for the determination of retroviral titers. J Biomed Sci 6, 342348.[Medline]
Thomas, M. J., Platas, A. A. & Hawley, D. K. (1998). Transcriptional fidelity and proofreading by RNA polymerase II. Cell 93, 627637.[Medline]
van Leeuwen, F. W., de Kleijn, D. P., van den Hurk, H. H. & 11 other authors (1998). Frameshift mutants of beta amyloid precursor protein and ubiquitin-B in Alzheimer's and Down patients. Science 279, 242247.
Young, M., Inaba, H., Hoyer, L. W., Higuchi, M., Kazazian, H. H., Jr. & Antonarakis, S. E. (1997). Partial correction of a severe molecular defect in hemophilia A, because of errors during expression of the factor VIII gene. Am J Hum Genet 60, 565573.[Medline]
Zhang, J. & Temin, H. M. (1993). Rate and mechanism of nonhomologous recombination during a single cycle of retroviral replication. Science 259, 234238.[Medline]
Received 2 March 2004;
accepted 7 May 2004.
HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
INT J SYST EVOL MICROBIOL | MICROBIOLOGY | J GEN VIROL |
J MED MICROBIOL | ALL SGM JOURNALS |