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Originally published In Press as doi:10.1074/jbc.M204774200 on July 31, 2002

J. Biol. Chem., Vol. 277, Issue 41, 38053-38061, October 11, 2002
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Mutational Analysis of HIV-1 Long Terminal Repeats to Explore the Relative Contribution of Reverse Transcriptase and RNA Polymerase II to Viral Mutagenesis*

Patrick K. O'NeilDagger §, Guoli Sun§||, Hong Yu**, Yacov Ron, Joseph P. DoughertyDaggerDagger, and Bradley D. PrestonDagger §§

From the Dagger  Department of Biochemistry and Radiation Oncology, Eccles Institute of Human Genetics, University of Utah, Salt Lake City, Utah 84112, the  Department of Molecular Genetics, Microbiology and Immunology, University of Medicine and Dentistry of New Jersey-Robert Wood Johnson Medical School, Piscataway, New Jersey 08854, and the || Graduate Program in Microbiology and Molecular Genetics, Rutgers University, New Brunswick, New Jersey 08903

Received for publication, May 15, 2002, and in revised form, July 22, 2002

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

HIV-1 evolves rapidly, which is thought to result from one or more error-prone steps in the virus life cycle. Because HIV-1 reverse transcriptase (RT) does not possess 3'- to 5'-exonucleolytic proofreading activity and because RT has been shown to be error-prone in cell free systems, it should be an important contributor to the high rate of HIV-1 mutation. However, because RNA polymerase II (pol II) synthesizes viral RNA, it might also contribute significantly to HIV-1 mutagenesis. To assess the relative contributions of RT and RNA pol II to HIV-1 mutagenesis, a system was established to study the rate and nature of mutations in HIV-1 long terminal repeats (LTRs). Owing to the unique nature of replication at the ends of the viral genome, mutational analysis of retroviral LTRs provides an opportunity to evaluate the relative contribution of HIV-1 RT and RNA pol II to viral mutagenesis. Mutational analysis was performed on both LTRs of 215 proviruses, restricted to a single cycle of replication, employing single-stranded conformational polymorphism and DNA sequencing allowing direct identification of mutations in the absence of selection and within autologous viral sequences. A total of 21 independent mutations was identified. Ten mutations were observed in both LTRs, which could have been introduced by either RT or RNA pol II, whereas the other eleven mutations were only present in a single LTR and could only have been introduced by RT. This provides the first direct evidence that HIV-1 RT contributes significantly to HIV-1 mutagenesis and is likely to be the primary engine for HIV-1 mutagenesis. Moreover, mutations were observed at the U3-R border, but the nature of the mutations and their frequency differed from experiments performed using cell-free systems suggesting that other viral and/or cellular factors contribute to fidelity at the ends of the viral genome.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

HIV-11 genomes evolve rapidly (1-3) with genetic diversity having been extensively documented in sequential isolates taken from patients (4-6). Sequence analyses revealed that virus isolates consist of multiple genomic subclasses, which fluctuate substantially during the course of infection, so HIV-1 exists as complex genetically heterogeneous populations termed "swarms" or "quasispecies" (7, 8). This characteristically imperfect replication process is responsible for the generation of genetic diversity, which is advantageous for responding to selective forces. However, this genetic diversity imposes serious hurdles for the treatment of AIDS.

Theoretically, retroviral mutations can be introduced during transcription by RNA polymerase II (pol II), minus- or plus-strand DNA synthesis by RT, or provirus replication by cellular DNA polymerases. Due to the notable fidelity of cellular DNA replication (10-9 to 10-11 substitution per base pair) (9), it is unlikely that mutations occurring during cellular replication of the provirus contribute significantly to the high degree of HIV-1 diversity. Therefore, most mutations are introduced during reverse transcription and/or RNA pol II transcription of the provirus.

Unlike cellular replicative DNA polymerases, HIV-1 RT does not possess 3'- to 5'-exonucleolytic proofreading activity (10). Fidelity studies using purified HIV-1 RT in cell-free systems indicate that nucleotide misincorporation frequencies are quite high (2.5 × 10-4 to 5.8 × 10-4 per nucleotide) (11-14), although it is noteworthy that they are typically greater than the overall rate of mutation observed for HIV-1 during virus replication indicating that other factors contribute to the overall fidelity in vivo (14-17). Nevertheless, based upon these studies, it has been proposed that RT is responsible for the high genetic variability seen for HIV-1 (14, 18). However, RNA pol II might also contribute significantly to HIV-1 sequence variation during replication, and it is plausible that RNA pol II is an important contributor to HIV-1 mutagenesis.

To gain insight into the relative contribution of HIV-1 RT and cellular RNA pol II to HIV-1 mutagenesis, mutational analysis of autologous HIV-1 long terminal repeats (LTRs) was performed after restricting replication of HIV-1-derived vectors to a single cycle. Due to unique aspects of replication of the viral LTRs, a mutation that is found in a single LTR could only be introduced by RT, whereas the same mutation found in both LTRs could be introduced by either RT or RNA pol II (see Fig. 1). Thus, by examining the LTRs, insight can be gleaned concerning the contributions of RT and RNA pol II to HIV-1 mutagenesis. Moreover, direct examination of autologous HIV-1 sequence allows determination of HIV-1 mutation rates of authentic HIV-1 sequences in contrast to the mutation rate of exogenous, non-viral marker sequences (19-21). Lastly, analysis of the autologous LTR sequence also allows one to address whether there is a mutational hotspot at the U3-R border of the HIV-1 LTR. Cell-free studies using purified HIV-1 RT indicated that, when DNA synthesis reaches the end of the viral RNA template, non-templated additions of at least one nucleotide occur with a frequency approaching 50%, which would result in the introduction of one or more base pairs at the U3-R junctions of an integrated provirus (Fig. 1) (22, 23).


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  		Fig. 1.   Mutations within the viral LTRs can yield information related to the stage of their occurrence during reverse transcription. Depicted in this figure is the accepted mechanism of retroviral reverse transcription. Also noted are the consequences of introducing mutations during different steps in the life cycle in relation to their occurrence in the LTRs. If a mutation occurs during RNA pol II transcription within the viral RNA sequence utilized as template for synthesis of the viral LTRs, then the same mutation ends up in both LTRs of the progeny provirus (open circle ). If a mutation occurs early during reverse transcription such as during minus strand primer synthesis, this mutation will also be found in both LTRs (). However, if a mutation occurs later during reverse transcription, such as during plus strand primer synthesis () or during the last stage of reverse transcription when the LTRs are completely synthesized (triangle ), it will only appear in one LTR. Thus, mutations found in both LTRs might have been incorporated either by RNA pol II or RT, whereas mutations found in one LTR would have been incorporated by RT alone. LTR, long terminal repeat; ppt, polypurine tract; pbs, primer binding site; U3, unique sequence at the 3'-end of the viral RNA; R, terminal direct repeat; U5, unique sequence at the 5'-end of the viral RNA.


    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Plasmid Construction-- pHIV-gpt and NL4-3 proviral DNA plasmids were obtained from the AIDS Research and Reference Reagent Program. pHIV-gpt is an HIV-1 vector based on the HXB2 strain (GenBankTM accession number K03455), which has deletions, mutations, and disruptions in the env, nef, vif, and vpr genes (24, 25). NL4-3gpt was based on HIV-1 strain NL4-3 (GenBankTM accession number AF070521) (26). To make NL4-3gpt, a 1.2-kbp portion of the envelope gene was deleted from the NdeI site at HIV nucleotide 6402 to the BglII site at HIV nucleotide 7620. A 1.1-kb fragment from pSV2gpt (27) from PvuII to DraI was inserted at the deletion site. The inserted fragment includes the simian virus 40 (SV40) origin of replication and promoter and coding sequences for the gpt gene. pTIRevEnv was constructed by inserting HIV rev and env coding sequences (nucleotides 5538 to 8607) into pUHD10-3 (28) between the EcoRI and BamHI sites. The inserted HIV rev and env coding sequences were under the control of the tetracycline-responsive inducible promoter (Fig. 2).

Cell Culture-- The human embryonal kidney cell line, 293T, was grown in Dulbecco's modified Eagles' medium (DMEM) supplemented with 10% fetal bovine serum (HyClone, SH300071.03). The #69TIRevEnv packaging cell line (29) was grown in DMEM supplemented with 10% fetal bovine serum, 0.2 mg/ml G418 (Sigma-Aldrich Inc.), 0.1 mg/ml hygromycin (Calbiochem Chem. Co.), and 2 µg/ml tetracycline. The HeLaT4 cell line was grown in DMEM supplemented with 10% fetal bovine serum and 0.1 mg/ml hygromycin.

Transfections and Infections-- Plasmid DNA was transfected by the modified calcium phosphate precipitation method (30) into the human 293T cell line. The producer cells containing HIV-1 vector were plated at 2 × 105 cells per 60-mm diameter dish with 2 µg/ml tetracycline (Sigma-Aldrich Inc.) and were induced by withdrawal of tetracycline 24 h after plating. HIV-1 vector virus was harvested 5 days past induction. Infection of HeLaT4 cells (3×105) was performed by first treating with 2 ml of media containing 2 µg/ml Polybrene for 30 min after which HeLaT4 were inoculated with 0.3 ml of 10-fold serial dilutions of virus supernatant for 2 h. Cells were then selected for xanthine-guanine phosphoribosyl transferase resistance with 0.25 mg/ml xanthine (Sigma-Aldrich Inc.), 15 µg/ml hypoxanthine (Aldrich Chem. Co.), plus 7 µg/ml mycophenolic acid (Sigma-Aldrich Inc.) to obtain target cell clones.

PCR and Gross Rearrangement Screening-- All polymerase chain reactions in this study used 50 mM KCl, 10 mM Tris-HCl (pH 8.0), 1.5 mM MgCl2, 200 µM of each dNTP, 5 µM of each primer, 0.7 mg of genomic DNA, and 2.5 units of Taq DNA polymerase (Roche Molecular Biochemicals) in a final volume of 40 µl.

The initial PCR reactions were used to screen each proviral LTR on agarose gels for gross rearrangements. Primer names are prefixed with a letter indicating whether the primer is specific for HXB2 (H), NL4-3 (N), or both (B), followed by a number indicating the coordinate of the 5'-end of each oligonucleotide and a "+" or "-" to denote the sense or antisense primer, respectively. The primer pairs used to amplify 5'-LTRs were B8+ (5'-GCTAATTCACTCCCAAAGAAGC-3') and B732- (5'-CCCTCGCCTCTTGCCGTGC-3') producing a normal amplicon length of 725 bp. Amplification conditions were 94 °C for 30 s, 55 °C for 40 s, and 72 °C for 50 s, carried out for 30 cycles. Amplification of 3'-LTRs was performed using primers B9017+ (5'-ACCTTTAAGACCAATGACAAGG-3') and B634- (5'-TGCTAGAGATTTTCCACACTGAC-3') anticipated to generate a product of 702 bp. Amplification conditions for this PCR were: 95 °C for 30 s, 60 °C for 40 s, and 72 °C for 50 s, for 30 cycles.

Single-stranded Conformation Polymorphism-- PCR fragments used for SSCP were restricted to between 150 and 180 bp in length. Each PCR reaction contained 0.2 mCi of [alpha -32P]dCTP, and the reaction conditions used were the same as those described above for the 5'-LTR. The following primer pairs were used for screening the LTRs by SSCP: SSCP1 (H8+ (5'-GCTAATTCACTCCCAAAGAAG-3') and H167- (5'-CTTCTGGCTCAACTGGTACTAG-3') or N8+ (5'-GCTAATTTGGTCCCAAAAAAG-3') and N168- (5'-CTTGCTCTGGTTCAACTGGTAC-3')), SSCP2 (B123+ (5'-CCTTTGGATGGTGCTACAAGCTAG-3') and B275- (5'-GCGGCTGTCAAACCTCCACTCTAAC-3')), SSCP3 (B244+ (5'-GAGAAGTGTTAGAGTGGAGGTTTGACAG-3') and B400- (5'-CCAGTCCCGCCCAGGCCACGC-3'), SSCP4 (B347+ (5'-ACTTTCCGCTGGGGACTTTCCAGG-3') and B507- (5'-CCCTAGTTAGCCAGAGAGCTCCCAGG-3')), and SSCP5 (B482+ (5'-CCTGGGAGCTCTCTGGCTAACTAGGG-3') and B634- (5'-TGCTAGAGATTTTCCACACTGACTAAAAG-3')).

PCR products were mixed with 6× loading dye (95% formamide, 20 mM EDTA, 0.05% xylene cyanol) (31), heated at 94 °C for 3 min, then placed on ice for 5 min before loading onto the gel. The non-denaturing gels either contained 6% polyacrylamide and 5% glycerol or were mutation detection enhancement gels made according to the manufacturer's instructions (FMC Bioproducts, Rockland, ME). SSCP gels were run at 32 watts at 4 °C until the xylene cyanol dye front was within 50 to 80 mm from the bottom of the gel. Typical running times were ~6.5 h. Then the gels were dried and exposed in PhosphorImager cassettes overnight before scanning (Molecular Dynamics Model 400E). Candidate mutations detected by SSCP were confirmed by independent PCR amplification and DNA sequencing.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Protocol for the Study of HIV-1 Mutation Rate in a Single Cycle System-- Mutational events were studied by examining provirus sequences after a single cycle of virus replication. Two similar HIV-1 vectors, HIV-gpt (24) and NL4-3gpt, derived from the HXB2 and NL4-3 strains, respectively, were employed in these experiments (Fig. 2). They were constructed by deleting a large part of env and inserting the gpt resistance gene under control of the SV40 early gene promoter. The respective deletions only disrupted the env reading frame, leaving the other coding sequences intact (Fig. 2).


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  		Fig. 2.   HIV-1 vectors and packaging cell constructs. The two HIV-1 vectors were derived from HIV-1 strains HXB2 and NL4-3. Approximately 1.1 kb of env was deleted and the xanthine-guanine phosphoribosyl transferase (gpt) gene under control of the simian virus 40 (SV40) early gene promoter was inserted. Boxes interrupted by jagged lines contain partial deletions. tetO, heptamerized tetracycline operator fused to a minimal cytomegalovirus promoter; SV-gpt, the gpt gene under control of the SV40 early gene promoter; SV40 pA, SV40 polyadenylation signal; *, defective genes.

The vector virus was subjected to a single cycle of replication followed by molecular analyses of the LTRs according to the protocol outlined in Fig. 3. The HIV-1 vector plasmid (HIV-gpt or NL4-3gpt) was first cotransfected with an amphotropic murine leukemia virus env expression plasmid pEnvAm (32) into human 293T cells to produce pseudotyped HIV-1 vector virus (Fig. 3). 2-3 days later, pseudotyped HIV-1 vector virus was harvested and utilized to inoculate an HIV-1 env inducible cell line, which is CD4-negative at a low multiplicity of infection (29). Individual cell clones harboring a HIV-1 vector provirus were selected, isolated, expanded, and designated Step 2 cell clones (Fig. 3). Because these cell clones resulted from infection at a low multiplicity of infection, each was anticipated to harbor a single provirus, which was confirmed by Southern blotting (data not shown). Within the Step 2 cell clones, expression HIV-1 rev and env was controlled by a tetracycline-responsive promoter. After tetracycline removal, Rev and Env production ensued initiating vector virus production. The vector virus produced from the Step 2 cell clones were then used to infect CD4-positive HeLaT4 cells followed by selection with gpt, with subsequent isolation and expansion of these Step 3 cell clones. Fourteen Step 2 cell clones were utilized to generate 215 Step 3 cell clones, averaging ~15 progeny Step 3 clones derived from each Step 2 cell clone. From Step 2 clones to Step 3 clones, the vector virus was restricted to a single cycle of replication. Vector virus produced by Step 2 clones could not re-infect the producer cells, because they were CD4-negative, and the HeLaT4-based Step 3 cells could not complement the defective vector, because they do not express Env (Fig. 3). Proviral DNA extracted from Step 3 clones was subject to further molecular analyses.


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  		Fig. 3.   Protocol for HIV-1 mutation rate study. The viral vector was cotransfected with a plasmid, which expresses amphotropic MLV env, into 293T cells, a human embryonic kidney cell line (Step 1 cells). Pseudotyped vector virus was used to infect a CD4-negative HIV-1 env inducible cell line (Step 2 cells). Upon induction, virus was harvested and used to infect CD4-positive HeLaT4 target cells, followed by selection and isolation of cell clones (Step 3 cells). The proviruses in these Step 3 cells were further analyzed. Replication from Step 2 cells to Step 3 cells is confined to a single cycle of replication. mpro, MLV U3 promoter; ampho-env, amphotropic MLV envelope gene; SV-gpt, SV40 early gene promoter and coding sequences for the gpt gene; tetO, heptamerized tetracycline operator fused to a minimal cytomegalovirus promoter.

Molecular Analyses of Viral LTRs-- The impetus for examining the viral LTRs was to gain insight into the relative contribution to viral mutagenesis by HIV-1 RT and RNA pol II (Fig. 1). Identical mutations found within both LTRs could have been introduced by either RNA pol II or RT, but mutations found in only one LTR would have been introduced by RT alone (Fig. 1).

Molecular analyses of Step 3 provirus LTRs were carried out employing PCR amplification combined with agarose gel electrophoresis, single-stranded conformation polymorphism (SSCP), and automated DNA sequencing. This allowed identification of mutations in the context of autologous HIV-1 sequence without the need for selection. To detect gross genetic rearrangements, the proviral LTRs were first amplified using appropriate primers. The 5'-LTR was amplified using a primer pair composed of one oligonucleotide with homology to the 5' most proviral sequence and the other with homology to sequence just downstream of the primer binding site (see "Experimental Procedures" for specific coordinates). This primer pair was anticipated to yield a 725-bp product. The 3'-LTRs were amplified with one primer corresponding to the 3' most end of the provirus and the other just upstream of the polypurine tract and was expected to yield a 702-bp product. The PCR samples were then subjected to electrophoresis in a 1.2% agarose gel and visualized by staining with ethidium bromide. Of the 430 LTRs analyzed, corresponding to 215 vector proviruses, only five did not display the anticipated mobilities (Fig. 4A).


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  		Fig. 4.   A, a representative example of mutational screening by PCR of the 5'-LTRs of Step 3 proviruses. The band of clone 8 is downshifted compared with the control, indicating a deletion. B, SSCP analysis of a portion of the LTRs in Step 3 proviruses. The alpha -32P-labeled PCR-amplified products were electrophoresed through a non-denaturing gel. The single-strand DNA conformers are indicated as well as the double-strand DNA. The band in lane 10 is shifted compared with control, indicating a mutation.

To detect single base mutations, the SSCP assay was employed. This is a relatively simple procedure for detecting single base mutations in a nucleic acid. The PCR product is denatured and electrophoresed in a non-denaturing polyacrylamide gel. Mutated fragments will typically display an altered mobility. Conditions were employed that allowed detection of ~80% of point mutations (33). Preliminary experiments were performed with five samples, each with a single base substitution within the LTR that had been identified by direct DNA sequencing. It was possible to identify four of five mutants via SSCP using the conditions described under "Experimental Procedures" and is in agreement with previous publications attesting to the mutational scoring efficiency of this procedure (31, 34, 35) (data not shown). It is noteworthy that SSCP is a more efficient method for scoring mutations relative to utilizing marker gene systems where it has been estimated that ~40% of mutations are scored with the remainder being silent (20, 36, 37). Five primer pairs were designed to amplify both LTRs of proviral DNA. Each fragment spanned 150-180 bp with adjacent fragments overlapping each other. The PCR products were isotopically labeled with alpha -32P, and after amplification, products were denatured and electrophoresed in a non-denaturing polyacrylamide gel followed by visualization using a PhosphorImager (Amersham Biosciences). DNA fragments that displayed a shift in mobility were anticipated to contain mutations. A representative example is shown in Fig. 4B. Mutations were confirmed by automated sequencing. Any segment that yielded a mutation was amplified and sequenced a second time to confirm that the shift did not result from a mutation introduced during PCR amplification. Given that the frequency of mutation introduced by Taq polymerase is 1 × 10-4 per base pair (38), the chance that the same mutation would arise a second time in the same position then becomes 1 × 10-8, which is three to four logs lower than the mutation frequency observed in this analysis.

It should be noted that controls were performed to exclude the possibility that the mutations scored in the Step 3 progeny proviruses were actually fixed in the Step 2 cell proviruses during their development, which involved transfection and one cycle of replication. To that end, the DNA sequence of the LTRs of all 14 Step 2 cell proviruses was determined. The DNA sequence of 13 Step 2 proviruses corresponded exactly to the parental sequence. The DNA sequence of one of the Step 2 proviruses contained a single base substitution, and consequently that base substitution was not scored as a mutation when observed in all of the corresponding Step 3 cell progeny (Figs. 5 and 6). In no other case was an identical mutation observed in all of the Step 3 proviruses originating from a particular Step 2 cell clone. Thus, the mutations denoted in Figs. 5 and 6 occurred during replication from the Step 2 to Step 3 cells.


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  		Fig. 5.   LTR sequence alignment of HXB2 and NL4-3 strains of HIV-1 showing the locations of point mutations identified by SSCP and DNA sequencing. The mutations labeled above the sequences are those identified in HIV-gpt proviruses, whereas the ones underneath were found in NL4-3gpt proviruses. All the mutations shown are single base substitutions except for one deletion at nucleotide 456 and three consecutive base substitutions shown in parentheses from nucleotides 377 to 379. The mutations marked with superscript 1 occurred only in 5'-LTRs. The mutations marked with superscript 2 occurred in both LTRs, whereas the mutations marked with superscript 3 were found only in 3'-LTRs.


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  		Fig. 6.   Genetic Rearrangement. A, the sequences of the relevant segments of the rearrangements are shown. The rectangles indicate sequence overlap at the point of mutation. The boldface letters indicate a point mutation. B, schematic representation of the aforementioned mutations.

Observed Mutations and Rates-- As noted above, two HIV-1 vectors, HIV-gpt and NL4-3gpt, based on strains HXB2 and NL4-3, respectively, were used in these studies. This allows the comparison of mutation rates between two different strains one of which, HIV-gpt, contains mutations within some of the accessory genes, in particular Vpr, which has been reported to influence fidelity (Fig. 2) (19). These two vectors were separately subjected to replication through a single cycle. A total of 215 Step 3 cell clones were collected and analyzed, of which 99 were from the HIV-gpt vector and 116 were from the NL4-3gpt vector. In all, 21 mutations were identified with 10 in both LTRs and 11 in a single LTR. The mutation rates obtained with HIV-gpt and NL4-3gpt were quite close, 9.2 × 10-5 and 7.9 × 10-5 per base pair per replication cycle, respectively, with the overall rate of mutation being 8.5 × 10-5 per base pair per replication cycle (Table I).

                              
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Table I
Summary of mutation types and rates

The predominant type of mutation was single base substitutions, of which there were 14, comprising 67% of the total number of mutations (Fig. 5 and Tables II and III). Among them, 10 mutations were transitions (8 G right-arrow A and 2 C right-arrow T transitions), and 4 mutations were transversions (one of each of the following, T right-arrow A, G right-arrow T, A right-arrow T, and C right-arrow A). One mutant LTR harbored three consecutive base substitutions consisting of a change from GAG to ACC, so it contains a mix of both a transition and two transversions (Fig. 5 and Table II). In addition to the base substitutions, there was a single frameshift deletion near the U3-R border of one mutation (Fig. 5).

                              
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Table II
Mutations that occurred in either 5' or 3' LTRs

                              
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Table III
Mutations that occurred in both LTRs

There were also five larger mutations observed: three were duplications (H17, H168, and N85), one deletion (N26), and one insertion (H40) (Fig. 6 and Table II). H17 had a duplicated U5 and part of R near the end of an almost complete 3'-LTR (Fig. 6). More specifically, the mutation is 20 bp from the end of the parental LTR. H168 had a nearly complete 3'-LTR duplication, which began after the 16th base pair of U5 of the first LTR with the second LTR starting 13 bp from the beginning of the parental U3 (Fig. 6). Although N85 has a normal 3'-LTR, it also contained two additional bases, GT, as well as a partial U5 duplication (Fig. 6). The pattern of duplications is similar for all three mutants and is likely to share a common mechanism, which is elaborated upon under "Discussion."

Of the remaining two larger mutations, one had a deletion and the other an insertion. The deletion, N26, consisted of a 266-bp deletion within the 3'-LTR (Fig. 6). The deleted sequence included part of U3 and U5 as well as all of R (Fig. 6). CTT was common to both breakpoints suggesting a simple misalignment copy-choice mechanism of mutagenesis described in more detail under "Discussion." The insertion, H40 (Fig. 6), is an unusual mutation containing a reverse read of the entire tRNALys, followed first by insertion of 4 bp of unknown origin and then by 66 bp apparently of cellular origin, because it is identical in 65 of 66 bp to part of a CpG island found on human chromosomes 6, 14, and 17 (39). Another G of unknown origin was also added after the CpG island sequence. These insertions were then followed by a parental pbs as well as being continued from there by parental viral sequence. This is the first example of transduction of cellular sequence by HIV-1 and was described in more detail elsewhere (39).

    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

In this study, mutational analysis of HIV-1 LTRs was performed after confining replication to a single cycle. Molecular analysis of the LTRs was carried out employing physical mapping and sequencing of mutations that score more effectively for mutations than gene marker systems and allow direct examination of autologous HIV-1 sequence. Of the 21 mutations observed, ten were found in both LTRs. These ten mutations could have been introduced during either RNA transcription by pol II or minus-strand DNA primer synthesis by RT (Fig. 1 and Table III). The other eleven mutations were found in only one LTR and would have been incorporated during later stages of reverse transcription by RT (Fig. 1 and Table II). Thus, at least 52% of the mutations were introduced by RT clearly indicating that HIV-1 RT has an important influence upon the generation of viral diversity.

In light of the finding that the rate of mutation early during replication, when either RNA pol II or HIV-1 RT would have introduced the mutations, was equivalent to the rate late in replication when only HIV-1 RT would have introduced the mutations suggests that HIV-1 RT is the main engine of diversity and that the influence of RNA pol II is less pronounced. On the other hand, it might be argued that the rate of mutation by RT is different when it utilizes an RNA as opposed to a DNA template. If the rate of mutation was lower when utilizing the RNA template, then mutation by RNA pol II would account for the rates being similar during the early stages of replication when both can contribute and during the later stages when only RT can contribute. However, it should be noted that this argument is not supported by in vitro experiments in which purified HIV-1 RT was shown to have a similar fidelity when using either an RNA or DNA template. So it seems more plausible that the rates are similar during early and late synthesis, because RT introduces most of the mutations (40-42).

Additional support for the contention that mutagenesis is primarily driven by HIV-1 RT and not by RNA pol II is supplied by recent studies indicating that RNA pol II exhibits proofreading activity (43, 44). As noted earlier, HIV-1 RT does not contain 3'- to 5'-exonuclease activity essential for proofreading (10). In contrast, it was found that RNA pol II had 3'- to 5'-exonuclease activity and that in the presence of transcription elongation factor SII could cleave misincorporated nucleotides from nascent transcripts during rapid RNA synthesis (43). Further support for the notion that RNA pol II transcription has associated proofreading was supplied by crystallographic studies. These studies indicated that a misincorporated nucleotide would be destabilizing causing extrusion of the misincorporated nucleotide from the active site in a back-tracked complex allowing cleavage of the misincorporated nucleotide (44).

The approach utilized in these experiments also allowed examination of whether the U3-R border represents a mutational hotspot for non-templated addition of nucleotides. Previous reports also using purified RT in cell-free systems reported that there was a high degree of misincorporation of additional bases when the newly synthesized DNA reached the 5'-end of the RNA template with the misincorporation frequency reaching 50% (22, 45). These results predict that base pair insertions should be found with a similarly high frequency at the U3-R junction, because insertions that occur when the nascent DNA reaches the 5'-end of the RNA template before minus strand primer transfer would result in their incorporation into both LTRs at the U3-R border (Fig. 1C). Of the 430 LTRs examined, not one contained additional base pairs precisely at the U3-R junction. There was a small clustering of mutations near the U3-R border consisting of three G to A transitions found in both LTRs from three different proviruses (Fig. 5). Two mutations were located within R with one mutation being a single base pair away from the junction and the other mutation removed by 2 bp from the junction. The third mutation was found in U3 positioned 8 bp from the junction. It is noteworthy that the only single-base pair deletion was discovered within R, 2 bp of the U3-R junction (Fig. 5), but it was only found in the 5'-LTR, so it is not likely that it occurred when DNA synthesis reached the 5'-end of the RNA template. Inasmuch as the clustering of mutations suggests a slight increase in the rate of mutation when RT reaches the end of the RNA template, it is quite clear that the nature of the mutations found is different and the frequency is much lower than predicted by the cell-free system studies. Thus, this would appear to be another example in which other viral and/or cellular factors have an effect upon reverse transcription during viral replication, which was not recapitulated in cell-free systems (40).

The rate of mutation observed in the LTRs is a bit higher than was previously observed when an HIV-1-derived shuttle vector was utilized in which the target sequence consisted of the lacZ peptide gene (15). The rate in that previous study was 3.4 × 10-5, whereas in this study, it is 8.5 × 10-5 or 2.5 times higher. At least three factors may account for this modest difference. One is that the rate of mutation might be a little higher at the ends of the viral genome. Another possibility is that the differences in the primary sequence might result in a slightly elevated rate. Lastly, direct examination using SSCP might result in more effective scoring of mutations as opposed to phenotypic scoring using the lacZ peptide gene. At this juncture, we favor this last explanation as accounting for most of the difference, because the SSCP conditions used score ~80% of mutations, whereas the phenotypic assay scores roughly 40% (20, 36).

HIV-1 vectors based upon two different strains, HXB2 and NL4-3, were utilized in these experiments. The HIV-1 sequences of these two strains differ by ~3% throughout their genomes, and HXB2 does not express functional Vif, Vpr, or Nef (46). The rate and spectrum of mutations were quite similar for both strains with the rates being 9.2 × 10-5 and 7.9 × 10-5 for the HXB2 and NL4-3 strains, respectively (Tables II and III). This finding was unexpected, because it had been reported that Vpr augments HIV-1 RT fidelity by 4-fold (19). Thus, it was anticipated that the rate of mutation for the HXB2 (Vpr -)-based vector would be also about 4-fold higher than that of the NL4-3 (Vpr+)-derived vector. One possible explanation is that the fidelity of HXB2 RT is higher than that of NL4-3, compensating for the lack of Vpr, and would therefore imply that the fidelity of different strains of HIV-1 RT can vary during replication. Some evidence has been obtained supporting this contention. A previous report demonstrated that HIV-1 variants, which have developed resistance to 2',3'-dideoxy-3'-thiacytidine due to a M184V mutation within RT, might have greater fidelity for decreasing the mutation rate and retarding the emergence of variants thus enabling a more effective immune response (47). Support for this notion was obtained by examining the characteristics of the M184V mutant RT in cell-free systems. Significant decreases were observed both in the frequency of misinsertions and in the efficiency of polymerized extensions of mispairs by the M184V mutant (47-49). Experiments are in progress to ascertain the range of variation in the rate of mutation between different viral strains.

The predominant forms of mutation in this study were single base substitutions, which comprised 67% of the mutations, and more than 50% of these base substitutions were G to A transitions. This finding is supported by a previous report using an HIV-1 shuttle vector system in which the majority of mutations were also base substitutions with a predominance of G to A mutations (15). Of the dinucleotides associated with the eight G to A transitions, three were GpG, two were GpA, two were GpT, and one was GpC (Fig. 5). These dinucleotide contexts are quite similar to other reports (50, 51). The most accepted model states that a G to A transition is caused by an imbalance of the [dTTP]/[dCTP] ratio, with [dTTP] being higher than [dCTP], increasing the chance for a T to be inserted opposite the G such that upon completion of DNA synthesis a G to A transition would result (52-54). If this is true in our case, the G to A transition should happen during minus strand synthesis and would be observed in both LTRs. In accordance with this model, seven of eight G to A transitions did occur in both LTRs.

Genetic rearrangements also occurred with similar frequencies in the two separate studies with the exception of frameshift mutations: only one frameshift out of 21 (4.8%) versus 26% in the previous study (15). Factors that might contribute to the difference are: (i) the primary sequence is different and has been shown to affect the spectrum of mutations in vitro and in vivo (55, 56) and (ii) the spectrum of mutations might be influenced by their position within the genome, because here mutations were analyzed at the ends of the genome, whereas, in the previous study, mutations were analyzed in a marker gene between the viral LTRs (13, 57).

One surprising aspect of the mutations observed was that all five of the genetic rearrangements were found in a single LTR, indicating that they occurred late during plus strand synthesis and were introduced by RT. It is not clear why this is the case (Fig. 6). One possibility is that the sample size was not sufficiently large and that with an expanded collection of mutations this peculiarity would disappear. It is also conceivable that HIV-1 RT is more prone to duplications and deletions during the later stages of reverse transcription. However, this remains to be tested.

Mechanistically, the three duplications and the single deletion (Fig. 6) are likely to have occurred by a simple misalignment copy-choice mechanism with the leading edge dissociating from the template followed by reassociation with the template either upstream or downstream of the breakpoint as has been described previously (58). If this occurred late during DNA synthesis, the integrated proviruses would have had looped out structures, which the cellular DNA repair machinery would have needed to resolve, and to date there is no precedence for the repair of looped out structures approaching 600 bases as would have been the case for clone H168 (Fig. 6). Yet these results indicate that such a function exists. An alternative explanation is that after cell division there was a mixture of two proviruses one with the mutation and one with parental LTRs, and the mutants had a growth advantage. However, the first possibility seems more plausible, because it would have been expected that at least in one case a clone with a mixture of mutant and parental proviruses would have been observed. As for the mutation in which cellular sequence was transduced, additional experiments were performed to examine its mechanism of transduction, and that work is outlined in another study (39).

In summary, we studied the rate and nature of mutations in HIV-1 LTRs of 215 proviruses, restricted to a single cycle of replication. By analyzing the relationship between mutations and when they occur, the first direct evidence that HIV-1 RT is likely to be the major engine of HIV-1 mutagenesis was obtained. Moreover, the nature and frequency of mutations observed at the U3-R border during viral replication differ from the anticipated findings based upon experiments using cell-free systems suggesting that other viral and/or cellular factors contribute to RT fidelity in vivo.

    ACKNOWLEDGEMENTS

We thank Martin E. Adelson, Chiann-chyi Chen, Malvika Kaul, Sayandip Mukherjee, Annmarie L. Pacchia, Amariliz Rivera, Robert A. Smith, and Jianling Zhuang for helpful suggestions and discussions.

    FOOTNOTES

* This work was supported by Grants CA50777 and AI34834 from the National Institutes of Health.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ Both authors contributed equally to this work.

** Present address: Dept. of Oncology, School of Medicine, Johns Hopkins University, Baltimore, MD 21231.

§§ Present address: Dept. of Pathology, University of Washington, 1959 NE Pacific St., Seattle, WA 98195.

Dagger Dagger To whom correspondence should be addressed: Dept. of Molecular Genetics, Microbiology and Immunology, University of Medicine and Dentistry of New Jersey-Robert Wood Johnson Medical School, 675 Hoes Lane, Piscataway, NJ 08854. Tel.: 732-235-4588; Fax: 732-235-5223; E-mail: doughejp@umdnj.edu.

Published, JBC Papers in Press, July 31, 2002, DOI 10.1074/jbc.M204774200

    ABBREVIATIONS

The abbreviations used are: HIV-1, human immunodeficiency virus, type 1; RT, reverse transcriptase; pol, polymerase; LTR, long terminal repeat; DMEM, Dulbecco's modified Eagle's medium; SSCP, single-stranded conformation polymorphism; dTTP, deoxythymidine 5'-triphosphate; dCTP, 2'-deoxycytidine 5'-triphosphate; MLV, murine leukemia virus.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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