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Journal of Virology, May 2008, p. 5104-5108, Vol. 82, No. 10
0022-538X/08/$08.00+0     doi:10.1128/JVI.01897-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Mutations in the Human Immunodeficiency Virus Type 1 Polypurine Tract (PPT) Reduce the Rate of PPT Cleavage and Plus-Strand DNA Synthesis ^,

M. J. McWilliams,¹ J. G. Julias,^2, and S. H. Hughes^1*

HIV Drug Resistance Program, National Cancer Institute at Frederick,¹ Basic Research Program, SAIC-Frederick, Inc., Frederick, Maryland 21702-1201²

Received 30 August 2007/ Accepted 21 February 2008

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Previously, we analyzed the effects of point mutations in the human immunodeficiency virus type 1 (HIV-1) polypurine tract (PPT) and found that some mutations affected both titer and cleavage specificity. We used HIV-1 vectors containing two PPTs and the D116N integrase active-site mutation in a cell-based assay to measure differences in the relative rates of PPT processing and utilization. The relative rates were measured by determining which of the two PPTs in the vector is used to synthesize viral DNA. The results indicate that mutations that have subtle effects on titer and cleavage specificity can have dramatic effects on rates of PPT generation and utilization.

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Previously, we used real-time PCR to analyze the rates of plus-strand DNA synthesis; in human immunodeficiency virus type 1 (HIV-1)-based vectors with mutations in both RNase H and the polypurine tract (PPT), the rate of plus-strand DNA synthesis was significantly reduced. However, there were no detectable differences in the rates of PPT utilization for mutants which had mutations only in the PPT (4). We used vectors that contained two PPTs to compare the relative rates at which PPTs are utilized. Spleen necrosis virus (SNV)-based retroviral vectors with a second PPT inserted 5' of the normal PPT were used to measure the efficiency of plus-strand transfer (1). The normal PPT was the primary site for plus-strand DNA transfer; however, some plus-strand DNA transfer was detected from the 5' PPT, indicating that the 5' PPT was used, albeit inefficiently. Analysis of PPT usage was also done with murine leukemia virus vectors containing two adjacent PPTs. In the murine leukemia virus experiments, the relative usages of the two PPTs were compared by primer extension. Mutant PPTs were used less efficiently than the wild type (WT); however, this technique measured the relative amounts of the two plus-strand products rather than the relative rates at which the two plus strands were synthesized (7). Most of the mutant PPTs we analyzed have been previously described (3, 5, 6); however, we also analyzed the effect of substituting PPTs from other retroviruses (Fig. 1; also see the supplemental material).

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FIG. 1. Titers of PPT mutants and 2-LTR circle junctions produced by the SNV and PFV PPT mutants. The experiments represented by this figure involved vectors with a single PPT in the normal (3') position. The HIV-1 PPT was mutated or replaced by other PPTs in an HIV-1 vector that undergoes a single cycle of retroviral replication (2). The HIV-1 vector expresses the heat-stable antigen (Hsa) from the nef reading frame; virus titers were measured by infecting HOS cells with the viruses and staining the cells with anti-Hsa followed by fluorescence-activated cell sorting. The virus titers are from the linear range of virus dilution. (A) The name of the mutant is indicated in the leftmost column, and the sequence of the PPT of the mutant is indicated in the middle column. The relative virus titers, normalized to the amount of p24 antigen in the supernatant, are shown in the right column. (B) The 2-LTR circle junctions were derived by infecting cells with PPT mutants. The primer binding site (PBS) is indicated by a white box; the leader sequence downstream of the PBS is shown as a black box. The PPT is shown by a box with black horizontal bars, the U tract is shown by a gray box, and the sequences immediately upstream of the U tract are shown by a box with diagonal bars. "PPT + Short flank inserted" refers to the PPT plus less than 10 nucleotides from 5' of the PPT. HOS cells were infected with the WT HIV-1-based vector and with vectors in which the PPT was replaced with the PPT from SNV or PFV. The 2-LTR circle junctions were amplified by PCR and cloned, and the DNA was sequenced. The leftmost column shows the types of 2-LTR circle junctions that were obtained, and the other three columns show the numbers of the different types of 2-LTR circle junctions for the different vectors.

HIV-1 vector with two PPTs. We developed an HIV-1 vector containing an extra PPT flanked by 11 nucleotides of 5' sequence and 11 nucleotides of 3' sequence inserted just 5' of the normal PPT. Titers were measured in a single replication cycle by use of vectors with WT integrase (IN). The effects of mutations on the relative rates of PPT utilization were determined by sequencing the two-long terminal repeat (2-LTR) circle junctions from infections with vectors that had the D116N active-site mutation in IN to avoid any effects of integration on the population of viral DNAs that gave rise to the 2-LTR circles. Some of the PPT mutations affected the specificity of PPT cleavage. It is possible that some of the mutant PPTs give rise to linear DNAs that are difficult for the host cells to ligate, which could affect the assay.

Previous analysis indicated that a number of single or double mutations in the PPT do not cause substantial alterations in RNase H cleavage specificity in an HIV-1 vector (the T2-5 mutation is an exception). In order to measure differences in the relative rates of PPTs usage, we generated vectors that contained two PPTs. In the experiments with the two-PPT vectors, when the 3' PPT was used in plus-strand DNA transfer, a normal 2-LTR circle junction was produced; when the 5' PPT was used, the circle junction contained the intervening sequence between the PPTs (Fig. 2).

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FIG. 2. HIV vectors with two PPTs and the 2-LTR circular viral DNAs produced by the double-PPT vectors. Retroviral vectors containing an additional PPT were constructed using a BspMI cassette-based cloning strategy (see the supplemental material). The vectors contained the central polypurine tract (cPPT) and two other PPTs in the 3' end of the genome (indicated as the 5' PPT and 3' PPT). The 3' PPT is in the normal location; the 5' PPT is located 35 nucleotides upstream of the normal PPT. In order to maintain the appropriate context for the 5' PPT, the sequences adjacent to the 5' PPT were constructed so that the 11 nucleotides from the 5' end of U3 and the 11 nucleotides just 5' of the 5' PPT were identical to the 11 nucleotides that flank both sides of the normal PPT. The vectors that were used to isolate the 2-LTR circles and to compare the rates at which the PPTs were cleaved and extended also contained the D116N mutation in IN. Blocking integration increases the proportion of viral DNAs that form 2-LTR circles and eliminates any bias due to the preferential integration of one of the linear DNAs. The 2-LTR circles arising from the infections with the two PPT vectors differ based on which PPT was processed first by RNase H and was used to successfully initiate DNA synthesis and plus-strand DNA transfer [(+) ST]. When the 3' PPT is used, the 2-LTR circles contain the typical consensus junction resulting from joining RU5 to U3. When the 5' PPT was used, the 2-LTR circles contained the 3' PPT and the sequences between the 5' PPT and the 3' PPT (intervening sequence [ivs]) and the duplicated portion of U3. The vector is indicated at the top of the figure, and the 2-LTR circles are indicated at the bottom. (The figure is not to scale.)

Both PPTs are functional. Vectors that had mutations in only the 5' or the 3' PPT had titers that were the same as that of the parental vector with one PPT (Fig. 3); vectors with both PPTs mutated had lower titers. For example, when both WT PPTs were replaced by SNV or prototype primate foamy virus (PFV) PPTs, the virus titers were decreased to 5% or 7% of the WT titer, respectively. Both PPTs were functional, and the longer linear viral DNA was integrated efficiently (see the supplemental material).

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FIG. 3. Effects of one and two PPT mutations on the titers of vectors containing two PPTs. The effects of the PPT mutations on the titers of the vectors were determined in a single cycle of replication with a version of the vector that has two copies of the PPT and WT IN. Viruses containing the indicated mutation in the PPT were generated and used to infect HOS cells; the relative titers were normalized to the amount of p24 antigen in the supernatant. (A) The sequence context of the 5' and 3' PPTs in the 2-PPT constructs. (B) Titers of mutants containing G-to-A mutations at the second or at the second and fifth positions of the PPT. (C) Titers of mutants containing G-to-T mutations at the second or the fifth and at the second and fifth positions. (D) Titers of the mutants with the SNV or PFV PPTs.

Mutant PPTs are used more slowly than WT PPT. We sequenced the 2-LTR circle junctions derived from infections with IN-negative vectors with two PPTs to determine which PPT generated the DNA used for plus-strand transfer (Fig. 4). When the vector contained two WT PPTs, the downstream (3') PPT was used 96% of the time. Because the two PPTs had the same sequence and the surrounding nucleic acid sequences were the same, the rates of PPT cleavage should have been similar. When both PPTs are cleaved and extended at approximately the same rate, the upstream primer is at a disadvantage for strand transfer. The normal PPT is copied first, and it should be cleaved and extended first. Moreover, when the normal PPT is used and the resulting plus-strand DNA copies the tRNA primer, the tRNA primer is removed by RNase H. When the second plus strand cannot copy the tRNA, it cannot participate in plus-strand transfer. The fact that the 3' PPT was used 96% of the time shows that the 3' position is advantageous and suggests that the normal PPT is processed and extended very rapidly. When the A2 mutant (in which the second G in the G tract is converted to A) was present in the 3' PPT and the 5' PPT was the WT, the WT PPT was used 71% of the time and the mutant A2 PPT was used 29% of the time. This indicates that the WT PPT is processed and extended more rapidly than the A2 PPT. When the 5' PPT contained the A2 mutant and the 3' PPT was the WT, the WT PPT was strongly preferred, as expected. When A2 was present in both positions of the PPT, the 5' PPT was used 73% of the time. The fact that the A2 PPT in the 3' position was used only 27% of the time cannot simply reflect the fact that when both of the PPTs are used slowly, the positional advantage of the 3' PPT is diminished. When both PPTs are used slowly, the 3' PPT should be used 50% of the time. The data for the A2 mutant suggest that the 5' PPT has some particular advantage of sequence and/or structure (see the supplemental material). The analysis of the other mutant-plus-mutant double-PPT vectors (described below) showed that this preference for the 5' PPT is the exception and not the rule.

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FIG. 4. Proportions of 2-LTR circle junctions arising as the result of plus-strand transfer from either the 5' PPT or the 3' PPT in the vectors containing two PPTs (see the supplemental material for additional details). The type of 2-LTR circle junction is shown in the leftmost column. Consensus circle junctions are 2-LTR circle junctions that arise from linear viral DNAs with complete ends that are generated after correct processing of the replicative intermediates by use of RNase H. "tRNA" and "PPT" refer to 2-LTR circles that retained part (or all) of the tRNA or PPT primers that were used to initiate minus- or plus-strand DNA synthesis. "Insertion" refers to the presence of one or two additional nucleotides at the 2-LTR circle junction. "Small Deletion" refers to deletions of 10 bp or fewer at the 2-LTR circle junction. "Large Deletion" refers to deletions of more than 10 bp at the 2-LTR circle junction. Small and large deletions can arise at either the U5 or U3 end of the linear viral DNA that gives rise to the 2-LTR circle junction. (A) Results of PPT competition between the WT and the A2 mutant (G-to-A mutation at the second position of the PPT). (B) Results of PPT competition between the WT and the A2-5 mutant (G-to-A mutations at the second and fifth positions of the PPT). (C) Results of PPT competition between the WT and the T2 mutant (G-to-T mutation at the second position in the PPT). (D) Results of PPT competition between the WT and the T5 mutant (G-to-T mutation in the fifth position of the PPT). (E) Results of PPT competition between the WT and the T2-5 mutant (G-to-T mutation in the second and fifth positions in the PPT). (F) Results of PPT competition between the WT and the SNV mutant (HIV PPT sequence replaced by the SNV PPT sequence). (G) Results of PPT competition between the WT and the PFV mutant (HIV PPT sequence replaced by the PFV sequence).

The WT PPT was strongly preferred to the A2-5 (double) mutant in which both the second and fifth Gs of the G tract were converted to A even when the WT PPT was in the 5' position (89%). When the A2-5 PPT was in both locations, the upstream mutant PPT was preferred to the 3' mutant PPT (62%). The preference for the 5' A2-5 PPT was statistically significant (P < 0.0001). Because the A2-5 mutant contained the A2 mutation, this result is probably related to the unexpected advantage of the 5' PPT when the A2 mutation was present in both PPTs. When the A2 PPT and the A2-5 PPT are compared to the WT PPT, the results show that the A2 PPT was used more frequently than the A2-5 PPT, suggesting that A2 is used more rapidly than A2-5. However, in an HIV-1-based vector with one PPT, the relative titers were similar (the A2 titer was 56% of the WT titer, and the A2-5 mutant titer was 48% of the WT titer), suggesting that the difference in the rates had a modest impact on the titer.

Analysis of G-to-T mutations in the G tract showed that these mutations also had a significant effect on the relative rates of utilization of the PPT. When the WT PPT was present at the 5' position and the T2 and T5 PPTs were in the 3' position, the mutant PPTs were used in 44% and 23% of the plus-strand DNA transfers, respectively. When the mutant PPT was present in the 5' position, T2 was used 10% of the time but T5 was never used. The relative rates of plus-strand DNA priming and plus-strand DNA transfer were also determined for vectors with the mutated PPTs in both the 5' and 3' positions. In the mutant with T2 in both PPTs, each PPT was used about half of the time (the upstream site was used in 47% of the events). However, when the T5 mutation was in both PPTs, plus-strand synthesis was preferentially initiated from the 3' PPT; the 5' PPT was used 25% of the time. When the T2-5 mutant was in the 3' position and WT PPT was in the 5' position, there was a very strong preference for the WT PPT. When both PPTs contained the T2-5 mutations, there was a modest preference for the 3' PPT; the 3' PPT was used 60% of the time. Comparing the results of the WT plus T2-5 mutant to the results obtained with the WT plus T2 and the WT plus T5 mutant vectors suggests that T2-5 was used more slowly than either of the related single mutants T2 and T5.

Vectors containing a single copy of either the SNV PPT or the PFV PPT had the lowest titers of the vectors we tested (3% of the WT titer), and many of the 2-LTR circle junctions that arose from infections with these vectors had PPT insertions at the circle junction. When the mutant PPTs were present at both positions in the double-PPT vectors (PFV plus PFV and SNV plus SNV), the virus titers increased modestly to 7% and 5% of WT titers, respectively, for PFV and SNV. Analysis of the 2-LTR circle junctions that arose from the vectors containing either the SNV or the PFV PPT indicated that the WT PPT was strongly preferred.

Taken together, these data show that the wild type PPT is used almost immediately after it is copied into DNA. Almost all of the PPT mutants we tested had a measurable effect on the relative rates of PPT utilization; in some cases the effect was dramatic. However, in most cases, this dramatic effect on the rate of PPT utilization did not correlate with the more modest effect of the mutations on titers. This suggests that in the WT virus, the processing and extension of the PPT, and the subsequent plus-strand transfer, are not rate-limiting events and that a substantial reduction in the rates at which these events occur has little, if any, real impact on viral replication.

 
We thank Christie Vu for help in preparation of the figures, Dave Hoberman for his help with statistical analysis, and Terri Burdette for help in preparing the manuscript.

The research described in this publication was funded in part with federal funds from the National Cancer Institute, National Institutes of Health, under contract NO1CO-12400, and by the Intramural Research Program of the NIH, National Cancer Institute, Center for Cancer Research.

The content of this publication does not necessarily reflect the views or policies of the Department of Health and Human Services, and mention of trade names, commercial products, or organizations does not imply endorsement by the U.S. government.

 
* Corresponding author. Mailing address: HIV Drug Resistance Program, NCI at Frederick, P.O. Box B, Bldg. 539, Rm. 130A, Frederick, MD 21702-1201. Phone: (301) 846-1619. Fax: (301) 846-6966. E-mail: hughes{at}ncifcrf.gov

Published ahead of print on 5 March 2008.

Supplemental material for this article may be found at http://jvi.asm.org/.

Present address: Booz Allen Hamilton, Rockville, MD 20852.

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2. Julias, J. G., P. L. Boyer, M. J. McWilliams, W. G. Alvord, and S. H. Hughes. 2004. Mutations at position 184 of human immunodeficiency virus type-1 reverse transcriptase affect virus titer and viral DNA synthesis. Virology 322:13-21.[CrossRef][Medline]
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3. Julias, J. G., M. J. McWilliams, S. G. Sarafianos, E. Arnold, and S. H. Hughes. 2002. Mutations in the RNase H domain of HIV-1 reverse transcriptase affect the initiation of DNA synthesis and the specificity of RNase H cleavage in vivo. Proc. Natl. Acad. Sci. USA 99:9515-9520.[Abstract/Free Full Text]
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4. McWilliams, M. J., J. G. Julias, S. G. Sarafianos, W. G. Alvord, E. Arnold, and S. H. Hughes. 2006. Combining mutations in HIV-1 reverse transcriptase with mutations in the HIV-1 polypurine tract affects RNase H cleavages involved in PPT utilization. Virology 348:378-388.[CrossRef][Medline]
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5. McWilliams, M. J., J. G. Julias, S. G. Sarafianos, W. G. Alvord, E. Arnold, and S. H. Hughes. 2003. Mutations in the 5' end of the human immunodeficiency virus type 1 polypurine tract affect RNase H cleavage specificity and virus titer. J. Virol. 77:11150-11157.[Abstract/Free Full Text]
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6. Miles, L. R., B. E. Agresta, M. B. Khan, S. Tang, J. G. Levin, and M. D. Powell. 2005. Effect of polypurine tract (PPT) mutations on human immunodeficiency virus type 1 replication: a virus with a completely randomized PPT retains low infectivity. J. Virol. 79:6859-6867.[Abstract/Free Full Text]
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Journal of Virology, May 2008, p. 5104-5108, Vol. 82, No. 10
0022-538X/08/$08.00+0     doi:10.1128/JVI.01897-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

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This Article Abstract Full Text (PDF) Supplemental material Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by McWilliams, M. J. Articles by Hughes, S. H. Search for Related Content PubMed PubMed Citation Articles by McWilliams, M. J. Articles by Hughes, S. H.