JOURNAL OF VIROLOGY, May 2008, p. 5104–5108 Vol. 82, No. 100022-538X/08/$08.00�0 doi:10.1128/JVI.01897-07Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Mutations in the Human Immunodeficiency Virus Type 1 PolypurineTract (PPT) Reduce the Rate of PPT Cleavage and Plus-Strand

DNA Synthesis�†M. J. McWilliams,1 J. G. Julias,2‡ and S. H. Hughes1*

HIV Drug Resistance Program, National Cancer Institute at Frederick,1 and Basic Research Program,SAIC-Frederick, Inc.,2 Frederick, Maryland 21702-1201

Received 30 August 2007/Accepted 21 February 2008

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 usedHIV-1 vectors containing two PPTs and the D116N integrase active-site mutation in a cell-based assay tomeasure differences in the relative rates of PPT processing and utilization. The relative rates were measuredby determining which of the two PPTs in the vector is used to synthesize viral DNA. The results indicate thatmutations that have subtle effects on titer and cleavage specificity can have dramatic effects on rates of PPTgeneration and utilization.

Previously, we used real-time PCR to analyze the rates ofplus-strand DNA synthesis; in human immunodeficiency virustype 1 (HIV-1)-based vectors with mutations in both RNase Hand the polypurine tract (PPT), the rate of plus-strand DNAsynthesis was significantly reduced. However, there were nodetectable differences in the rates of PPT utilization for mu-tants which had mutations only in the PPT (4). We used vec-tors that contained two PPTs to compare the relative rates atwhich PPTs are utilized. Spleen necrosis virus (SNV)-basedretroviral vectors with a second PPT inserted 5� of the normalPPT were used to measure the efficiency of plus-strand transfer(1). The normal PPT was the primary site for plus-strand DNAtransfer; however, some plus-strand DNA transfer was de-tected from the 5� PPT, indicating that the 5� PPT was used,albeit inefficiently. Analysis of PPT usage was also done withmurine leukemia virus vectors containing two adjacent PPTs.In the murine leukemia virus experiments, the relative usagesof the two PPTs were compared by primer extension. MutantPPTs were used less efficiently than the wild type (WT); how-ever, this technique measured the relative amounts of the twoplus-strand products rather than the relative rates at which thetwo plus strands were synthesized (7). Most of the mutantPPTs we analyzed have been previously described (3, 5, 6);however, we also analyzed the effect of substituting PPTs fromother retroviruses (Fig. 1; also see the supplemental material).

HIV-1 vector with two PPTs. We developed an HIV-1 vectorcontaining an extra PPT flanked by 11 nucleotides of 5� se-quence and 11 nucleotides of 3� sequence inserted just 5� of thenormal PPT. Titers were measured in a single replication cycleby use of vectors with WT integrase (IN). The effects of mu-

tations on the relative rates of PPT utilization were determinedby sequencing the two-long terminal repeat (2-LTR) circlejunctions from infections with vectors that had the D116Nactive-site mutation in IN to avoid any effects of integration onthe population of viral DNAs that gave rise to the 2-LTRcircles. Some of the PPT mutations affected the specificity ofPPT cleavage. It is possible that some of the mutant PPTs giverise 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 doublemutations in the PPT do not cause substantial alterations inRNase H cleavage specificity in an HIV-1 vector (the T2-5mutation is an exception). In order to measure differences inthe relative rates of PPTs usage, we generated vectors thatcontained two PPTs. In the experiments with the two-PPTvectors, when the 3� PPT was used in plus-strand DNA trans-fer, a normal 2-LTR circle junction was produced; when the 5�PPT was used, the circle junction contained the interveningsequence between the PPTs (Fig. 2).

Both PPTs are functional. Vectors that had mutations in onlythe 5� or the 3� PPT had titers that were the same as that of theparental vector with one PPT (Fig. 3); vectors with both PPTsmutated had lower titers. For example, when both WT PPTs werereplaced 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 linearviral DNA was integrated efficiently (see the supplemental mate-rial).

Mutant PPTs are used more slowly than WT PPT. Wesequenced the 2-LTR circle junctions derived from infectionswith IN-negative vectors with two PPTs to determine whichPPT 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 thesame sequence and the surrounding nucleic acid sequenceswere the same, the rates of PPT cleavage should have beensimilar. When both PPTs are cleaved and extended at approx-imately the same rate, the upstream primer is at a disadvantage

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

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

‡ Present address: Booz Allen Hamilton, Rockville, MD 20852.� Published ahead of print on 5 March 2008.

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for strand transfer. The normal PPT is copied first, and itshould be cleaved and extended first. Moreover, when thenormal PPT is used and the resulting plus-strand DNA copiesthe tRNA primer, the tRNA primer is removed by RNase H.When the second plus strand cannot copy the tRNA, it cannotparticipate in plus-strand transfer. The fact that the 3� PPT wasused 96% of the time shows that the 3� position is advanta-geous and suggests that the normal PPT is processed andextended very rapidly. When the A2 mutant (in which thesecond G in the G tract is converted to A) was present in the3� 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 extendedmore rapidly than the A2 PPT. When the 5� PPT contained theA2 mutant and the 3� PPT was the WT, the WT PPT wasstrongly preferred, as expected. When A2 was present in bothpositions of the PPT, the 5� PPT was used 73% of the time. Thefact that the A2 PPT in the 3� position was used only 27% ofthe time cannot simply reflect the fact that when both of thePPTs are used slowly, the positional advantage of the 3� PPT is

diminished. When both PPTs are used slowly, the 3� PPTshould be used 50% of the time. The data for the A2 mutantsuggest that the 5� PPT has some particular advantage ofsequence and/or structure (see the supplemental material).The analysis of the other mutant-plus-mutant double-PPT vec-tors (described below) showed that this preference for the 5�PPT is the exception and not the rule.

The WT PPT was strongly preferred to the A2-5 (double)mutant in which both the second and fifth Gs of the G tractwere converted to A even when the WT PPT was in the 5�position (89%). When the A2-5 PPT was in both locations, theupstream mutant PPT was preferred to the 3� mutant PPT(62%). The preference for the 5� A2-5 PPT was statisticallysignificant (P � 0.0001). Because the A2-5 mutant containedthe A2 mutation, this result is probably related to the unex-pected advantage of the 5� PPT when the A2 mutation waspresent in both PPTs. When the A2 PPT and the A2-5 PPT arecompared to the WT PPT, the results show that the A2 PPTwas used more frequently than the A2-5 PPT, suggesting thatA2 is used more rapidly than A2-5. However, in an HIV-1-

FIG. 1. Titers of PPT mutants and 2-LTR circle junctions produced by the SNV and PFV PPT mutants. The experiments represented by thisfigure 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 vectorthat 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 cellsorting. The virus titers are from the linear range of virus dilution. (A) The name of the mutant is indicated in the leftmost column, and thesequence of the PPT of the mutant is indicated in the middle column. The relative virus titers, normalized to the amount of p24 antigen in thesupernatant, are shown in the right column. (B) The 2-LTR circle junctions were derived by infecting cells with PPT mutants. The primer bindingsite (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 blackhorizontal 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-basedvector 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 andcloned, and the DNA was sequenced. The leftmost column shows the types of 2-LTR circle junctions that were obtained, and the other threecolumns show the numbers of the different types of 2-LTR circle junctions for the different vectors.

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based vector with one PPT, the relative titers were similar (theA2 titer was 56% of the WT titer, and the A2-5 mutant titerwas 48% of the WT titer), suggesting that the difference in therates had a modest impact on the titer.

Analysis of G-to-T mutations in the G tract showed thatthese mutations also had a significant effect on the relativerates of utilization of the PPT. When the WT PPT was presentat the 5� position and the T2 and T5 PPTs were in the 3�position, the mutant PPTs were used in 44% and 23% of theplus-strand DNA transfers, respectively. When the mutantPPT was present in the 5� position, T2 was used 10% of thetime but T5 was never used. The relative rates of plus-strandDNA priming and plus-strand DNA transfer were also deter-mined for vectors with the mutated PPTs in both the 5� and 3�positions. In the mutant with T2 in both PPTs, each PPT wasused about half of the time (the upstream site was used in 47%of the events). However, when the T5 mutation was in bothPPTs, plus-strand synthesis was preferentially initiated fromthe 3� PPT; the 5� PPT was used 25% of the time. When theT2-5 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 amodest preference for the 3� PPT; the 3� PPT was used 60% ofthe time. Comparing the results of the WT plus T2-5 mutant tothe results obtained with the WT plus T2 and the WT plus T5mutant vectors suggests that T2-5 was used more slowly thaneither of the related single mutants T2 and T5.

Vectors containing a single copy of either the SNV PPT orthe PFV PPT had the lowest titers of the vectors we tested (3%of the WT titer), and many of the 2-LTR circle junctions thatarose from infections with these vectors had PPT insertions atthe circle junction. When the mutant PPTs were present atboth positions in the double-PPT vectors (PFV plus PFV andSNV plus SNV), the virus titers increased modestly to 7% and5% of WT titers, respectively, for PFV and SNV. Analysis of

FIG. 2. HIV vectors with two PPTs and the 2-LTR circular viralDNAs produced by the double-PPT vectors. Retroviral vectors con-taining an additional PPT were constructed using a BspMI cassette-based cloning strategy (see the supplemental material). The vectorscontained the central polypurine tract (cPPT) and two other PPTs inthe 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 nucleotidesupstream of the normal PPT. In order to maintain the appropriatecontext for the 5� PPT, the sequences adjacent to the 5� PPT wereconstructed so that the 11 nucleotides from the 5� end of U3 and the11 nucleotides just 5� of the 5� PPT were identical to the 11 nucleotidesthat flank both sides of the normal PPT. The vectors that were used toisolate the 2-LTR circles and to compare the rates at which the PPTswere cleaved and extended also contained the D116N mutation in IN.Blocking integration increases the proportion of viral DNAs that form2-LTR circles and eliminates any bias due to the preferential integra-tion of one of the linear DNAs. The 2-LTR circles arising from theinfections with the two PPT vectors differ based on which PPT wasprocessed first by RNase H and was used to successfully initiate DNAsynthesis and plus-strand DNA transfer [(�) ST]. When the 3� PPT isused, the 2-LTR circles contain the typical consensus junction resultingfrom joining RU5 to U3. When the 5� PPT was used, the 2-LTR circlescontained the 3� PPT and the sequences between the 5� PPT and the3� PPT (intervening sequence [ivs]) and the duplicated portion of U3.The vector is indicated at the top of the figure, and the 2-LTR circlesare indicated at the bottom. (The figure is not to scale.)

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 ofthe 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. Virusescontaining the indicated mutation in the PPT were generated and used to infect HOS cells; the relative titers were normalized to the amount ofp24 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-Amutations 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 thefifth and at the second and fifth positions. (D) Titers of the mutants with the SNV or PFV PPTs.

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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 containingtwo PPTs (see the supplemental material for additional details). The type of 2-LTR circle junction is shown in the leftmost column. Consensus circlejunctions are 2-LTR circle junctions that arise from linear viral DNAs with complete ends that are generated after correct processing of the replicativeintermediates 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 toinitiate minus- or plus-strand DNA synthesis. “Insertion” refers to the presence of one or two additional nucleotides at the 2-LTR circle junction. “SmallDeletion” 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 circlejunction. 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) Resultsof 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 betweenthe 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 theT2 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 mutationin 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 positionsin the PPT). (F) Results of PPT competition between the WT and the SNV mutant (HIV PPT sequence replaced by the SNV PPT sequence). (G) Resultsof PPT competition between the WT and the PFV mutant (HIV PPT sequence replaced by the PFV sequence).

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the 2-LTR circle junctions that arose from the vectors contain-ing either the SNV or the PFV PPT indicated that the WT PPTwas strongly preferred.

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

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

The research described in this publication was funded in part withfederal funds from the National Cancer Institute, National Institutes ofHealth, under contract NO1CO-12400, and by the Intramural Re-search Program of the NIH, National Cancer Institute, Center forCancer Research.

The content of this publication does not necessarily reflect the viewsor policies of the Department of Health and Human Services, and

mention of trade names, commercial products, or organizations doesnot imply endorsement by the U.S. government.

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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-1reverse transcriptase affect virus titer and viral DNA synthesis. Virology322:13–21.

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 transcrip-tase affect the initiation of DNA synthesis and the specificity of RNase Hcleavage in vivo. Proc. Natl. Acad. Sci. USA 99:9515–9520.

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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 immunodefi-ciency virus type 1 polypurine tract affect RNase H cleavage specificity andvirus titer. J. Virol. 77:11150–11157.

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 immuno-deficiency virus type 1 replication: a virus with a completely randomized PPTretains low infectivity. J. Virol. 79:6859–6867.

7. Robson, N. D., and A. Telesnitsky. 1999. Effects of 3� untranslated regionmutations on plus-strand priming during Moloney murine leukemia virusreplication. J. Virol. 73:948–957.

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