Effect of Polypurine Tract (PPT) Mutations on Human Immunodeficiency Virus Type 1 Replication: a Virus with a Completely Randomized PPT Retains Low Infectivity

Miles, Lesa; Agresta, Beth E.; Khan, Mahfuz; Tang, Shixing; Levin, Joseph; Powell, Michael D.

doi:10.1128/jvi.79.11.6859-6867.2005

Cited by 23 publications

(29 citation statements)

References 76 publications

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“…19 This scheme was further predicated on the notion that the PPT deletion could alter the RT process, directing the plus-strand initiation site to cryptic sites ( Figure 2); indeed, previous in vitro and in vivo studies with HIV-1 have indicated that, in the course of RT, HIV-1 second-strand synthesis can be initiated from non-PPT sites. 20,[30][31][32] Furthermore, premised on earlier studies 18 suggesting that linear DNA is more prone to integrase-independent integration than circular DNA, we postulated that the decrease in the formation of linear genomes would correspond with a decrease in illegitimate integration of vector genomes. Indeed, the data presented here indicated a threefold reduction in linear genomes after deletion of the PPT, with a comparable reduction in illegitimate integration of HIV vectors packaged without functional integrase.…”

Section: Discussionmentioning

confidence: 99%

Notable Reduction in Illegitimate Integration Mediated by a PPT-deleted, Nonintegrating Lentiviral Vector

Kantor

Bayer

et al. 2011

Molecular Therapy

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Nonintegrating lentiviral vectors present a means of reducing the risk of insertional mutagenesis in nondividing cells and enabling short-term expression of potentially hazardous gene products. However, residual, integrase-independent integration raises a concern that may limit the usefulness of this system. Here we present a novel 3' polypurine tract (PPT)-deleted lentiviral vector that demonstrates impaired integration efficiency and, when packaged into integrase-deficient particles, significantly reduced illegitimate integration. Cells transduced with PPT-deleted vectors exhibited predominantly 1-long terminal repeat (LTR) circles and a low level of linear genomes after reverse transcription (RT). Importantly, the PPT-deleted vector exhibited titers and in vitro and in vivo expression levels matching those of conventional nonintegrating lentiviral vectors. This safer nonintegrating lentiviral vector system will support emerging technologies, such as those based on transient expression of zinc-finger nucleases (ZFNs) for gene editing, as well as reprogramming factors for inducing pluripotency.

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Section: Discussionmentioning

confidence: 99%

Notable Reduction in Illegitimate Integration Mediated by a PPT-deleted, Nonintegrating Lentiviral Vector

Kantor

Bayer

et al. 2011

Molecular Therapy

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“…Consistent with preferred nucleotides spanning positions +1 through −14 for HIV-1 RNase H, both the 5′ and 3′ ends of the PPT are important for viral replication in vivo Miles et al, 2005). In the co-crystal structure of HIV-1 reverse transcriptase with a PPT RNA/DNA (Sarafianos et al, 2001), the + 1 RNA base makes hydrogen-bonding contacts with Arg448, and the −2 RNA guanine makes hydrogen-bonding contacts with Gln475.…”

Section: Generation Of Ppt Primermentioning

confidence: 92%

RNase H activity: Structure, specificity, and function in reverse transcription

2008

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This review compares the well-studied RNase H activities of human immunodeficiency virus, type 1 (HIV-1) and Moloney murine leukemia virus (MoMLV) reverse transcriptases. The RNase H domains of HIV-1 and MoMLV are structurally very similar, with functions assigned to conserved subregions like the RNase H primer grip and the connection subdomain, as well as to distinct features like the C-helix and loop in MoMLV RNase H. Like cellular RNases H, catalysis by the retroviral enzymes appears to involve a two-metal ion mechanism. Unlike cellular RNases H, the retroviral RNases H display three different modes of cleavage: internal, DNA 3′ end-directed, and RNA 5′ enddirected. All three modes of cleavage appear to have roles in reverse transcription. Nucleotide sequence is an important determinant of cleavage specificity with both enzymes exhibiting a preference for specific nucleotides at discrete positions flanking an internal cleavage site as well as during tRNA primer removal and plus-strand primer generation. RNA 5′ end-directed and DNA 3′ end-directed cleavages show similar sequence preferences at the positions closest to a cleavage site. A model for how RNase H selects cleavage sites is presented that incorporates both sequence preferences and the concept of a defined window for allowable cleavage from a recessed end. Finally, the RNase H activity of HIV-1 is considered as a target for anti-virals as well as a participant in drug resistance. KeywordsRNase H; reverse transcriptase; human immunodeficiency virus; type 1 (HIV -1); Moloney murine leukemia virus (MoMLV); reverse transcription; polypurine tract (PPT)

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“…We did two sets of experiments in which we asked whether mutations in the PPT caused similar defects in RNase H cleavages in vitro and in vivo; we were able to show that there is good agreement in the in vivo and in vitro results (8,23). Miles et al reported that substituting Cs either for the last four or all six Gs in the G tract had almost as great an effect on the titer as replacing the entire PPT with a non-PPT sequence (16). We sequenced the 2-LTR circle junctions that resulted from infections with vectors carrying these PPT mutations; these assays showed that the A3, A5, T2, T5, A2-5, C2-5, and T2-5 mutations affected cleavage specificity (the number represents the position or positions in the G tract that were mutated, and the letter designates the nucleotide substitution).…”

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confidence: 94%

“…In the analysis of mutations in the 5Ј end of the PPT, substituting one or two C residues for the A residues at the 5Ј end of the PPT had modest effects on the titer of an HIV-1 vector (two-to fivefold) and, using the 2-LTR circle junction assay, on the specificity of RNase H cleavage. Miles et al reported that a complex multiple mutation in the 5Ј end of the HIV-1 PPT had a relatively modest effect on the titer (about 40% of the wild type [WT]) (16). This region has also been analyzed using in vitro assays; the results imply that weak base pairing plays a role in determining cleavage specificity (3,13,35).…”

mentioning

confidence: 99%

The Effects of Alternate Polypurine Tracts (PPTs) and Mutations of Sequences Adjacent to the PPT on Viral Replication and Cleavage Specificity of the Rous Sarcoma Virus Reverse Transcriptase

Chang

Alvord

et al. 2008

J Virol

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We previously reported that a mutant Rous sarcoma virus (RSV) with an alternate polypurine tract (PPT), DuckHepBFlipPPT, had unexpectedly high titers and that the PPT was miscleaved primarily at one position following a GA dinucleotide by the RNase H of reverse transcriptase (RT). This miscleavage resulted in a portion of the 3 end of the PPT (5-ATGTA) being added to the end of U3 of the linear viral DNA. To better understand the RNase H cleavage by RSV RT, we made a number of mutations within the DuckHepBFlipPPT and in the sequences adjacent to the PPT. Deleting the entire ATGTA sequence from the DuckHepBFlipPPT increased the relative titer to wild-type levels, while point mutations within the ATGTA sequence reduced the relative titer but had minimal effects on the cleavage specificity. However, mutating a sequence 5 of ATGTA affected the relative titer of the virus and caused the RNase H of RSV RT to lose the ability to cleave the PPT specifically. In addition, although mutations in the conserved stretch of thymidine residues upstream of the PPT did not affect the relative titer or cleavage specificity, the mutation of some of the nucleotides immediately upstream of the PPT did affect the titer and cleavage specificity. Taken together, our studies show that the structure of the PPT in the context of the cognate RT, rather than a specific sequence, is important for the proper cleavage by RSV RT.During retroviral infection, the virally encoded enzyme reverse transcriptase (RT) converts the single-stranded RNA genome of the virus into a linear double-stranded DNA that can be integrated into the host genome (31, 32). RT has two enzymatic activities: a DNA polymerase that can copy either an RNA or DNA template and an RNase H that cleaves RNA if, and only if, it is part of an RNA-DNA hybrid. Both activities are required for the synthesis of the linear viral DNA. Like many other DNA polymerases, RT cannot initiate DNA synthesis de novo. A host tRNA, hybridized to the viral RNA genome near the 5Ј end of the genome, is used to prime the synthesis of minus-strand DNA. As the polymerase activity of RT synthesizes minus-strand DNA, it generates an RNA-DNA hybrid that is a substrate for RNase H. After RT has copied the short segment of the retroviral RNA genome between the tRNA primer and the 5Ј end of the viral genome and RNase H has degraded the RNA strand, minus-strand DNA synthesis is translocated to the 3Ј end of the viral genome via the repeat or R sequence found at both the 5Ј and 3Ј ends of the viral RNA genome. The synthesis of minus-strand DNA is then able to proceed toward the 5Ј end of the RNA genome. The viral RNA genome contains a sequence called the polypurine tract (PPT), which is relatively resistant to degradation by RNase H. Because the PPT is relatively stable, it is used to prime plusstrand DNA synthesis. In general, the cleavages made by RNase H are thought to be relatively nonspecific; however, there are several steps during reverse transcription in which the cleavages are known to be specific. One s...

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Effect of Polypurine Tract (PPT) Mutations on Human Immunodeficiency Virus Type 1 Replication: a Virus with a Completely Randomized PPT Retains Low Infectivity

Cited by 23 publications

References 76 publications

Notable Reduction in Illegitimate Integration Mediated by a PPT-deleted, Nonintegrating Lentiviral Vector

Notable Reduction in Illegitimate Integration Mediated by a PPT-deleted, Nonintegrating Lentiviral Vector

RNase H activity: Structure, specificity, and function in reverse transcription

The Effects of Alternate Polypurine Tracts (PPTs) and Mutations of Sequences Adjacent to the PPT on Viral Replication and Cleavage Specificity of the Rous Sarcoma Virus Reverse Transcriptase

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