Integration of Rous Sarcoma Virus DNA: a CA Dinucleotide Is Not Required for Integration of the U3 End of Viral DNA

Oh, Jangsuk; Chang, Kevin W.; Hughes, Stephen H.

doi:10.1128/jvi.01353-08

Cited by 5 publications

(8 citation statements)

References 24 publications

(22 reference statements)

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“…The conserved CA/GT sequence located 2 bp from the DNA end (immediately 5Ј of the scissile phosphodiester bond) comprises an invariant feature of the terminal inverted repeats. Surprisingly, some studies have shown that even these conserved dinucleotide pairs are not absolutely essential for IN-mediated DNA processing in vitro (4) or integration in vivo (5,6). Others have demonstrated cross-recognition of heterologous LTRs by retroviral IN proteins (7), which is typically accounted for by limited sequence identity between the cognate and heterologous DNA tips.…”

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confidence: 99%

Retroviral Integrases Promote Fraying of Viral DNA Ends

Katz

Merkel

Andrake

et al. 2011

Journal of Biological Chemistry

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In the initial step of integration, retroviral integrase (IN) introduces precise nicks in the degenerate, short inverted repeats at the ends of linear viral DNA. The scissile phosphodiester bond is located immediately 3 of a highly conserved CA/GT dinucleotide, usually 2 bp from the ends. These nicks create new recessed 3 -OH viral DNA ends that are required for joining to host cell DNA. Previous studies have indicated that unpairing, "fraying," of the viral DNA ends by IN contributes to end recognition or catalysis. Here, we report that end fraying can be detected independently of catalysis with both avian sarcoma virus ( Within hours after infection of a host cell, a colinear DNA copy of the retroviral RNA genome becomes integrated into the host chromosome, through a highly coordinated series of enzymatic events. The steps are carried out by the virus-encoded reverse transcriptase, which makes a double-stranded copy of the viral RNA genome, and the viral integrase (IN), 2 which catalyzes the integration of this newly synthesized DNA into the DNA of the host cell. Following integration, an essential step for virus replication, the viral DNA is permanently associated with the genome of the host cell and all daughters and serves as a template for viral gene transcription and the production of progeny virus.The underlying chemical steps in retroviral DNA integration are generally understood (1). The reaction can be divided into two steps, which can be reproduced in vitro using purified IN and model DNA substrates. (i) The nuclease activity of IN introduces nicks, 3Ј of the highly conserved CA dinucleotide, usually 2 bp from the DNA terminus, in a "processing" step that releases a dinucleotide and generates new, recessed 3Ј-OH ends. (ii) In the second step, these newly exposed 3Ј-oxygen atoms are joined to staggered backbone phosphates on opposite strands of the host DNA, via concerted cleavage and ligation. Because the target phosphates are staggered, this "joining" step leaves gaps in the opposite strands of host DNA, and host mechanisms are required to repair the gaps and complete the covalent joining (for review, see Ref.2). These combined viral and host-catalyzed enzymatic steps produce the characteristic sequence signatures of retroviral integration: loss of the terminal 2 bp from the linear viral DNA ends and the duplication, depending on the virus, of 4 -6 bp of host DNA flanking the integration site.Substrate Recognition by IN Does Not Depend on Sequence Alone-Sequences at the tips of the blunt-ended retroviral DNA (10 -20 bp) are sufficient for IN recognition, processing, and joining. However, the mechanism by which such substrates are recognized is not completely understood, and it has been difficult to detect sequence-specific binding to viral DNA in vitro (3). Sequence identity of the generally imperfect terminal inverted repeats that are embedded in the U3 and U5 regions of the long terminal repeat (LTR) at the ends of each viral DNA can range from 16/18 for murine leukemia virus, to 12/18 for avian s...

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confidence: 99%

Retroviral Integrases Promote Fraying of Viral DNA Ends

Katz

Merkel

Andrake

et al. 2011

Journal of Biological Chemistry

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“…However, as has already been discussed, it is possible to create conditions under which IN can insert only one end of the viral DNA. When this happens, host enzymes insert the second viral DNA end into the host genome with an efficiency that can be as high as 30 % of the rate at which a normal viral DNA end can be inserted by IN [ 12 ]. This produces what at first appears to be a complex array of host/virus DNA junctions, in which the viral DNA sequences are often (but not always) truncated.…”

Section: Discussionmentioning

confidence: 99%

“…It should be clear that this homologous recombination can take place on either side of the initial IN-mediated event. Thus the host-mediated insertion reaction can lead to a deletion in the flanking host DNA if it takes place on one side of the initial IN-mediated integration event; if it takes place on the other side, it will cause a duplication of the flanking host DNA, as was shown in experiments done using ASLV-based vectors [ 11 , 12 , 34 ].…”

Section: Discussionmentioning

confidence: 99%

“…In the initial experiments, done using an ASLV vector, we made mutations in the viral genome that led to the synthesis of linear viral DNAs that had one end that was not a good substrate for IN. To our surprise, this had, in some cases, only a modest impact on the titer [ 12 ]. As was discussed earlier, we showed, by isolating the resulting proviruses that, if one end of the viral DNA was inserted into the host genome by IN, the other could be efficiently inserted into the genome by the host machinery.…”

Section: Discussionmentioning

confidence: 99%

“…Generally speaking, these aberrant HIV integrations are quite similar to the aberrant integrations that arose in experiments done with avian sarcoma leukoisis virus (ASLV) vectors. In the ASLV experiments, one of the ends of the viral DNA was mutated in a way that caused it to be a poor substrate for IN-mediated integration [ 11 , 12 ]. The “good” end of the viral DNA was integrated normally, by IN, whereas the second (mutant) end was apparently inserted by host enzymes.…”

Section: Introductionmentioning

confidence: 99%

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Drug resistant integrase mutants cause aberrant HIV integrations

et al. 2016

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Background HIV-1 integrase is the target for three FDA-approved drugs, raltegravir, elvitegravir, and dolutegravir. All three drugs bind at the active site of integrase and block the strand transfer step of integration. We previously showed that sub-optimal doses of the anti-HIV drug raltegravir can cause aberrant HIV integrations that are accompanied by a variety of deletions, duplications, insertions and inversions of the adjacent host sequences.ResultsWe show here that a second drug, elvitegravir, also causes similar aberrant integrations. More importantly, we show that at least two of the three clinically relevant drug resistant integrase mutants we tested, N155H and G140S/Q148H, which reduce the enzymatic activity of integrase, can cause the same sorts of aberrant integrations, even in the absence of drugs. In addition, these drug resistant mutants have an elevated IC50 for anti-integrase drugs, and concentrations of the drugs that would be optimal against the WT virus are suboptimal for the mutants.ConclusionsWe previously showed that suboptimal doses of a drug that binds to the HIV enzyme integrase and blocks the integration of a DNA copy of the viral genome into host DNA can cause aberrant integrations that involve rearrangements of the host DNA. We show here that suboptimal doses of a second anti-integrase drug can cause similar aberrant integrations. We also show that drug-resistance mutations in HIV integrase can also cause aberrant integrations, even in the absence of an anti-integrase drug. HIV DNA integrations in the oncogenes BACH2 and MKL2 that do not involve rearrangements of the viral or host DNA can stimulate the proliferation of infected cells. Based on what is known about the association of DNA rearrangements and the activation of oncogenes in human tumors, it is possible that some of the deletions, duplications, insertions, and inversions of the host DNA that accompany aberrant HIV DNA integrations could increase the chances that HIV integrations could lead to the development of a tumor.Electronic supplementary materialThe online version of this article (doi:10.1186/s12977-016-0305-6) contains supplementary material, which is available to authorized users.

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Avian Sarcoma and Leukemia Virus (ASLV) integration in vitro: Mutation or deletion of integrase (IN) recognition sequences does not prevent but only reduces the efficiency and accuracy of DNA integration

Moreau

Charmetant²,

Gallay³

et al. 2009

Virology

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Integrase (IN) is the enzyme responsible for provirus integration of retroviruses into the host cell genome. We used an Avian Sarcoma and Leukemia Viruses (ASLV) integration assay to investigate the way in which IN integrates substrates mutated or devoid of one or both IN recognition sequences. We found that replacing U5 by non-viral sequences (U5del) or U3 by a mutated sequence (pseudoU3) resulted in two and three fold reduction of two-ended integration (integration of the two ends from a donor DNA) respectively, but had a slight effect on concerted integration (integration of both ends at the same site of target DNA). Further, IN was still able to integrate the viral ends of the double mutant (pseudoU3/U5del) in a two-ended and concerted integration reaction. However, efficiency and accuracy (i.e. fidelity of size duplication and of end cleavage) of integration were reduced.

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Integration of Rous Sarcoma Virus DNA: a CA Dinucleotide Is Not Required for Integration of the U3 End of Viral DNA

Cited by 5 publications

References 24 publications

Retroviral Integrases Promote Fraying of Viral DNA Ends

Retroviral Integrases Promote Fraying of Viral DNA Ends

Drug resistant integrase mutants cause aberrant HIV integrations

Avian Sarcoma and Leukemia Virus (ASLV) integration in vitro: Mutation or deletion of integrase (IN) recognition sequences does not prevent but only reduces the efficiency and accuracy of DNA integration

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