Alternate Polypurine Tracts Affect Rous Sarcoma Virus Integration In Vivo

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

doi:10.1128/jvi.00361-06

Cited by 17 publications

(22 citation statements)

References 11 publications

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“…Genomic DNA was isolated from infected DF-1 (24,25) cells that survived zeocin selection using a QIAamp DNA blood maxikit (Qiagen). To recover full-length proviruses, 100 to 200 g of genomic DNA was digested with DraI overnight at 37°C.…”

Section: Methodsmentioning

confidence: 99%

Rous Sarcoma Virus (RSV) Integration In Vivo: a CA Dinucleotide Is Not Required in U3, and RSV Linear DNA Does Not Autointegrate

Chang

Wierzchoslawski

et al. 2008

J Virol

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The sequences required for integration of retroviral DNA have been analyzed in vitro. However, the in vitro experiments do not agree on which sequences are required for integration: for example, whether or not the conserved CA dinucleotide in the 3 end of the viral DNA is required for normal integration. At least a portion of the problem is due to differences in the experimental conditions used in the in vitro assays. To avoid the issue of what experimental conditions to use, we took an in vivo approach. We made mutations in the 5 end of the U3 sequence of the Rous sarcoma virus (RSV)-derived vector RSVP(A)Z. We present evidence that, in RSV, the CA dinucleotide in the 5 end of U3 is not essential for appropriate integration. This result differs from the results seen with mutations in the U5 end, where the CA appears to be essential for proper integration in vivo. In addition, based on the structure of circular viral DNAs smaller than the full-length viral genome, our results suggest that there is little, if any, integrase-mediated autointegration of RSV linear DNA in vivo.The retroviral life cycle is characterized by the conversion of the single-stranded RNA genome found in virions into a double-stranded linear DNA that is subsequently integrated into the host cell genome. Viral DNA synthesis takes place in the cytoplasm of the infected cell. The RNA genome of the virus is copied into DNA by a virally encoded enzyme, reverse transcriptase (RT). RT contains two enzymatic activities, a DNA polymerase that can copy either an RNA or a DNA template, and an RNase H that cleaves RNA only if the RNA is part of an RNA/DNA hybrid. Like many other DNA polymerases, RT requires both a template and a primer. First (minus)-strand DNA synthesis is initiated from a cellular tRNA primer that is base paired at the primer binding site (PBS), which is near the 5Ј end of the viral genomic RNA. During the reverse transcription process, this tRNA primer is removed from the end of the minus-strand DNA by RNase H; this defines the right (U5) end of the linear viral DNA. Second (plus)-strand DNA synthesis is initiated from a polypurine tract (PPT) primer adjacent to U3. The RNase H cleavages that generate and remove PPT primer define the left (

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Section: Methodsmentioning

confidence: 99%

Rous Sarcoma Virus (RSV) Integration In Vivo: a CA Dinucleotide Is Not Required in U3, and RSV Linear DNA Does Not Autointegrate

Chang

Wierzchoslawski

et al. 2008

J Virol

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“…DNA end processing prior to SSC formation could account for the different apparent rates of U5 versus U3 cleavage during HIV-1 infection (31) and is consistent with integrase-mediated integration of the sole wild type ends of single LTR end mutant viruses (128,129). However, 3′ processing and strand transfer of single viral DNA ends can occur in the context of the SSC in vitro (120).…”

Section: Integrase Functions As a Multimermentioning

confidence: 99%

“…Titers of viruses that contained one mutated LTR end were modestly reduced, whereas double end-mutant viruses were essentially dead (11,12,127). Sequence analysis revealed large insertions or deletions of cellular DNA associated with the integration of mutant LTR ends (128,129). Though virus replication normally proceeds through the pairwise insertion of both LTRs (Fig.…”

Section: Integrase Functions As a Multimermentioning

confidence: 99%

Retroviral Integrase Structure and DNA Recombination Mechanism

Engelman

Cherepanov

2014

Microbiol Spectr

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SUMMARYDue to the importance of human immunodeficiency virus type 1 (HIV-1) integrase as a drug target, the biochemistry and structural aspects of retroviral DNA integration have been the focus of intensive research during the past three decades. The retroviral integrase enzyme acts on the linear double-stranded viral DNA product of reverse transcription. Integrase cleaves specific phosphodiester bonds near the viral DNA ends during the 3′ processing reaction. The enzyme then uses the resulting viral DNA 3′-OH groups during strand transfer to cut chromosomal target DNA, which simultaneously joins both viral DNA ends to target DNA 5′-phosphates. Both reactions proceed via direct transesterification of scissile phosphodiester bonds by attacking nucleophiles: a water molecule for 3′ processing, and the viral DNA 3′-OH for strand transfer. X-ray crystal structures of prototype foamy virus integrase-DNA complexes revealed the architectures of the key nucleoprotein complexes that form sequentially during the integration process and explained the roles of active site metal ions in catalysis. X-ray crystallography furthermore elucidated the mechanism of action of HIV-1 integrase strand transfer inhibitors, which are currently used to treat AIDS patients, and provided valuable insights into the mechanisms of viral drug resistance.

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“…Consistent with the observation that the NTD works in trans with the CCD to catalyze 3Ј-processing activity (27,28), Li et al (24) demonstrated that a single HIV-1 DNA end can moreover be processed and integrated in the context of the intasomal tetramer in vitro. Such asymmetry may very well account for the different rates at which the U3 versus U5 ends of the HIV-1 LTR are processed during acute infection (3), as well as for the reasonable titers of single LTR end mutant viruses (53,54), where IN-mediated integration of the wild-type end presumably templates subsequent host-mediated integration of the mutant viral DNA end (55,56). Skalka and co-workers (57) have interestingly reported a novel "reaching dimer" structure for avian sarcoma virus IN protein based on small-angle x-ray scattering and chemical cross-linking that mimics the extended inner monomers of the PFV intasome.…”

Section: Retroviral In-dna Nucleoprotein Complexesmentioning

confidence: 99%

Retroviral Integrase Proteins and HIV-1 DNA Integration

Krishnan

Engelman

2012

Journal of Biological Chemistry

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Retroviral integrases catalyze two reactions, 3-processing of viral DNA ends, followed by integration of the processed ends into chromosomal DNA. X-ray crystal structures of integrase-DNA complexes from prototype foamy virus, a member of the Spumavirus genus of Retroviridae, have revealed the structural basis of integration and how clinically relevant integrase strand transfer inhibitors work. Underscoring the translational potential of targeting virus-host interactions, small molecules that bind at the host factor lens epithelium-derived growth factor/ p75-binding site on HIV-1 integrase promote dimerization and inhibit integrase-viral DNA assembly and catalysis. Here, we review recent advances in our knowledge of HIV-1 DNA integration, as well as future research directions.Retroviral replication proceeds through an obligate proviral or integrated DNA recombination intermediate. Integration provides a favorable environment for efficient gene expression, ensures inheritance of the virus in both daughter cells during mitosis, and forms the basis for latent HIV-1 reservoirs that persist in the face of highly active antiretroviral therapy. The early events of retroviral replication take place within the context of nucleoprotein complexes that are derived from the core of the infecting virus particle (1, 2). Within the confines of the reverse transcription complex (RTC), 2 the reverse transcriptase enzyme copies single-stranded viral RNA into a linear double-stranded DNA molecule containing a copy of the LTR sequence at each end. The viral integrase (IN) engages the LTR ends prior to catalyzing two spatially and temporally distinct chemical reactions. Soon after their synthesis (3), IN site-specifically processes each LTR end adjacent to an invariant CA dinucleotide (4, 5), yielding CA OH 3Ј-hydroxyl groups that serve as the nucleophiles for the second reaction, DNA strand transfer. Because the viral replication intermediate at this point gains the ability to catalyze DNA strand transfer activity, 3Ј-processing operationally marks the transition of the RTC to the pre-integration complex (PIC) (Fig. 1). In the strand transfer step, IN utilizes the 3Ј-oxygen atoms to cut chromosomal DNA in a staggered fashion and simultaneously join the viral DNA ends to the 5Ј-phosphates of the target DNA (4, 6, 7). The resulting DNA recombination intermediate, with unjoined viral DNA 5Ј-ends, is repaired by host cell enzymes to yield the integrated provirus flanked by the duplication of the sequence of the staggered DNA cut (Fig. 1). The spumaviruses, which compose one of seven Retroviridae genera, are an exception to this generalized scheme, as DNA synthesis is largely complete prior to target cell infection (8). Research on the functionality of the active nucleoprotein complexes that mediate HIV-1 DNA integration has led to the development of antiviral inhibitors that target the IN and block integration. IN Domain Structure and Reaction MechanismRetroviral INs belong to a superfamily of proteins known as the retroviral IN superfam...

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Alternate Polypurine Tracts Affect Rous Sarcoma Virus Integration In Vivo

Cited by 17 publications

References 11 publications

Rous Sarcoma Virus (RSV) Integration In Vivo: a CA Dinucleotide Is Not Required in U3, and RSV Linear DNA Does Not Autointegrate

Rous Sarcoma Virus (RSV) Integration In Vivo: a CA Dinucleotide Is Not Required in U3, and RSV Linear DNA Does Not Autointegrate

Retroviral Integrase Structure and DNA Recombination Mechanism

Retroviral Integrase Proteins and HIV-1 DNA Integration

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