Isolation and Analysis of HIV-1 Preintegration Complexes

Engelman, Alan

doi:10.1007/978-1-59745-170-3_10

Cited by 22 publications

(30 citation statements)

References 35 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…This report details a nested qPCR strategy for measuring HIV-1 PIC-associated integration activity in vitro. The major advantage and significance of this method is the requirement of relatively low amounts of PIC preparation per reaction 4,5,19 . The most critical steps of this method are the generation of a high-titer virus for infection and the handling of PIC preparation.…”

Section: Discussionmentioning

confidence: 99%

“…This protocol is derived from the methods described by Engelman et al 19,20 . HIV-1 PICs are nucleoprotein complexes that are assembled in infected cells and are comprised of the reverse-transcribed vDNA, viral proteins, and cellular factors 19,20 . The method described in this report uses the cytoplasmic extracts of HIV-1-infected cells as the source of PIC-associated integration activity.…”

Section: Isolation Of Hiv-1 Picsmentioning

confidence: 99%

“…This method utilizes a nested qPCR-based strategy for measuring PIC-associated IVI activity, and the procedures are adapted, with modifications, from protocols published by the Engelman laboratory 19,20,21 . In this method, the PIC-associated integration activity is initiated by providing a heterologous linear target DNA 21 , and the DNA products resulting from this integration reaction are purified and used as the starting material for subsequent PCR-based quantification.…”

Section: In Vitro Integration Activity Of Hiv-1 Picsmentioning

confidence: 99%

“…Though this method provides a direct measure of integration events, relatively high amounts of PIC preparation are required, which remains a technically challenging endeavor. To circumvent this, nested PCR-based assays requiring only modest amounts of PICs were developed 4,5,19 .…”

Section: Introductionmentioning

confidence: 99%

See 3 more Smart Citations

Measurement of <em>In Vitro</em> Integration Activity of HIV-1 Preintegration Complexes

Muthukumar

Davids

Addai

et al. 2017

JoVE

View full text Add to dashboard Cite

HIV-1 envelope proteins engage cognate receptors on the target cell surface, which leads to viral-cell membrane fusion followed by the release of the viral capsid (CA) core into the cytoplasm. Subsequently, the viral Reverse Transcriptase (RT), as part of a namesake nucleoprotein complex termed the Reverse Transcription Complex (RTC), converts the viral single-stranded RNA genome into a double-stranded DNA copy (vDNA). This leads to the biogenesis of another nucleoprotein complex, termed the pre-integration complex (PIC), composed of the vDNA and associated virus proteins and host factors. The PIC-associated viral integrase (IN) orchestrates the integration of the vDNA into the host chromosomal DNA in a temporally and spatially regulated two-step process. First, the IN processes the 3' ends of the vDNA in the cytoplasm and, second, after the PIC traffics to the nucleus, it mediates integration of the processed vDNA into the chromosomal DNA. The PICs isolated from target cells acutely infected with HIV-1 are functional in vitro, as they are competent to integrate the associated vDNA into an exogenously added heterologous target DNA. Such PIC-based in vitro integration assays have significantly contributed to delineating the mechanistic details of retroviral integration and to discovering IN inhibitors. In this report, we elaborate upon an updated HIV-1 PIC assay that employs a nested realtime quantitative Polymerase Chain Reaction (qPCR)-based strategy for measuring the in vitro integration activity of isolated native PICs.

show abstract

Section: Discussionmentioning

confidence: 99%

Section: Isolation Of Hiv-1 Picsmentioning

confidence: 99%

Section: In Vitro Integration Activity Of Hiv-1 Picsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

Measurement of <em>In Vitro</em> Integration Activity of HIV-1 Preintegration Complexes

Muthukumar

Davids

Addai

et al. 2017

JoVE

View full text Add to dashboard Cite

show abstract

“…In addition to cellular proteins, both complexes include viral RNA or DNA and several proteins, such as reverse transcriptase, integrase, and the viral protein R or Vpr [46]. These proteins are closely positioned together within these complexes, enabling mutual interactions between the proteins.…”

Section: Inhibition Of Rt By Peptides Derived From Vprmentioning

confidence: 98%

Therapeutic Potential of Peptide Motifs Against HIV-1 Reverse Transcriptase and Integrase

Andréola

2009

CPD

View full text Add to dashboard Cite

Multiple clinical benefits have been obtained thanks to the combination of drugs targeting several steps of the HIV-1 replication. However, despite such combination therapy, complete eradication of the virus cannot be attained. Moreover, emergence of resistance observed under treatment and the lengthening life expectancy of treated patients highlight the need for new anti-HIV agents. Peptide-based compounds that exhibit anti RT and anti integrase activities were particularly described. Active peptides have been obtained from several ongoing approaches. The study of interaction between viral proteins inside the preintegration complex, and the growing knowledge of interactions between viral proteins and cellular partners, have generated a useful source of data for the development of peptide inhibitors. Recent data were also obtained from the observation that viral enzymes such as RT and integrase are fully active when they are in a dimeric (RT) or oligomeric state. Peptides derived from the interface of dimers are also of interest. The obtention of efficient small molecules as competitive oligomerization inhibitors is problematic, but anyway, improved cellular uptake and chemical modifications that were obtained in the past ten years allowed numerous peptide drugs to reach the clinic. Finally, a new promising class of peptide inhibitors is emerging called "shiftides", which interfere with the ability of IN to adopt an oligomeric active state.

show abstract

Quantitative analysis of HIV-1 preintegration complexes

Engelman

Öztop

Vandegraaff

et al. 2009

Methods

Self Cite

View full text Add to dashboard Cite

Retroviral replication proceeds through the formation of a provirus, an integrated DNA copy of the viral RNA genome. The linear cDNA product of reverse transcription is the integration substrate and two different integrase activities, 3′ processing and DNA strand transfer, are required for provirus formation. Integrase nicks the cDNA ends adjacent to phylogenetically-conserved CA dinucleotides during 3′ processing. After nuclear entry and locating a suitable chromatin acceptor site, integrase joins the recessed 3′-OHs to the 5′-phosphates of a double-stranded staggered cut in the DNA target. Integrase functions in the context of a large nucleoprotein complex, called the preintegration complex (PIC), and PICs are analyzed to determine levels of integrase 3′ processing and DNA strand transfer activities that occur during acute virus infection. Denatured cDNA end regions are monitored by indirect end-labeling to measure the extent of 3′ processing. Native PICs can efficiently integrate their viral cDNA into exogenously added target DNA in vitro, and Southern blotting or nested PCR assays are used to quantify the resultant DNA strand transfer activity. This study details HIV-1 infection, PIC extraction, partial purification, and quantitative analyses of integrase 3′ processing and DNA strand transfer activities.

show abstract

Isolation and Analysis of HIV-1 Preintegration Complexes

Cited by 22 publications

References 35 publications

Measurement of <em>In Vitro</em> Integration Activity of HIV-1 Preintegration Complexes

Measurement of <em>In Vitro</em> Integration Activity of HIV-1 Preintegration Complexes

Therapeutic Potential of Peptide Motifs Against HIV-1 Reverse Transcriptase and Integrase

Quantitative analysis of HIV-1 preintegration complexes

Contact Info

Product

Resources

About