Quantitative analysis of HIV-1 preintegration complexes

Abstract

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.

Differential Effects of Human Immunodeficiency Virus Type 1 Capsid and Cellular Factors Nucleoporin 153 and LEDGF/p75 on the Efficiency and Specificity of Viral DNA Integration

“…The Q-PCR primers and probes for total HIV-1 detection have been described (39). The BBL-PCR primers and probes were also as described (22), except that AE3014 and AE3013 replaced the first-and secondround primers AE2257 and AE989, respectively, to increase the specificities of the amplifications (44).…”

LTR-mediated retroposition as a mechanism of RNA-based duplication in metazoans

“…LTRs should coexist in the retroposed product because the integrase has to recognize 15-to 20-bp sequences located at each terminal (Hindmarsh and Leis 1999;Engelman et al 2009). Consistently, we found in the assembled sequences of nine (60%) retrocopies, the presence of the 5 ′ LTR (the data are unclear for the other retrocopies because the assembled sequences are too short).…”

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

“…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.…”

“…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.…”

“…This method uses the cytoplasmic extracts of HIV-1-infected target cells as a source of endogenous PIC activity, which is measured with a real-time quantitative Polymerase Chain Reaction (qPCR). The procedures for isolating PICs and measuring their integration activities are adapted, with modifications, from protocols published by the Engelman laboratory 20 . In this method, the PIC-associated integration activity is initiated by providing an exogenous 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.…”