Abstract
Integration, one of the hallmarks of retrovirus replication, is mediated by a nucleoprotein complex called the preintegration complex (PIC), in which viral DNA is associated with many protein components that are required for completion of the early phase of infection. A striking feature of the PIC is its powerful integration activity in vitro. The PICs from a freshly isolated cytoplasmic extract of infected cells are able to insert viral DNA into exogenously added target DNA in vitro. Therefore, a PIC-based in vitro assay is a reliable system for assessing protein factors influencing retroviral integration. In this study, we applied a microtiter plate-based in vitro assay to a screening study using a protein library that was produced by the wheat germ cell-free protein synthesis system. Using a library of human E3 ubiquitin ligases, we identified RFPL3 as a potential stimulator of human immunodeficiency virus, type 1 (HIV-1) PIC integration activity in vitro. This enhancement of PIC activity by RFPL3 was likely to be attributed to its N-terminal RING domain. To further understand the functional role of RFPL3 in HIV infection, we created a human cell line overexpressing RFPL3. Immunoprecipitation analysis revealed that RFPL3 was associated with the human immunodeficiency virus, type 1 PICs in infected cells. More importantly, single-round HIV-1 infection was enhanced significantly by RFPL3 expression. Our proteomic approach displays an advantage in the identification of new cellular proteins affecting the integration activity of the PIC and, therefore, contributes to the understanding of functional interaction between retroviral integration complexes and host factors.
Highlights
The retroviral preintegration complex (PIC) exhibits full fidelity of integration in vitro
EcoRI-digested pUC19 (2.7 kb) was covalently immobilized on a Covalink 96-well microtiter plate as the target DNA, and HIV-1 PICs were produced in 293T cells with the infection of a vesicular stomatitis virus G protein (VSV-G)-pseudotyped lentiviral vector, LTR-Tat-IRES-GFP (20)
The copy number of viral DNA detected from the reaction using 25 l of active HIV-1 PICs was 5957.6 Ϯ 427.1, which was about 34 times higher than that of the background DNA detected with an inactivated PIC (180.8 Ϯ 12.7 copies, Fig. 1A)
Summary
The retroviral preintegration complex (PIC) exhibits full fidelity of integration in vitro. An alternative approach taken for the study of a host cofactor of the PIC is the in vitro reconstitution of salt-disrupted PIC integration activity by engaging purified recombinant proteins (14 –16) These conventional biochemical procedures targeting a particular protein or proteins are often laborious, presenting technical obstacles for high-throughput screening studies using a wide variety of proteins for comprehensive functional analysis of cellular factors involved in PIC integration. As a novel aspect of our study, the microtiter plate-based PIC integration assay was combined with a library of human proteins that had been produced using wheat germ cell-free technology to screen for new cellular modulators of HIV-1 PIC integration activity in vitro (18). By exploiting the high-throughput capabilities of both the microtiter plate-based PIC integration assay and the wheat germ cell-free protein production system, we performed a screening study for the identification of cellular proteins that regulate the integration activity of the HIV-1 PIC in vitro. Our work offers a proof of concept approach for the identification of novel host modulators of PIC function, which allows us to better understand the molecular aspects of HIV-1 integration
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