title: Identification of RFPL3 protein as a novel E3 ubiquitin ligase modulating the integration activity of human immunodeficiency virus, type 1 preintegration complex using a microtiter plate-based assay.
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.
authors.affiliation: Department of Microbiology, Yong Loo Lin School of Medicine, National University of Singapore, 14 Medical Drive, #15-02, Singapore 117599, Singapore.
authors.forename: Beng Hui
authors.initials: BH
authors.lastname: Tan
authors.affiliation: Department of Microbiology, Yong Loo Lin School of Medicine, National University of Singapore, 14 Medical Drive, #15-02, Singapore 117599, Singapore.
authors.forename: Yasutsugu
authors.initials: Y
authors.lastname: Suzuki
authors.affiliation: Department of Microbiology, Yong Loo Lin School of Medicine, National University of Singapore, 14 Medical Drive, #15-02, Singapore 117599, Singapore.
authors.forename: Hirotaka
authors.initials: H
authors.lastname: Takahashi
authors.affiliation: Veterinary Bioscience, Life Sciences and Chemical Technology, Ngee Ann Polytechnic, 535 Clementi Road, Singapore 599489, Singapore.
authors.forename: Pamela Ho Rui
authors.initials: PHR
authors.lastname: Ying
authors.affiliation: Department of Microbiology, Yong Loo Lin School of Medicine, National University of Singapore, 14 Medical Drive, #15-02, Singapore 117599, Singapore.
authors.forename: Chikako
authors.initials: C
authors.lastname: Takahashi
authors.affiliation: Department of Microbiology, Yong Loo Lin School of Medicine, National University of Singapore, 14 Medical Drive, #15-02, Singapore 117599, Singapore.
authors.forename: Qi'En
authors.initials: Q
authors.lastname: Han
authors.affiliation: Department of Microbiology, Yong Loo Lin School of Medicine, National University of Singapore, 14 Medical Drive, #15-02, Singapore 117599, Singapore.
authors.forename: Wei Xin
authors.initials: WX
authors.lastname: Chin
authors.affiliation: Expression Engineering Group, Bioprocessing Technology Institute, Agency for Science, Technology and Research (A*STAR), 20 Biopolis Way, #06-01 Centros, Singapore 138668, Singapore.
authors.forename: Sheng-Hao
authors.initials: SH
authors.lastname: Chao
authors.affiliation: Proteo-Science Center, Ehime University, 3 Bunkyo-cho, Matsuyama, Ehime 790-8577, Japan, and.
authors.forename: Tatsuya
authors.initials: T
authors.lastname: Sawasaki
authors.affiliation: Department of Microbiology, Yong Loo Lin School of Medicine, National University of Singapore, 14 Medical Drive, #15-02, Singapore 117599, Singapore,. Electronic address: micny@nus.edu.sg.
authors.forename: Naoki
authors.initials: N
authors.lastname: Yamamoto
authors.affiliation: Department of Microbiology, Yong Loo Lin School of Medicine, National University of Singapore, 14 Medical Drive, #15-02, Singapore 117599, Singapore,; Program in Emerging Infectious Diseases, Duke-NUS Graduate Medical School, 8 College Road, Singapore 169857, Singapore. Electronic address: californiacircle@gmail.com.
authors.forename: Youichi
authors.initials: Y
authors.lastname: Suzuki
mesh_terms: D002352:Carrier Proteins / Q000502:physiology*; D057809:HEK293 Cells; D015497:HIV-1 / Q000502:physiology*; D006801:Humans; D008979:Moloney murine leukemia virus / Q000502:physiology; D011485:Protein Binding; D017186:Titrimetry; D044767:Ubiquitin-Protein Ligases / Q000502:physiology*; D016662:Virus Integration
chemical_list: D002352:Carrier Proteins; C400219:RFPL3 protein, human; D044767:Ubiquitin-Protein Ligases
keywords: E3 Ubiquitin Ligase; HIV-1 Protease; Host-Pathogen Interaction; Microtiter Plate-based Integration Assay; Preintegration Complex; Protein Screening; Recombinant Protein Expression; Retrovirus; Wheat Germ Cell-free Protein Production System
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