國家衛生研究院 NHRI:Item 3990099045/7429
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    题名: Differential expression levels of polymerase from HIV-1 subtypes prevalent in Taiwan via reporter assays
    作者: Tseng, FC;Wang, SW;Wang, JC;Lin, YC;Wu, CJ;Cheng, L;Ko, WC
    贡献者: Division of Infectious Diseases
    摘要: Introduction: Molecular characterization of HIV-1 has been mostly performed on sub-genotype B, which only accounts for 10% of the infected population. Among HIV-1 subtypes, transmission efficiency and AIDS progressionrate aswell as response to antiretroviral therapy (ART) can differ substantially. Objectives: In Taiwan, 3 prevalent subtypes are associated with different transmission routes: B', CRF07_BC and CRF01_AE were mostly identified from men who have sex with men, injection drug users and persons with heterosexual risks, respectively. In our clinical observations, patients with CRF07_BC usually had milder progression as surrogated by longer time intervals to the need for ART initiation, which contrastedwith the faster decline in CD4 counts after diagnoses among patients infected with the other two subtypes. Methods: In order to examine relative replication levels of the 3 subtypes, we incorporated luciferase (Luc) or green fluorescence protein (GFP) reporters into a 3rd generation lentivector system, and single stable clones of Jurkat T cells carrying the reporter were obtained by selection markers and limiting dilutions. Full-length uninterrupted 3Kb polymerase genes from CRF07_BC, CRF01_AE and B' strains were cloned, and replaced into the homologous region of a wild-type B HIV-1 plasmid, pNL4–3. HIV-1 recombinant viruses of each subtype harvested from the transfection of 293T cells were titrated by p24 ELISA and reverse transcriptase (RT) assay. Jurkatreporter cells were infected with equal amount of each recombinant viruses at MOI=1, and viral replication levels were measured via the reporters. Results: By the Luc reporter at 48 to 96 hours post-infection, the B' clone expressed the highest levels similar to the wild-type B, and the CRF07_BC clones consistently had the lowest levels. GFP levels were quantified by microscopy and flow cytometry, and similar patterns were observed. Intracellular HIV-1 Gag and Nef RNA expression as determined by real-time RT-PCR at 24-, 48- and 72- hour post-infection also demonstrated the same order, wild type = B' > CRF01_AE > CRF07_BC. In western blot analyses, recombinant viruses of the CRF07_BC had lower RT levels and the CRF01_AE had more unprocessed Gag as compared with other subtypes. Our results suggested that, at controlling for the same viral co-receptors and cell tropisms, polymerase activities of the CRF07_BC were significantly lower than those of the CRF01_AE and the B', which agreed with the disease progression observed in patients. Conclusion: Our reporter system is proven to differentiate levels of polymerase activities, and can potentially be developed to measure susceptibility to ART among these subtypes.
    日期: 2013-06
    關聯: International Journal of Antimicrobial Agents. 2013 Jun;42(Supl. 2):S129.
    Link to: http://dx.doi.org/10.1016/S0924-8579(13)70517-X
    JIF/Ranking 2023: http://gateway.webofknowledge.com/gateway/Gateway.cgi?GWVersion=2&SrcAuth=NHRI&SrcApp=NHRI_IR&KeyISSN=0924-8579&DestApp=IC2JCR
    Cited Times(WOS): https://www.webofscience.com/wos/woscc/full-record/WOS:000321447400399
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