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KMID : 0364820090450030246
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Korean Journal of Microbiology
2009 Volume.45 No. 3 p.246 ~ p.250
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Construction of a Fluorescently Labeled Infectious R Peptide-Less Moloney MLV Molecular Clone for Analysis of Syncytium
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Lee Yong-Jin
Park Jin-Woo
Lee Kyu-Jun
Bae Eun-Hye
Park Sung-Han
Lim Ji-Hyun
Kim Sae-Ro-Mi
Jung Yong-Tae
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Abstract
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Retroviruses enter host cells by membrane fusion between the viral Env proteins on the virus membrane and a
virus receptor on the cellular membrane. The envelope protein of the ecotropic Moloney murine leukemia virus is synthesized as a gp85 precursor and is proteolytically cleaved into an extracellular surface unit (SU) and the transmembrane protein (TM). The cytoplasmic tail (16 amino acid; R peptide) of the TM protein is further cleaved by the viral protease during virion maturation. Unlike the wild type Env protrin bearing the R peptide, R peptide-truncated Envelope induces syncytia in susceptible cells. To understand the mechanism of R peptidetruncated Env in syncytium formation, R peptide-truncated Env expressing full-length molecular clone containing EGFP in PRR (proline rich region) of Env was constructed. This molecular clone induced syncytia in transfected NIH3T3 cells, fluorescence was detected in the cytoplasm and at the plasma membrane, while the nuclei did not stain and appeared black by fluorescence microscopy. Interestingly, virions with truncated envelope produced from transfected NIH3T3 cells induced syncytia in NIH3T3 cells, but fluorescence was not detected in the same infected cells. It is believed that cell-free viruses direct the fusion of neighboring cells without infection. Our data suggests that use of EGFP-tagged envelope for monitoring syncytium is a sensitive and convenient method. We also found that virion incorporated the R peptide-truncated Env is able to induce the formation of syncytia by fusion from without.
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KEYWORD
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envelope protein, murine leukemia virus, R peptide, syncytium
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