a large extension to hiv-1 gag, like pol, has negative impacts on virion assembly大扩展hiv - 1插科打诨,像波尔,对病毒粒子的组装有负面影响.pdfVIP

A Large Extension to HIV-1 Gag, Like Pol, Has Negative Impacts on Virion Assembly 1 2 2,3,4 1 Hiyori Haraguchi , Takeshi Noda , Yoshihiro Kawaoka , Yuko Morikawa * 1 Kitasato Institute for Life Sciences, Kitasato University, Minato-ku, Tokyo, Japan, 2 Institute of Medical Science, University of Tokyo, Minato-ku, Tokyo, Japan, 3 ERATO Infection-Induced Host Responses Project, Japan Science and Technology Agency, Saitama, Japan, 4 Influenza Research Institute, Department of Pathological Sciences, University of Wisconsin-Madison, Madison, Wisconsin, United States of America Abstract The GagPol protein of HIV-1 harbors viral enzymes, such as protease (PR), reverse transcriptase, and integrase, that are all crucial for virion infectivity. Previous studies have suggested that expression of GagPol alone does not produce viral particles and that the budding defect is caused by the presence of the Pol region. However, it has remained unknown why GagPol fails to produce viral particles. We show here that HIV-1 GagPol is incapable of membrane binding and subsequent particle assembly. Our confocal data indicated that, despite full N-myristoylation, GagPol protein failed to target plasma membrane with diffuse distribution in the cytoplasm. Membrane flotation analysis confirmed these findings. Progressive C- terminal truncation of GagPol to give GagPR allowed for plasma membrane targeting but still not for particle production. Conversely, the C-terminal addition of a noncognate protein, such as ß-galactosidase or 4 tandem GFP, to Gag impaired the membrane affinity, indicating that the Pol region, a large extension to Gag, inhibits membrane binding in the context of GagPol. The addition of the 10 N-terminal amino acids of Fyn kinase [Fyn(10)], a