Low level expression of the HIV-1 Nef protein in transiently transfected COS-1 cells in culture.
Nef (negative factor) is a membrane-associated cytoplasmic accessory gene product of HIV-1 that induces increased viral load and pathogenesis in the host. In the present study, we have utilized a transient co-transfection assay with the HIV-1lai nef gene and the gene for green fluorescent protein (GFP) in the monkey kidney cell line, COS-1, to analyze Nef expression. We have observed significantly lower level expression of the Nef protein relative to expression of the GFP reporter protein, even when identical vector constructs and similar plasmid DNA concentrations were used. Moreover, Northern blot analysis have indicated that comparable levels of both the Nef and GFP transcripts were transcribed from the co-transfected nef and gfp plasmid constructs, respectively. Western blot analysis have also indicated that the intracellular accumulation of the Nef protein was not visibly affected by proteolytic inhibitors of two major subcellular compartments, lysosomes and proteosomes. In addition, the Nef protein does not appear to be secreted into the culture medium up to 24 hr post-transfection. Our results indicate the possible selective translational expression or post-translational down-regulation of the Nef protein in cultured COS-1 cells that were transiently co-transfected with the nef and gfp genes. However, the mechanism(s) underlying this phenomenon has not been determined.
Hartz
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PA
Department of Biology, Morgan State University, 1700 East Cold Spring Lane, Baltimore, MD 21251, USA.
McMiller
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T
Scott-Wright
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D
Samuel
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KP
D000818:Animals; D019556:COS Cells; D002522:Chlorocebus aethiops; D003001:Cloning, Molecular; D003546:Cysteine Endopeptidases / Q000378:metabolism; D015693:Gene Products, nef / Q000235:genetics / Q000378:metabolism*; D049452:Green Fluorescent Proteins; D015497:HIV-1 / Q000502:physiology*; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism*; D008247:Lysosomes / Q000378:metabolism; D009097:Multienzyme Complexes / Q000378:metabolism; D046988:Proteasome Endopeptidase Complex; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D014779:Virus Replication / Q000502:physiology*; D054311:nef Gene Products, Human Immunodeficiency Virus
D015693:Gene Products, nef; D008164:Luminescent Proteins; D009097:Multienzyme Complexes; D012333:RNA, Messenger; D054311:nef Gene Products, Human Immunodeficiency Virus; D049452:Green Fluorescent Proteins; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex