Role of protein disulfide isomerase and other thiol-reactive proteins in HIV-1 envelope protein-mediated fusion.
Cell-surface protein disulfide isomerase (PDI) has been proposed to promote disulfide bond rearrangements in HIV-1 envelope protein (Env) that accompany Env-mediated fusion. We evaluated the role of PDI in ways that have not been previously tested by downregulating PDI with siRNA and by overexpressing wild-type or variant forms of PDI in transiently and stably transfected cells. These manipulations, as well as treatment with anti-PDI antibodies, had only small effects on infection or cell fusion mediated by NL4-3 or AD8 strains of HIV-1. However, the cell-surface thiol-reactive reagent 5, 5'-dithiobis(2-nitrobenzoic acid) (DTNB) had a much stronger inhibitory effect in our system, suggesting that cell-surface thiol-containing molecules other than PDI, acting alone or in concert, have a greater effect than PDI on HIV-1 Env-mediated fusion. We evaluated one such candidate, thioredoxin, a PDI family member reported to reduce a labile disulfide bond in CD4. We found that the ability of thioredoxin to reduce the disulfide bond in CD4 is enhanced in the presence of HIV-1 Env gp120 and that thioredoxin also reduces disulfide bonds in gp120 directly in the absence of CD4. We discuss the implications of these observations for identification of molecules involved in disulfide rearrangements in Env during fusion.
Ou
Wu
W
Laboratory of Molecular Microbiology, National Institute of Allergy and Infectious Disease, National Institutes of Health, Building 4, Room 336, Bethesda, MD 20892, USA. wou@niaid.nih.gov
Silver
Jonathan
J
D001483:Base Sequence; D002459:Cell Fusion; D002460:Cell Line; D017931:DNA Primers; D015497:HIV-1 / Q000502:physiology*; D006801:Humans; D008969:Molecular Sequence Data; D019704:Protein Disulfide-Isomerases / Q000378:metabolism*; D034741:RNA, Small Interfering / Q000235:genetics; D013438:Sulfhydryl Compounds / Q000378:metabolism; D014162:Transfection; D014759:Viral Envelope Proteins / Q000502:physiology*
D017931:DNA Primers; D034741:RNA, Small Interfering; D013438:Sulfhydryl Compounds; D014759:Viral Envelope Proteins; D019704:Protein Disulfide-Isomerases