Intrinsic activity of human immunodeficiency virus type 1 protease heterologous fusion proteins in mammalian cells.
We have generated various mammalian expression constructs that produce fusion proteins of human immunodeficiency virus type 1 (HIV-1) protease (PR) with the HIV-1 Nef protein. The expression of these proteins is inducible by the HIV-1 Tat protein. High-level expression of proteolytically active PR was produced from PR imbedded into Nef coding sequences, flanked by PR cleavage sites. The fusion protein was cleaved nearly to completion and did not exhibit the regulated processing that is seen with the virally encoded PR. No cytotoxic effect of PR expression was detected. The self-cleavage of PR could be inhibited by a specific inhibitor of HIV-1 PR (U75875). Elimination of the aminoterminal PR cleavage site did not have a measurable effect on cleavage of the precursor fusion protein. The cleaved fusion proteins appeared to be extremely unstable in the transfected cells. These findings demonstrate the intrinsic activity of HIV-1 PR in mammalian cells, in the context of a heterologous fusion protein.
Arrigo
S J
SJ
Department of Microbiology and Immunology, Medical University of South Carolina, Charleston 29425-2230.
Haines
J K
JK
Huffman
K M
KM
D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D015967:Gene Expression Regulation, Viral; D015693:Gene Products, nef / Q000235:genetics*; D015696:Gene Products, tat / Q000235:genetics; D005822:Genetic Vectors / Q000235:genetics; D016333:HIV Protease / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D017320:HIV Protease Inhibitors / Q000494:pharmacology; D015497:HIV-1 / Q000378:metabolism*; D006801:Humans; D008322:Mammals; D008969:Molecular Sequence Data; D011499:Protein Processing, Post-Translational / Q000187:drug effects; D012333:RNA, Messenger / Q000096:biosynthesis; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000378:metabolism / Q000633:toxicity; D017384:Sequence Deletion / Q000502:physiology; D054311:nef Gene Products, Human Immunodeficiency Virus; D054322:tat Gene Products, Human Immunodeficiency Virus
D015693:Gene Products, nef; D015696:Gene Products, tat; D017320:HIV Protease Inhibitors; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D054311:nef Gene Products, Human Immunodeficiency Virus; D054322:tat Gene Products, Human Immunodeficiency Virus; D016333:HIV Protease