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Constitutive expression of HIV-1 reverse transcriptase heterodimer in a human cell line.
Lari MA; Gibbs RA; Baylor College of Medicine Houston, TX.
December 30, 1995
Natl Conf Hum Retroviruses Relat Infect (1st). 1993 Dec 12-16;:125.

Human Immunodeficiency virus type-1 (HIV-1) reverse transcriptase (RT) is a heterodimer composed of 51 and 66 kDa subunits (p51 and p66). In order to generate a human cell line that constitutively expresses an active HIV RT heterodimer, the entire 66 kDa RT region was first cloned in a mammalian expression vector (pCMV66). Transient transfection of pCMV66 into the HT-1080 human fibrosarcoma cell line led to the expression of p66 without the generation of p51, and only a low level of RT activity could be detected. Stable pCMV66 cell lines also only expressed the 66 kDa subunit; thus, it appears that endogenous cellular proteases are not capable of cleaving p66 to generate the 51 kDa subunit. Attempts to establish a stable cell line expressing protease-RT region of HIV-1 were hampered by an apparent intolerance of HT-1080 cells to the HIV protease expression. Hence, to generate p51 independent of the HIV protease expression, the 51 kDa subunit was cloned separately (pSV51). Cotransfection of pSV51 and pCMV66 led to coexpression of the p51 and p56 subunits, with a dramatic increase in the RT activity. Stable HT-1080 cell lines expressing both the 51 kDa and the 66 kDa subunits exhibited on average a 17 fold increase in RT activity compared to the HT-1080 parental cell line. Immunohistochemical staining revealed a diffuse cytoplasmic expression of p51 and p66. This high level of HIV RT activity in the HT-1080 cell line is stable for at least 30 cell generations. These constructs are being used for studies of drug resistance.

Cell Line Drug Resistance, Microbial Human HIV Protease/GENETICS HIV-1/DRUG EFFECTS/*ENZYMOLOGY *RNA-Directed DNA Polymerase/*GENETICS Tumor Cells, Cultured ABSTRACT