Reconstitution and properties of homologous and chimeric HIV-1.HIV-2 p66.p51 reverse transcriptase.
Metal chelate affinity chromatography has been used to follow reconstitution of the 66- and 51-kDa human immunodeficiency (HIV)-1 and HIV-2 reverse transcriptase (RT) subunits into heterodimer, as well as chimeric enzymes comprised of heterologous subunits. By adding a small N-terminal polyhistidine extension to the 51-kDa subunit of either enzyme, reconstituted RT could be recovered from a cell lysate by chromatography on Ni(2+)-nitrilotriacetic acid-Sepharose. Homologous RT subunits rapidly associated to form the respective heterodimers (1-p66.1-p51 and 2-p66.2-p51) when bacterial lysates containing the individual components were mixed. Under the same conditions, association of p66 HIV-2 and p51 HIV-1 RT was inefficient and could be improved slightly by prolonged incubation of the respective p66 and p51 subunits. In contrast, HIV-1 p66 RT rapidly associated with the 51-kDa subunit of the HIV-2 enzyme. RNA-dependent DNA polymerase activity was associated with all reconstituted enzymes, and the response of each chimeric RT to an inhibitor selective for the HIV-1 enzyme indicated that sensitivity to inhibition was determined by the source of its 66-kDa subunit.
Howard
K J
KJ
Division of Infectious Diseases, Case Western Reserve University School of Medicine, Cleveland, Ohio 44106.
Frank
K B
KB
Sim
I S
IS
Le Grice
S F
SF
D002678:Chimera; D002850:Chromatography, Gel; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D054303:HIV Reverse Transcriptase; D015497:HIV-1 / Q000201:enzymology*; D015498:HIV-2 / Q000201:enzymology*; D055503:Protein Multimerization; D012194:RNA-Directed DNA Polymerase / Q000302:isolation & purification / Q000378:metabolism*; D018894:Reverse Transcriptase Inhibitors
D018894:Reverse Transcriptase Inhibitors; C072416:reverse transcriptase, Human immunodeficiency virus 2; D054303:HIV Reverse Transcriptase; D012194:RNA-Directed DNA Polymerase