FIG. 2.
Decreased synthesis of cDNA products during endogenous RT assay with Leu74Val RT. Virion-associated RT lysates with equivalent RT activity (2.5 × 104 methyl-3H cpm) were incubated at 37°C in the presence of dCTP, dGTP, dTTP, and [α-32P]dATP, and reactions were stopped at 1, 5, 10, 20, 40, and 60 min. Endogenous synthesized DNA products were digested with pronase, extracted with a phenol-chloroform mixture, and precipitated with ethanol. DNA products were boiled and separated on an alkaline agarose gel. The gel was dried and exposed to autoradiography. (A) Autoradiograph. Lanes: 1 to 6, RT lysates of pNL4-3 (WT) virus; 7 to 12, RT lysates of Leu74Val virus; 13, RT lysate of Leu74Val virus containing 1.5-fold-higher RT activity (4.0 × 104 methyl-3H cpm). (B) Quantitation of DNA products. The intensity of the total cDNA synthesized in each lane was quantified with ImageQuant (Molecular Dynamics) software and plotted in relation to time.