Human Immunodeficiency Virus Reverse Transcriptase-associated RNase H Activity

M C Starnes,Y C Cheng

doi:10.1016/s0021-9258(18)83542-6

Abstract

Biochemical characteristics of the RNase H activity associated with immunoaffinity purified human immunodeficiency virus (HIV) reverse transcriptase (RT) were examined. Glycerol gradient centrifugation of HIV RT resulted in a single peak of RNase H, associated with RT activity, with an apparent molecular weight of 110,000. HIV RNase H exhibited a marked substrate preference for poly(dC).[3H]poly(rG) compared to poly(dT).[3H]poly(rA). It did not hydrolyze single-stranded RNA or the DNA component of DNA.RNA hybrids. Products of the HIV RT-associated RNase H reaction consisted primarily of monomers, dimers, and trimers with 3' OH groups. This reaction was Mg2+ dependent, with greater than 90% of maximum activity at MgCl2 concentrations between 4 and 12 mM. The optimum KCl concentration for HIV RT catalyzed polymerization with a poly(rA).(dT)10 template. The optimum pH for HIV RNase H activity was between 8.0 and 8.5, in contrast to an optimum pH of 7.5 to 8.0 for HIV RT activity. The association of RNase H activity with the p66 component of HIV RT was demonstrated by activity gel analysis. These results indicate that HIV RT has an integral RNase H activity; however, some of its properties are different from those of RNase H associated with other retroviral RT's, and optimal assay conditions are different than those for HIV RT catalyzed DNA polymerization.

Highlights

Biochemical characteristics of the RNase H activity dition, retroviral reverse transcriptase (RT) typically catalyzes hydrolysis of the RNA associated with immunoaffinity purified humanim- component of DNA
Was demonstrated by activity gel analysis. These results indicate that H activmunodeficiencyvirus (HIV) RT has an integral RNase H activity;,some of its properties are different from those of RNase H associated with other retroviral RT’s, and optimal assay conditions are different than those for HIV RT catalyzed DNA polymerization
All three activitiesco-migrated in a glycerol gradient activity gel in B contains ( a ) HIV RT, ( b )MML RT, and (c) E. coli during ultracentrifugation

Summary

Enzyme Assays

DNA- and RNA-dependent DNA Polymerase-Standard reaction mixtures (50 pl) contained 50 mM Tris-HC1,pH 8.0,2mM DTT, 100 pg/ml BSA, 100mM KC1,8 mM MgClz;0.5 AZmunits/ml of poly(rA). ( d T h , poly(rC). (dG)lZ-la, or poly(dC). (dG)L2-lsa;nd 10 p M (20 pCi/ ml) of [3H]TTPor [3H]dGTP.Reactions were initiated with 2-5pl of enzyme and incubated at 37 "C for 30 min. 40-pl aliquots were spotted on glass fiber filters (Whatman GF/A) and processed for determination of trichloroacetic acid-insoluble radioactivity. The remainder of the gel,which contained enzymes for activity recovery, wasshaken gently at room temperature for 1h in 2 changes (1liter each) of 50 mM Tris-HC1, pH 8.0, 2 mM DTT, 20% glycerol, followed by 16 h in 2 changes (1 liter each) of the same buffer plus. 50 mM KC1 and 8 mM MgCL The gel was shaken for another 8 h in two changes (I liter each) of 50 mM Tris-HC1, pH 8.0, 50 mM KC1, 2 mM DTT, 8 mMMgC12, in the absence of glycerol, followedby 16 h in the same buffer at 37 "C without shaking This was followed by gentle shaking for 4 h in (1liter) changes of cold 5% trichloroacetic acid, 10 mM pyrophosphate. Reactions were terminated by transfer towet ice followed byaddition of 50 pl of ice-

RESULTS

TRlS HEPES pH

Findings

DISCUSSION