Purification and characterization of the DNA polymerase and RNase H activities in Moloney murine sarcoma-leukemia virus

Abstract

Two RNase H (RNA-DNA hybrid ribonucleotidohydrolase, EC 3.1.4.34) activities separable by Sephadex G-100 gel filtration were identified in lysates of Moloney murine sarcoma-leukemia virus (MSV). The larger enzyme, which we have called RNase H-I, represented about 10% of the RNase H activity in the virion. RNase H-I (i) copurified with RNA-directed DNA polymerase from the virus, (ii) had a sedimentation coefficient of 4.4S (corresponds to an apparent mol wt of 70,000), (iii) required Mn2+ (2 mM optimum) for activity with a [3H ]poly(A) .poly(dT) substrate, (iv) eluted from phosphocellulose at 0.2 M KCl, and (v) degraded [3H]poly(A) .poly(dT) and [3H]poly(C) .poly(dG) at approximately equal rates. The smaller enzyme, designated RNase H-II, which represented the majority of the RNase H activity in the virus preparation, was shown to be different since it (i) had no detectable, associated DNA polymerase activity, (ii) had a sedimentation coefficient of 2.6S (corresponds to an apparent mol wt of 30,000), (iii) preferred Mg2+ (10 to 15 mM optimum) over Mn2+ (5 to 10 mM optimum) 2.5-fold for the degradation of [3H]poly(A) .poly(dT), and (iv) degraded [3H]poly(A) .poly(dT) 6 and 60 times faster than [3H]poly(C) .poly(dG) in the presence of Mn2+ and Mg2+, respectively. Moloney MSV DNA polymerase (RNase H-I), purified by Sephadex G-100 gel filtration followed by phosphocellulose, poly(A) oligo(dT)-cellulose, and DEAE-cellulose chromatography, transcribed heteropolymeric regions of avian myeloblastosis virus 70S RNA at a rate comparable to avian myeloblastosis virus DNA polymerase purified by the same procedure.

Further characterization of the Friend murine leukemia virus reverse transcriptase-RNase H complex

“…ing a murine enzyme preparation consisting of a single polypeptide around 80,000 molecular weight (8,12); in a third case this polypeptide appears among others (2). In these three studies natural RNA transcription has been observed.…”

“…The size of the largest murine viral enzyme polypeptide ranges between the size of the two avian enzyme subunits, leaving open the question of whether it corresponds to a or /3. There is evidence in favor of either: the ability to transcribe native RNA observed in some cases (2,8,12) resembles the properties of /, whereas absence of RNA-dependent DNA synthesis (1,14) is similar to a. The RNase H activity has been characterized as a random exonuclease (3) identical to that exhibited by the avian a fragments (3), and the RNase H digestion products (12) have been described as being larger than expected by analogy to a /3-containing enzyme preparation.…”

Human T-Cell Leukemia Virus Type 1 Reverse Transcriptase (RT) Originates from the pro and pol Open Reading Frames and Requires the Presence of RT-RNase H (RH) and RT-RH-Integrase Proteins for Its Activity

“…The Pol domains of the various classes of viruses encode either three or four enzyme activities-PR, RT, RNase H (RH), and integrase (IN)-depending on whether the PR region is part of the pol gene, is encoded by the pro gene, or is located at the 3Ј end of the gag ORF. The mature RTs from different virus families have different subunit structures; avian virus RTs are ␣/␤ heterodimers (RT-RH-RT-RH-IN) (11), MuLV enzymes are monomers (RT-RH) (10,48), and the RT of HIV-1 is a heterodimer (RT-RH-RT; p66/p51) (7,25).…”

Reverse transcription of the HIV‐1 pandemic

“…However, the association rate of homodimer formation is slow and occurs with low affinity, while the dissociation constant of heterodimer formation is below 1 nM, which suggests that cleavage occurs before dimer formation (34). Furthermore, RTs from retroviruses are known to have two catalytic activities: DNA polymerization and an associated RNase H activity (RNA/DNA heteroduplex RNA degradation) (35)(36)(37). The formation of a p66/p51 functional heterodimer for HIV-1 RT evoked questions as to whether p66 and/or p51 possessed both DNA polymerase and RNase H activities.…”