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

Abstract

The purified reverse transcriptase-RNase H complex from Friend murine leukemia virus consists of a single polypeptide of 84,000 molecular weight, which after mild protease treatment in vitro or after intentional degradation during the purification procedure allows the generation of several additional polypeptides. Degradation destroys the RNA-dependent DNA polymerase activity with native RNA templates and reduces RNase H but does not affect response to synthetic template primers such as poly(rA)oligo(dT). The properties of the intact murine enzyme consisting of a single polypeptide of 84,000 molecular weight are compared to those of the avian a subunit and the avian a/3 enzyme complex. The intact murine enzyme resembles the avian /3-containing enzyme complex and is different from a in the following respects: (i) it binds to native RNA templates; (ii) it transcribes native RNA templates into DNA, a reaction which can be inhibited by actinomycin D; (iii) RNase H activity behaves like a processive exonuclease; and (iv) analysis of the RNase H digestion products reveals oligonucleotides approximately four bases in length.

Mutant of B-tropic murine leukemia virus synthesizing an altered polymerase molecule

“…In this avian mutant, a smaller gag-pol precursor is present in cells, and a 57K protein precipitating with anti-pol serum is present in virions. It is interesting that a mammalian and an avian polymerase mutant should be so similar since the normal processing of the 180K precursor leads, in the avian system, to the a and /8 polypeptides (19), whereas murine reverse transcriptase consists of only a single subunit of approximately 85K (27,42). Future experiments designed to delineate the pathway of processing in these mutants will be of great interest.…”

“…Furthermore, this band is precipitated more strongly by anti-polymerase serum than by anti-whole virus serum. Thus, on the basis of immunoreactivity and molecular weight, as determined by SDS-gel electrophoresis (27,42), the band at 82K can be identified as reverse transcriptase and is labeled "pol" in the figure. The absence of a normal pol band in the mutant virions strongly suggests that a polymerase of the usual size or antigenic characteristics is not made.…”

Multiple RNase H activities in mammalian type C retravirus lysates

“…No information to 90,000 have been examined in detail (10, 21). was presented concerning RNase H activity de-The mode of action of lower-molecular-weight void of polymerase activity (16). Final proof of mammalian RNase H species such as those identhe parent-product relationship between Mtified in calf thymus (2) have not been estab-MLV RNase H I and RNase H IH wil await lished.…”

“…It Interestingly, M-MSV(MLV) RNase H HI was is possible that during this period proteolytic inhibited at substantially lower antiserum con-cleavage of DNA polymerase-RNase H catacentrations than was RNase H I ( Fig. 8): 50% lyzed by proteases resident in virion preparainhibition occurred at approximately 5 and 50 tions (16) occurs. Such proteolysis could generpg of anti-MLV sera with RNase H II and I, ate RNase H I.…”

Functional organization of the murine leukemia virus reverse transcriptase: characterization of a bacterially expressed AKR DNA polymerase deficient in RNase H activity

“…Although polymerase and RNase H activities can be separated in vitro, this situation may not represent normal biological activity. Thus, the truncated proteins having RNase H activity but no polymerase activity have lost the ability to act in a processive manner (5,12,28). In the case of the human immunodeficiency virus reverse transcriptase, removal of C-terminal sequences (12,14,23), even as few as 23 amino acids (14), drastically reduces polymerase activity.…”