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

Abstract

The functional organization of the murine leukemia virus reverse transcriptase was investigated by expressing a molecular clone containing AKR MuLV reverse transcriptase-coding sequences in Escherichia coli. A purified preparation of the expressed enzyme (pRT250 reverse transcriptase) consisted primarily of a 69-kilodalton protein that has normal levels of murine leukemia virus polymerase activity but 10-fold-reduced levels of RNase H compared with the viral enzyme. The deficit in RNase H activity was correlated with the absence of 60 to 65 amino acids normally present at the carboxyl end of murine leukemia virus reverse transcriptase. The results provide additional experimental evidence for the localization of polymerase and RNase H domains to the Nand C-terminal regions of reverse transcriptase, respectively.

Cassette mutagenesis of the reverse transcriptase of human immunodeficiency virus type 1

“…Although these mutations are in the polymerase domain, it is possible that they distort the folding of RT in a manner that specifically affects RNase H activity and not the polymerase activity. In contrast to the wellstudied RT of murine leukemia virus, which has polymerase and RNase H domains that are almost completely separate genetically (16, 22,39), the polymerase and RNase H domains of both HIV-1 and HIV-2 RT appear to interact. Analysis of deletion and insertion mutations has shown that disruption of either the RNase H or the polymerase domain of HIV RT usually disrupts both activities (10-12, 30).…”

Human immunodeficiency virus type 1 nucleocapsid protein reduces reverse transcriptase pausing at a secondary structure near the murine leukemia virus polypurine tract

“…Recently, it has been shown that compounds which oxidize the sulfur atoms in the zinc finger can inactivate HIV-1 (65) or MuLV (64) virions and dramatically inhibit virus replication. These compounds would also inactivate RT in an in vitro assay, since the presence of a reducing agent like dithiothreitol is required for activity (52,76). To solve this problem, we tested the activity of HIV-1 NC proteins which were modified chemically with either 3 or 6 equivalents of NEM and then purified by HPLC to remove any residual free NEM (data not shown).…”

Translational readthrough of the murine leukemia virus gag gene amber codon does not require virus-induced alteration of tRNA

“…The cells were passaged in parallel in Dulbecco modified Eagle medium containing 10% fetal bovine serum (GIBCO Laboratories), and the fluid was changed 1 day before they reached confluence. On the next day, fluid samples were taken for tests of infectivity (1) and reverse transcriptase activity (21). (Typically.…”