Multiple RNase H activities in mammalian type C retravirus lysates

Self Cite

The mechanism of action of Moloney murine leukemia virus RNase H III was studied, utilizing the model substrate (A)n. (dT)n and polyacrylamide gel electophoresis to assay enzyme activity. Examination by electrophoresis on 15% polyacrylamide gels in 7 M urea and on DEAE-cellulose paper in 7 M urea revealed that, early in a reaction with [3H](A)n (dT). as substrate, RNase H III generated products ranging in length from 80 to 90 nucleotides to <10 nucleotides and that after extended incubation the limit digest products generated were 3 to 15 nucleotides long. Product oligomers were of the following configuration: [5'-P, 3'-OH](A)0. RNase H III was shown to be an exonuclease requiring free ends in its substrate for activity by the inability to degrade RNA inserted in Escherichia coli ColEl plasmid DNA. The enzyme was capable of attacking RNA in RNA-DNA hybrids in the 5' to 3' and 3' to 5' directions as demonstrated by the use of [3H, 5'-32P](A)wi. (dT)n and cellulose-[3H](A)n-(dT),. RNase H III was random in its mode of action because addition of excess unlabeled (A)n-(dT)n to an ongoing reaction with [3H](A)n.(dT)n as substrate resulted in immediate inhibition of enzyme activity.

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 86%

Mechanism of action of Moloney murine leukemia virus RNase H III

Gerard¹

1981

Self Cite

“…As mentioned above, the MuLV reverse transcriptase molecule is a single polypeptide which has both RNase H and polymerase activities (30,42,61). These activities are thought to have different active sites (6,11,15,18,29,40,41,60), but the location of the sites has not been identified. The present work on clone 23 indicates that both of these sites, as well as the antigenic determinants of the enzyme recognized by heterologous goat antisera (16), lie within the N-terminal portion of the polymerase protein.…”

Section: Nw I Pmentioning

confidence: 99%

“…Although polymerase and RNase H reside on the same polypeptide, each of these activities is thought to have a different active site. This conclusion is based on the differential sensitivities of the two activities to various types of inhibitors (6,11,18,40,41,60) and on the fact that proteolytic cleavage of reverse transcriptase gives rise to a protein product that has RNase H but not polymerase activity (15,29). It has been suggested (28,57) that an MuLV-associated endonuclease protein of approximately 40K caltons (28,43), which has properties similar to those of the avian retrovirus-encoded endonuclease pp32 (20) and which shares methionine-containing tryptic peptides with MuLV polymerase precursor molecules (28), may also be encoded by the pol gene.…”

mentioning

confidence: 99%

Murine leukemia virus mutant with a frameshift in the reverse transcriptase coding region: implications for pol gene structure

Levin¹,

Hu²,

Rein³

et al. 1984

The mQlecular defect in the nonconditional B-tropic MuLV pol mutant, clone 23 (Gerwin et al., J. Virol. 31:741-751, 1979), has been characterized by recombinant DNA technology. The entire mutant genome was cloned from an EcoRI digest of integrated cellular DNA into bacteriophage A Charon 4A and then subcloned at the EcoRI site of pBR322. NIH-3T3 cells transfected with the plasmid clone, termed pRTM (RTM, reverse transcriptase mutant), reproduced the properties of clone 23 virus-infected cells. In vivo ligation experiments involving cotransfection of subclones of pRTM and wild-type murine leukemia virus localized the defect in the clone 23 genome to an approximately 400-base-pair region in the pol gene between the Sail and XhoI sites.

“…1978, S76, p. 225). Although direct evidence to show that this second RNase H is a viral protein is not available, several published reports (4,5,12,21) indicate that this reverse transcriptase-free RNase H constitutes the major RNase H activity in mammalian retroviruses. Serologically, this activity seems to be unrelated to the reverse transcriptase-associated RNase H (4,12).…”

Section: Inhibition Of Rnase H Ofmentioning

confidence: 99%

Inhibition by RNA of RNase H Activity Associated with Reverse Transcriptase in Rauscher Murine Leukemia Virus Cores

Sarngadharan

Kalyanaraman

Gallo

1978

We reported earlier that core preparations of Rauscher murine leukemia virus, when separated on an isopycnic sucrose gradient, did not contain detectable levels of RNase H activity, while retaining high levels of reverse transcriptase activity. We reexamined this phenomenon, and the earlier observation was found to be reproducible. However, when doubly banded preparations of viral cores were solubilized and reverse transcriptase was isolated by ion-exchange chromatography, a coincident peak of a nuclease activity with the specificity of RNase H was observed, which indicated that RNase H was selectively inhibited in the core fractions. By direct activity measurements using the purified reverse transcriptase-RNase H from cores, this endogenous inhibitor has been identified as the viral RNA. Viral 70S RNA strongly inhibited RNase H activity purified either from whole virions or from prefractionated cores. Other RNAs tested that had inhibitory effects were yeast tRNA, polyadenylic acid, and polyguanylic acid. Polyuridylic acid and polyadenylic acid were moderately inhibitory, and polycytidylic acid did not inhibit the RNase H. A rabbit anti-reverse transcriptase immunoglobulin G inhibited both the reverse transcriptase and RNase H activities of the enzyme purified from cores. These data provide a rational explanation for the failure to detect RNase H activity in core preparations of Rauscher murine leukemia virus. Furthermore, these data are consistent with the idea that the RNase H and reverse transcriptase activities purified from cores reside on the same protein molecule. Possible biological implications of the observed inhibition of RNase H by RNA is discussed.