DNA synthesis exhibited by the reverse transcriptase of mouse mammary tumor virus : Processivity and fidelity of misinsertion and mispair extension

Abstract

We have recently expressed in bacteria an enzymatically active reverse transcriptase (RT) of mouse mammary tumor virus (MMTV), a mammalian retrovirus with a typical B-type morphology [Taube, R., Loya, S., Avidan, O., Perach, M. & Hizi, A. (1998) Biochemical J. 329, 579Ϫ587]. The purified recombinant protein was shown to possess the catalytic activities characteristic of retroviral reverse transcriptases. In the present study, we have analyzed two basic parameters characteristic of the DNA polymerase activity of the novel MMTV RT, namely the processivity and the fidelity of DNA synthesis. Two features related to fidelity were studied, the capacity to misinsert wrong nucleotides at the 3′ end of the nascent DNA strand and the ability to extend 3′ mispairs. The studied properties of MMTV RT were compared with those of the RT purified from virions of avian myeloblastosis virus (AMV), since AMV RT shows a relatively high sequence similarity to MMTV RT. MMTV RT shows a relative processivity of DNA synthesis which is as high as the reference AMV RT. Regarding fidelity of DNA synthesis, MMTV RT shows a fidelity of misinsertion lower than that of AMV RT, whereas its capacity to elongate mispaired DNA is lower than that of AMV RT indicating a somewhat higher fidelity. These fidelity properties are discussed also in the context of the RTs of lentiviruses, especially those of HIV, which were reported to exhibit an exceptionally low fidelity of DNA synthesis. It is clear that MMTV RT has a fidelity higher than that of lentiviral RTs.Keywords : mouse mammary tumor virus; avian myeloblastosis virus; reverse transcriptase ; DNA synthesis; processivity.The replication of retroviruses is initiated by the reverse transcription of the viral single-stranded RNA genome into a double-stranded linear DNA, which is subsequently integrated into the host cell chromosomal DNA. This critical step in the early life cycle of the viruses, is catalyzed by the viral reverse transcriptase (RT). The plus-strand viral RNA is copied by the RNA-dependent DNA polymerase activity of RT, producing RNA · DNA hybrids. Subsequently, the ribonuclease H activity of the RT specifically degrades the viral RNA in the RNA · DNA heteroduplex. Finally, a double-stranded linear DNA is obtained by the synthesis of the second DNA strand, catalyzed by DNA-dependent DNA polymerase activity of the same enzyme [1Ϫ4].Mouse mammary tumor virus (MMTV) is unique among retroviruses in its ability to replicate and function as a carcinogenic agent in mammary epithelial cells, resulting from transcriptional regulation by steroid hormones. Moreover, MMTV is the only infectious retrovirus with a distinct type-B morphology that was isolated both as an endogenous and an exogenous virus [5Ϫ8]. Therefore, it was of interest to study the reverse transcriptase of this retrovirus. We have recently expressed in bacteria the enzymatically active recombinant RT derived from the BR6 strain of milk-transmitted MMTV [9] by constructing a plasmid that induces in bacteria the synthesis of this e...

Biased mutational pattern and quasispecies hypothesis in H5N1 virus

“…A very similar bias was recently reported on SRAS, although to a lower extent with A > G and T > C substitutions making about 60% of all mutations observed (Eckerle et al, 2010). This bias was never reported for in vitro activity of AMV reverse transcriptase despite extensive misincorporation survey (Operario et al, 2005;Skinner and Eperon, 1986;Taube et al, 1998). This strong mutational bias is suggestive of the activity of a family of host cell proteins known as Adenosine Deaminases that Act on RNA or ADAR (Bass, 2002).…”

The processivity and fidelity of DNA synthesis exhibited by the reverse transcriptase of bovine leukemia virus

“…All our previous studies with a variety of retroviral RTs have shown that the parameters for fidelity of DNA synthesis in vitro (i.e. 3′ end misinsertion and the extension of the performed 3′ end mispaired primers) depend primarily on the sequences of nucleic acids copied, rather than whether DNA or RNA templates were copied [16,18,20,23]. Subsequently, in the present study we have analysed DNA templates as representing both DNA and RNA substrates.…”

“…the amount of total 32 P‐labelled primer extended per minute in the conditions used. The V max and K m values were calculated from the double‐reciprocal linear plots of velocity vs. dNTP concentrations [16,18].…”

Mutation Rates and Intrinsic Fidelity of Retroviral Reverse Transcriptases

“…Bovine leukemia virus (BLV) RT showed higher misinsertion fidelity than HIV-1 RT but lower than that of MLV RT, although the patterns of mispair elongation by the BLV enzyme suggested that its fidelity was similar to that reported for HIV-1 RT [105]. The MMTV RT was about 2- to 4-fold more error prone than AMV RT in misinsertion assays and showed a higher ability to extend mismatched template-primers [106]. On the other hand, misinsertion and mispair extension ratios obtained with RTs from porcine endogenous retrovirus were roughly similar to those obtained with MLV RT [107].…”