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. 1991 Aug;65(8):4387–4397. doi: 10.1128/jvi.65.8.4387-4397.1991

Abortive reverse transcription by mutants of Moloney murine leukemia virus deficient in the reverse transcriptase-associated RNase H function.

N Tanese 1, A Telesnitsky 1, S P Goff 1
PMCID: PMC248878  PMID: 1712862

Abstract

The reverse transcriptase enzymes of retroviruses are multifunctional proteins containing both DNA polymerase activity and a nuclease activity, termed RNase H, specific for RNA in RNA-DNA hybrid form. To determine the role of RNase H activity in retroviral replication, we constructed a series of mutant genomes of Moloney murine leukemia virus that encoded reverse transcriptase enzymes that were specifically altered to retain polymerase function but lack RNase H activity. The mutant genomes were all replication defective. Analysis of in vitro reverse transcription reactions carried out by mutant virions showed that minus-strand strong-stop DNA was formed but did not efficiently translocate to the 3' end of the genome; rather, the DNA was stably retained in RNA-DNA hybrid form. Plus-strand strong-stop DNA was not detected. These results suggest that RNase H normally promotes strong-stop translocation, perhaps by exposing single-stranded DNA sequences for base pairing. Four new DNA species were also detected among the reaction products. Analysis of these DNAs suggested that they were minus-strand DNAs formed from VL30 RNAs encoded by the mouse genome. We suggest that reverse transcriptase can initiate DNA synthesis at any one of four alternate tRNA primer-binding sites near the 5' ends of VL30 RNAs.

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