Characterization of RNA Strand Displacement Synthesis by Moloney Murine Leukemia Virus Reverse Transcriptase

Abstract

The RNase H activity of reverse transcriptase (RT) is presumably required to cleave the RNA genome following minus strand synthesis to free the DNA for use as a template during plus strand synthesis. However, since RNA degradation by RNase H appears to generate RNA fragments too large to spontaneously dissociate from the minus strand, we have investigated the possibility that RNA displacement by RT during plus strand synthesis contributes to the removal of RNA fragments. By using an RNase H ؊ mutant of Moloney murine leukemia virus (M-MuLV) RT, we demonstrate that the polymerase can displace long regions of RNA in hybrid duplex with DNA but that this activity is approximately 5-fold slower than DNA displacement and 20-fold slower than non-displacement synthesis. Furthermore, we find that although certain hybrid sequences seem nearly refractory to the initiation of RNA displacement, the same sequences may not significantly impede synthesis when preceded by a single-stranded gap. We find that the rate of RNA displacement synthesis by wild-type M-MuLV RT is significantly greater than that of the RNase H ؊ RT but remains less than the rate of non-displacement synthesis. M-MuLV nucleocapsid protein increases the rates of RNA and DNA displacement synthesis approximately 2-fold, and this activity appears to require the zinc finger domain.Retroviral replication requires the single-stranded RNA genome of the virus to be converted into double-stranded DNA through a complex series of reactions termed reverse transcription. This process appears to be catalyzed solely by the viral reverse transcriptase (RT) 1 that possesses the following two distinct enzymatic activities: a polymerase activity that synthesizes DNA using either RNA or DNA templates, and an RNase H activity that cleaves RNA in hybrid duplex with DNA (1, 2).The current model of reverse transcription proposes that the RNase H activity of RT is critical for several steps including degradation of the 5Ј end of the RNA genome following minusstrong-stop DNA synthesis to facilitate the first jump, specific cleavage at the polypurine tract to create the plus strand primer, and removal of the plus and minus strand primers (3). Additionally, it is presumed that the RNase H activity is required to degrade the RNA genome following minus strand synthesis to free the minus strand DNA for use as a template during plus strand synthesis (reviewed in Ref. 3). In vitro studies, however, suggest that RNA fragments that are too large to spontaneously dissociate from the minus strand remain following cleavage of the genome (4 -11). Furthermore, evidence that plus strand synthesis in several retroviral systems is discontinuous demonstrates that stably annealed RNA fragments persist in vivo (3,(12)(13)(14). This raises the interesting possibility that reverse transcription requires a mechanism for the displacement of genomic RNA fragments during plus strand synthesis.Most replicative polymerases require accessory proteins such as helicases and single-strand binding proteins (SSBs) ...

Unique progressive cleavage mechanism of HIV reverse transcriptase RNase H

“…Recently, a study using murine leukemia virus RT showed that the wild-type enzyme supports a greater amount of synthesis on a DNA template with an annealed downstream RNA than the RNase Hminus mutant (22). This result indicates that the RT with RNase H activity has a higher efficiency for the removal of downstream RNA.…”

RNA Degradation and Primer Selection by Moloney Murine Leukemia Virus Reverse Transcriptase Contribute to the Accuracy of Plus Strand Initiation

“…SP6S1 (5Ј-AGCTATTTAGGTGACACTA-TAGAATACGCATG-3Ј) and SP6S2 (5Ј-CGTATTCTATAGTGTCACCT-AAAT-3Ј) were used to insert an SP6 promoter into M13INT. Amplification primers T7M13 and TermM13, used to generate M13LTR1, have been described (22). Construction of plasmids pGEMLTR2 and M13LTR2 has been described previously (22); pGEMLTR1 was similarly generated but contains a 738-bp insert from the Mo-MLV LTR (genome position 7758 -8332 joined to 69 -231 (23)) that, in addition to downstream sequences, includes the PPT and 68 nt of upstream sequence.…”

“…Amplification primers T7M13 and TermM13, used to generate M13LTR1, have been described (22). Construction of plasmids pGEMLTR2 and M13LTR2 has been described previously (22); pGEMLTR1 was similarly generated but contains a 738-bp insert from the Mo-MLV LTR (genome position 7758 -8332 joined to 69 -231 (23)) that, in addition to downstream sequences, includes the PPT and 68 nt of upstream sequence. M13LTR1 was constructed by cloning a polymerase chain reaction-amplified 782-bp region of pGEMLTR1, including the LTR insert, into M13mp7 DNA.…”

“…Denaturing polyacrylamide gels (8.3 M urea) were prepared with Sequagel reagents from National Diagnostics. Synthesis and characterization of native Mo-MLV NC and the NCdd mutant (zinc finger replaced by a Gly-Gly linker) have been described (21,22) and were generously provided by J. L. Darlix.…”

“…Single-stranded phage and phagemid DNAs were isolated using established procedures (24). Single-stranded DNA inserts LTRi (807 nt), LTR⌬PPTi (709 nt), and N-LTRi (738 nt) were excised and recovered from M13LTR1, M13LTR2, and M13INT, respectively, as described (22) except that the restriction enzyme digestion was with BamHI.…”