The Reverse Transcriptase of the Tf1 Retrotransposon Has a Specific Novel Activity for Generating the RNA Self-Primer That Is Functional in cDNA Synthesis

ABSTRACT The Tf1 retrotransposon of Schizosaccharomyces pombe represents a group of eukaryotic long terminal repeat (LTR) retroelements that, based on their sequences, were predicted to use an RNA self-primer for initiating reverse transcription while synthesizing the negative-sense DNA strand. This feature is substantially different from the one typical to retroviruses and other LTR retrotransposons that all exhibit a tRNA-dependent priming mechanism. Genetic studies have suggested that the self-primer of Tf1 can be generated by a cleavage between the 11th and 12th bases of the Tf1 RNA transcript. The in vitro data presented here show that recombinant Tf1 reverse transcriptase indeed introduces a nick at the end of a duplexed region at the 5′ end of Tf1 genomic RNA, substantiating the prediction that this enzyme is responsible for generating this RNA self-primer. The 3′ end of the primer, generated in this manner, can then be extended upon the addition of deoxynucleoside triphosphates by the DNA polymerase activity of the same enzyme, synthesizing the negative-sense DNA strand. This functional primer must have been generated by the RNase H activity of Tf1 reverse transcriptase, since a mutant enzyme lacking this activity has lost its ability to generate the self-primer. It was also found here that the reverse transcriptases of human immunodeficiency virus type 1 and of murine leukemia virus do not exhibit this specific cleavage activity. In all, it is likely that the observed unique mechanism of self-priming in Tf1 represents an early advantageous form of initiating reverse transcription in LTR retroelements without involving cellular tRNAs.

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A Novel Leu92 Mutant of HIV-1 Reverse Transcriptase with a Selective Deficiency in Strand Transfer Causes a Loss of Viral Replication

Journal of Virology ◽

10.1128/jvi.00809-15 ◽

2015 ◽

Vol 89 (16) ◽

pp. 8119-8129 ◽

Cited By ~ 4

Author(s):

Eytan Herzig ◽

Nickolay Voronin ◽

Nataly Kucherenko ◽

Amnon Hizi

Keyword(s):

Life Cycle ◽

Viral Replication ◽

Reverse Transcriptase ◽

Dna Polymerase ◽

Reverse Transcription ◽

Polymerase Activity ◽

Rnase H ◽

Strand Transfer ◽

Hiv 1

ABSTRACTThe process of reverse transcription (RTN) in retroviruses is essential to the viral life cycle. This key process is catalyzed exclusively by the viral reverse transcriptase (RT) that copies the viral RNA into DNA by its DNA polymerase activity, while concomitantly removing the original RNA template by its RNase H activity. During RTN, the combination between DNA synthesis and RNA hydrolysis leads to strand transfers (or template switches) that are critical for the completion of RTN. The balance between these RT-driven activities was considered to be the sole reason for strand transfers. Nevertheless, we show here that a specific mutation in HIV-1 RT (L92P) that does not affect the DNA polymerase and RNase H activities abolishes strand transfer. There is also a good correlation between this complete loss of the RT's strand transfer to the loss of the DNA clamp activity of the RT, discovered recently by us. This finding indicates a mechanistic linkage between these two functions and that they are both direct and unique functions of the RT (apart from DNA synthesis and RNA degradation). Furthermore, when the RT's L92P mutant was introduced into an infectious HIV-1 clone, it lost viral replication, due to inefficient intracellular strand transfers during RTN, thus supporting thein vitrodata. As far as we know, this is the first report on RT mutants that specifically and directly impair RT-associated strand transfers. Therefore, targeting residue Leu92 may be helpful in selectively blocking this RT activity and consequently HIV-1 infectivity and pathogenesis.IMPORTANCEReverse transcription in retroviruses is essential for the viral life cycle. This multistep process is catalyzed by viral reverse transcriptase, which copies the viral RNA into DNA by its DNA polymerase activity (while concomitantly removing the RNA template by its RNase H activity). The combination and balance between synthesis and hydrolysis lead to strand transfers that are critical for reverse transcription completion. We show here for the first time that a single mutation in HIV-1 reverse transcriptase (L92P) selectively abolishes strand transfers without affecting the enzyme's DNA polymerase and RNase H functions. When this mutation was introduced into an infectious HIV-1 clone, viral replication was lost due to an impaired intracellular strand transfer, thus supporting thein vitrodata. Therefore, finding novel drugs that target HIV-1 reverse transcriptase Leu92 may be beneficial for developing new potent and selective inhibitors of retroviral reverse transcription that will obstruct HIV-1 infectivity.

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Pausing during Reverse Transcription Increases the Rate of Retroviral Recombination

Journal of Virology ◽

10.1128/jvi.80.5.2483-2494.2006 ◽

2006 ◽

Vol 80 (5) ◽

pp. 2483-2494 ◽

Cited By ~ 24

Author(s):

Christian Lanciault ◽

James J. Champoux

Keyword(s):

Reverse Transcriptase ◽

Dna Synthesis ◽

Reverse Transcription ◽

Fluorescent Protein ◽

Leukemia Virus ◽

Primer Binding Site ◽

Infection Assay ◽

Green Fluorescent

ABSTRACT Retroviruses package two copies of genomic RNA into viral particles. During the minus-sense DNA synthesis step of reverse transcription, the nascent DNA can transfer multiple times between the two copies of the genome, resulting in recombination. The mechanism for this process is similar to the process of obligate strand transfers mediated by the repeat and primer binding site sequences. The location at which the DNA 3′ terminus completely transfers to the second RNA strand defines the point of crossover. Previous work in vitro demonstrated that reverse transcriptase pausing has a significant impact on the location of the crossover, with a proportion of complete transfer events occurring very close to pause sites. The role of pausing in vivo, however, is not clearly understood. By employing a murine leukemia virus-based single-cycle infection assay, strong pausing was shown to increase the probability of recombination, as reflected in the reconstitution of green fluorescent protein expression. The infection assay results were directly correlated with the presence of strong pause sites in reverse transcriptase primer extension assays in vitro. Conversely, when pausing was diminished in vitro, without changing the sequence of the RNA template involved in recombination, there was a significant reduction in recombination in vivo. Together, these data demonstrate that reverse transcriptase pausing, as observed in vitro, directly correlates with recombination during minus-sense DNA synthesis in vivo.

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

Journal of Virology ◽

10.1128/jvi.65.8.4387-4397.1991 ◽

1991 ◽

Vol 65 (8) ◽

pp. 4387-4397 ◽

Cited By ~ 46

Author(s):

N Tanese ◽

A Telesnitsky ◽

S P Goff

Keyword(s):

Reverse Transcriptase ◽

Reverse Transcription ◽

Murine Leukemia Virus ◽

Leukemia Virus ◽

Rnase H ◽

Moloney Murine Leukemia Virus ◽

Murine Leukemia

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Characterization of the Polymerase and RNase H Activities of Human Foamy Virus Reverse Transcriptase

Journal of Virology ◽

10.1128/jvi.78.12.6112-6121.2004 ◽

2004 ◽

Vol 78 (12) ◽

pp. 6112-6121 ◽

Cited By ~ 22

Author(s):

Paul L. Boyer ◽

Carolyn R. Stenbak ◽

Patrick K. Clark ◽

Maxine L. Linial ◽

Stephen H. Hughes

Keyword(s):

Reverse Transcriptase ◽

Leukemia Virus ◽

Foamy Virus ◽

Polymerase Activity ◽

Rnase H ◽

Immunodeficiency Virus ◽

Moloney Leukemia Virus ◽

Basic Loop

ABSTRACT Foamy virus (FV) replication, while related to that of orthoretroviruses, differs at a number of steps. Several of these differences involve the reverse transcriptase (RT). There appear to be fewer RTs present in FV than in orthoretroviruses; we previously proposed that the polymerase of FV RT was more active than orthoretroviral RTs to compensate for the numerical difference. Here we present further characterization of the RT of FV. The polymerase activity of FV RT was greater than that of human immunodeficiency virus type 1 RT in a variety of assays. We also examined the RNase H activity of FV RT, and we propose that FV RT has a basic loop in the RNase H domain. Although the sequence of the basic loop of FV RT is different from the basic loop of either Moloney leukemia virus RNase H or Escherichia coli RNase H, the FV RT basic loop appears to have a similar function.

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Mn2+ Suppressor Mutations and Biochemical Communication between Ty1 Reverse Transcriptase and RNase H Domains

Journal of Virology ◽

10.1128/jvi.02502-06 ◽

2007 ◽

Vol 81 (17) ◽

pp. 9004-9012 ◽

Cited By ~ 7

Author(s):

Robert M. Yarrington ◽

Jichao Chen ◽

Eric C. Bolton ◽

Jef D. Boeke

Keyword(s):

Reverse Transcriptase ◽

Ion Homeostasis ◽

Point Mutations ◽

Rnase H ◽

Avian Myeloblastosis Virus ◽

Manganese Ion ◽

Suppressor Mutations ◽

Suppressor Mutants ◽

Ty1 Retrotransposition

ABSTRACT Ty1 reverse transcriptase/RNase H (RT/RH) is exquisitely sensitive to manganese concentrations. Elevated intracellular free Mn2+ inhibits Ty1 retrotransposition and in vitro Ty1 RT-polymerizing activity. Furthermore, Mn2+ inhibition is not limited to the Ty1 RT, as this ion similarly inhibits the activities of both avian myeloblastosis virus and human immunodeficiency virus type 1 RTs. To further characterize Mn2+ inhibition, we generated RT/RH suppressor mutants capable of increased Ty1 transposition in pmr1Δ cells. PMR1 codes for a P-type ATPase that regulates intracellular calcium and manganese ion homeostasis, and pmr1 mutants accumulate elevated intracellular manganese levels and display 100-fold less transposition than PMR1 + cells. Mapping of these suppressor mutations revealed, surprisingly, that suppressor point mutations localize not to the RT itself but to the RH domain of the protein. Furthermore, Mn2+ inhibition of in vitro RT activity is greatly reduced in all the suppressor mutants, whereas RH activity and cleavage specificity remain largely unchanged. These intriguing results reveal that the effect of these suppressor mutations is transmitted to the polymerase domain and suggest biochemical communication between these two domains during reverse transcription.

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The Mauriceville plasmid of Neurospora crassa: characterization of a novel reverse transcriptase that begins cDNA synthesis at the 3' end of template RNA

Molecular and Cellular Biology ◽

10.1128/mcb.12.11.5131-5144.1992 ◽

1992 ◽

Vol 12 (11) ◽

pp. 5131-5144

Author(s):

H Wang ◽

J C Kennell ◽

M T Kuiper ◽

J R Sabourin ◽

R Saldanha ◽

...

Keyword(s):

Reverse Transcriptase ◽

Reverse Transcription ◽

Full Length ◽

Minus Strand ◽

Ribonucleoprotein Particle ◽

Cdna Synthesis ◽

Reverse Transcriptases ◽

In Vitro Transcripts

The Mauriceville and Varkud plasmids are retroid elements that propagate in the mitochondria of some Neurospora spp. strains. Previous studies of endogenous reactions in ribonucleoprotein particle preparations suggested that the plasmids use a novel mechanism of reverse transcription that involves synthesis of a full-length minus-strand DNA beginning at the 3' end of the plasmid transcript, which has a 3' tRNA-like structure (M. T. R. Kuiper and A. M. Lambowitz, Cell 55:693-704, 1988). In this study, we developed procedures for releasing the Mauriceville plasmid reverse transcriptase from mitochondrial ribonucleoprotein particles and partially purifying it by heparin-Sepharose chromatography. By using these soluble preparations, we show directly that the Mauriceville plasmid reverse transcriptase synthesizes full-length cDNA copies of in vitro transcripts beginning at the 3' end and has a preference for transcripts having the 3' tRNA-like structure. Further, unlike retroviral reverse transcriptases, the Mauriceville plasmid reverse transcriptase begins cDNA synthesis directly opposite the 3'-terminal nucleotide of the template RNA. The ability to initiate cDNA synthesis directly at the 3' end of template RNAs may also be relevant to the mechanisms of reverse transcription used by LINEs, group II introns, and other non-long terminal repeat retroid elements.

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