Reverse Transcriptase Jumps and Gaps

Journal of virology, 1990

Reverse transcription of the retroviral RNA genome begins with tRNA-primed synthesis of a minus-strand DNA, which subsequently acts as the template for the synthesis of plus-strand DNA. This plus-strand DNA is initiated at a unique location and makes use of a purine-rich RNA oligonucleotide derived by RNase H action on the viral RNA. To determine the variables that are relevant to successful specific initiation of plus-strand DNA synthesis, we have used nucleic acid sequences from the genome of Rous sarcoma virus along with three different sources of RNase H: avian myeloblastosis virus DNA polymerase, murine leukemia virus DNA polymerase, and the RNase H of Escherichia coli. Our findings include evidence that specificity is controlled not only by the nucleic acid sequences but also by the RNase H. For example, while the avian reverse transcriptase efficiently and specifically initiates on the sequences of the avian retrovirus, the murine reverse transcriptase initiates specifically ...

Journal of Cell Science, 1987

The retroviral reverse transcriptase is a multifunctional protein. Not only does it contain both RNA-and DNA-directed DNA synthesis activities but also it contains an endonuclease activity necessary for the integration of viral RNA and a RNase H. This latter activity can reduce to oligoribonucleotides viral RNA that has been reverse transcribed into minus-strand DNA. However, during avian retrovirus genome replication it does this in a highly specific manner so as to generate a specific 12-base primer for plus-strand DNA synthesis. Even though many other oligoribonucleotides are also made there is an efficient selection of the specific primer followed by its efficient utilization in plus-strand DNA synthesis, and subsequent removal. We have used a reconstructed system to gain an understanding of the factors that contribute towards these observed specificities.

Viruses, 2009

Although polypurine tract (PPT)-primed initiation of plus-strand DNA synthesis in retroviruses and LTR-containing retrotransposons can be accurately duplicated, the molecular details underlying this concerted series of events remain largely unknown. Importantly, the PPT 3' terminus must be accommodated by ribonuclease H (RNase H) and DNA polymerase catalytic centers situated at either terminus of the cognate reverse transcriptase (RT), and in the case of the HIV-1 enzyme, ∼70Å apart. Communication between RT and the RNA/DNA hybrid therefore appears necessary to promote these events. The crystal structure of the HIV-1 RT/PPT complex, while informative, positions the RNase H active site several bases pairs from the PPT/U3 junction, and thus provides limited information on cleavage specificity. To fill the gap between biochemical and crystallographic approaches, we review a multidisciplinary approach combining chemical probing, mass spectrometry, NMR spectroscopy and single molecul...

Mobile Genetic Elements, 2015

Journal of Biological …, 2000

1 The abbreviations used are: MMLV, Moloney murine leukemia virus; RT, reverse transcriptase; H Ϫ RT, the RNase H-deficient version of MMLV reverse transcriptase containing point mutations that destroy RNase H activity (Superscript II); RT⌬Pol, a version of MMLV reverse transcriptase that lacks the polymerase domain; RT⌬H, a version of MMLV reverse transcriptase that lacks the RNase H domain (Superscript); T7, T7 DNA polymerase (Sequenase); T4, T4 DNA polymerase; PPT, polypurine tract; HIV-1, human immunodeficiency virus type 1; nt, nucleotide(s); LTR, long terminal repeat; DTT, dithiothreitol.

Virus Research, 2008

Journal of Virology, 2005

polymerase and RNase H activities. During reverse transcription these activities are necessary for the programmed sequence of events that include template switching and primer processing. Integrase then inserts the completed cDNA into the genome of the host cell. The RT of the LTR-retrotransposon Tf1 was subjected to random mutagenesis, and the resulting transposons were screened with genetic assays to test which mutations reduced reverse transcription and which inhibited integration. We identified a cluster of mutations in the RNase H domain of RT that were surprising because they blocked integration without reducing cDNA levels. The results of immunoblots demonstrated that these mutations did not reduce levels of RT or integrase. DNA blots showed that the mutations did not lower the amounts of full-length cDNA. The sequences of the 3 ends of the cDNA revealed that mutations within the cluster in RNase H specifically reduced the removal of the polypurine tract (PPT) primer from the ends of the cDNA. These results indicate that primer removal is not a necessary component of reverse transcription. The residues mutated in Tf1 RNase H are conserved in human immunodeficiency virus type 1 and make direct contact with DNA opposite the PPT. Thus, our results identify a conserved element in RT that contacts the PPT and is specifically required for PPT removal.

Proceedings of the National Academy of Sciences, 1990

Retroviral transduction of cellular nucleic acid sequences requires illegitimate RNA or DNA recombination. To test a model that postulates transduction via efficient illegitimate recombination during reverse transcription of viral and cellular RNAs, we have measured the ability of Harvey sarcoma viruses (HaSVs) with artificial 3' termini to recover a retroviral 3' terminus from helper Moloney virus (MoV) by illegitimate and homologous recombination. For this purpose, mouse NIH 3T3 cells were transformed with Harvey proviruses and then superinfected with MoV. The proviruses lacked the 3' long terminal repeat and an untranscribed region of the 5' long terminal repeat to prevent virus regeneration from input provirus. Only 0-11 focus-forming units of HaSV were generated upon MoV superinfection of 3 x 106 cells transformed by Harvey proviruses with MoV-unrelated termini. This low frequency is consistent with illegitimate DNA recombination via random Moloney provirus integration 3' of the transforming viral ras gene in the 10'-kilobase mouse genome. When portions of murine viral envelope (env) genes were attached 3' of ras, 102-105 focus-forming units of HaSV were generated, depending on the extent of homology with env of MoV. These recombinants all contained HaSV-specific sequences 5' and MoV-specific sequences 3' of the common env homology. They were probably generated by recombination during reverse transcription rather than by recombination among either input or secondary proviruses, since (i) the yield of recombinants was reduced by a factor of 10 when the env sequence was flanked by splice signals and (ii) HaSV RNAs without retroviral 3' Abbreviations: HaSV, Harvey sarcoma virus; MoV, Moloney virus; LTR, long terminal repeat; ffu, focus-forming unit(s); pfu, plaqueforming unit(s).

The Retroviridae, 1992

The retrovirus family encompasses a diverse group of metazoan viruses that have a replication step whereby DNA is synthesized from virion RNA in a process designated reverse transcription (Temin and Baltimore, 1972) (Fig. 1; Table I) (see Chapter 1). Molecular mechanisms in the virus life cycle are reviewed in this chapter, and the focus is on retroviruses containing genes for virion proteins but lacking genes that regulate viral expression. Retroviruses with simple genomes express the polyproteins (i.e., precursor polypeptides) encoded by the following genes: gag for group-specific antigen in the virion core, pol for RNAdependent DNA polymerase, and env for the viral envelope glycoprotein (Fig. 2). This genome organization is a feature of three genera in the retrovirus family, and both horizontally transmitted exogenous viruses and vertically transmitted endogenous viruses are included (Table II) (see Chapters 1 and 2) (Coffin, 1982b; Coffin and Stoye, 1985). Retroviruses with complex genomes (i.e., lentiviruses, spumaviruses, and certain oncoviruses) encode regulatory genes as well as virion proteins;

Journal of Virology, 1996

At the 3' end of all retroviral genomes there is a short, highly conserved sequence known as the polypurine tract (PPT), which serves as the primer for plus-strand DNA synthesis. We have identified the determinants for in vitro priming by the human immunodeficiency virus type 1 (HIV-1) PPT. We show that when the PPT is removed and placed into different nucleotide contexts, new priming sites are produced at the precise 3' end of the PPT. In addition, we find that a hybrid consisting of a 15- or 20-nucleotide RNA primer annealed to a 35-nucleotide DNA template is competent for initiation of plus-strand synthesis with HIV-1 reverse transcriptase. Thus, no cis-acting elements appear to be required for priming activity. Changes at the 5' end of the PPT have no effect on primer function, whereas the identity of bases at the 3' end is crucial. A primer containing only the 6 G residues from the 3' end of the wild-type PPT sequence and 9 bases of random sequence at the 5&...