Reverse Transcriptase

5 Key excerpts on "Reverse Transcriptase"

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  eBook - ePub

  Viruses: More Friends Than Foes

  • Karin Moelling(Author)
  • 2016(Publication Date)
  • WSPC

    (Publisher)

  Interestingly, the RT can perform two reactions: RNA to DNA and DNA to DNA, and between these two the original RNA, after it has been copied, has to be removed by the RNase H. The final result is double-stranded DNA.

  )

  The discovery of the reverse flow of information came as a great surprise. As a consequence the DNA can then be integrated into the host genomic DNA and be transmitted as long as the cell lives and divides as a cellular gene. Retroviruses normally do not lyse or destroy their host cells but “bud” out of the cells’ membranes. If we believe that RNA came first and that DNA came later during evolution, then “reverse” transcriptase is in fact the wrong name: the step from RNA to DNA is not a reverse one but a straight-ahead one. A strictly correct name would therefore be “real transcriptase”! Indeed, this would allow the same abbreviation to be used — however, nobody is interested in such a change.

  Now that, figuratively speaking, every possible genome has been sequenced, the latest surprise has been that there are numerous Reverse Transcriptases around. They are present in many organisms, in all eukaryotes (animals and plants), and also in archaea, in bacteria, in spliceosomes, in retrotransposons, in a strange chimeric multi-satellite msDNA, and in human and bacterial immune systems. In bacteria alone there are more than 1000 different kinds of Reverse Transcriptases. What are they all there for? In mammalian cells we know about the retrotransposons, which code for Reverse Transcriptases necessary for the “copy-and-paste” mechanism of cellular DNA described below (retrotransposons are reminiscent of simplified retroviruses). How unexpected this was can be described by an anecdote. In 1978 one of the co-discoverers of the Reverse Transcriptase David Baltimore got up in a meeting when someone described the existence of the Reverse Transcriptase in flies: “To my knowledge flies do not have retroviruses.” Only much later now we know the answer — also flies have Reverse Transcriptases not from retroviruses but from their relatives, the retrotransposons, which are precursors or truncated “crippled” retroviruses and widely distributed including flies, they are extremely abundant. The retroviruses as special case happened to be discovered first!

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  Enzymes of Nucleic Acid Synthesis and Modification

  Volume 1: DNA Enzymes

  • Samson T. Jacob(Author)
  • 2018(Publication Date)
  • CRC Press

    (Publisher)

  Table 1 ). Recent discoveries indicate that in addition to the RNase H and DNA polymerase activities of Reverse Transcriptase, the polymerase gene of retroviruses codes for a DNA endonuclease activity which may or may not be found associated with Reverse Transcriptase. This chapter will focus on the enzymatic and nucleic acid binding properties of these proteins and the role they play in the replication of retroviruses, and will attempt to place this analysis in perspective by making comparisons, where possible, with other better understood replicative systems.

  Retroviruses and their resident Reverse Transcriptases have been characterized from many different vertebrates. Most of our knowledge concerning Reverse Transcriptase and the replication of retroviruses is derived from studies with avian leukosis-sarcoma viruses (ALSV) and the enzyme from the avian myeloblastosis virus (AMV) complex of viruses, and to a lesser extent with murine leukemia virus (MuLV) and Moloney MuLV Reverse Transcriptase. This discussion will be confined to these two virus systems, which should portray a general picture applicable to most retroviruses and their enzymes.

  B. Background

  In order to understand the role of Reverse Transcriptase in the retrovirus life cycle a certain amount of background is essential.

  1. Life Cycle of Retroviruses

  The first event in the retrovirus life cycle is adsorption of the virion to the cell surface, followed by penetration of the virion into the cell cytoplasm. Uncoating of the virus particle to expose the internal core20 - 23 probably takes place within the cytoplasm the first 1 to 2 hr after infection.24 Viral RNA in the core is transcribed within the cytoplasm into linear duplex DNA16 , 25 - 27 which is transported to the nucleus where it can be converted to covalently closed circular DNA.28 Mature duplex DNA is integrated into the host genome.29 , 30 The mechanism of integration and the structure of the precursor to integration are not yet established. Subsequently, the integrated viral DNA is transcribed by cellular RNA polymerase31 and transcripts are processed and transported to the cytoplasm. Viral mRNA is translated into a series of precursor polyproteins which encapsidate 35S viral RNA32 and a specific subset population of cell tRNAs33 and the maturing particles move to the cell surface, from which they bud and acquire an outer envelope.32

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  Viruses

  From Understanding to Investigation

  • Susan Payne(Author)
  • 2017(Publication Date)
  • Academic Press

    (Publisher)

  Chapter 36 Family Retroviridae Abstract Retroviruses are enveloped viruses with RNA genomes. They are one of two families of animal viruses that reverse transcribe their genomes. Reverse transcription is the term used to describe the synthesis of a DNA copy of an RNA molecule. Reverse Transcriptase (RT) is the polymerase that catalyzes the process (RT is an RNA-dependent DNA polymerase). An older name for the group of viruses we now call retroviruses was “RNA tumor virus” because many are associated with transmissible cancers in mammals and birds. Retroviruses can transform cells because they must insert or integrate their genomes into the host cell chromosome, an activity that is inherently mutagenic. But retroviruses are much more than just cancer agents. About 8% of the human genome is comprised of retroviral sequences; retroviruses have shaped our genomes and driven our evolution. The human immunodeficiency virus (HIV), discovered only a few decades ago, infects millions of people worldwide. It causes disease primarily by damaging of a subset of T-lymphocytes that are central regulators of the immune response

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  Principles of Virology

  • Jane Flint, Vincent R. Racaniello, Glenn F. Rall, Theodora Hatziioannou, Anna Marie Skalka(Authors)
  • 2020(Publication Date)
  • ASM Press

    (Publisher)

  Chapter 8 ). The other two polymerases produce stable RNAs, such as ribosomal RNAs (rRNAs) and transfer RNAs (tRNAs). Synthesis of these stable “housekeeping” RNAs must be calibrated to match the rates of cell growth and division. But regulation of mRNA synthesis is crucial for orderly development and differentiation in eukaryotes, as well as for the responses of cells to their environment. The evolution of RNA polymerases with distinct transcriptional responsibilities appears to be a device for maximizing opportunities for control of mRNA synthesis while maintaining a constant and abundant supply of the RNA species essential for the metabolism of all cells.

  PRINCIPLES Synthesis of RNA from DNA templates

  • Transcription is the first viral biosynthetic reaction following infection of cells by double-stranded DNA viruses.
  • To form a template suitable for transcription, gapped, double-stranded or single-stranded DNA genomes are converted to double-stranded DNA molecules by cellular enzymes; retroviral RNA genomes are converted to double-stranded proviral DNA that is integrated into the cellular genome by viral enzymes.
  • Studies of viral transcription led to the identification of elements in DNA that direct pre-mRNA or mRNA synthesis, including promoters and enhancers that are binding sites for cellular proteins that mediate and regulate transcription.
  • The cellular transcriptional machinery alone is sufficient to transcribe some viral DNA templates.
  • Viral proteins can stimulate transcription of their own transcriptional unit to establish a positive autoregulatory loop or activate transcription of different viral genes.
  • Transcription of subsets of viral genes in distinct temporal periods (phases) is a characteristic feature of the reproductive cycles of all viruses with DNA genomes, including bacteriophages.
  • Viral proteins that regulate transcription may bind to viral promoter sequences directly or indirectly in association with cellular proteins.
  • Some viruses, including the herpesviruses, establish latent infections in which transcription of lytic genes is inhibited and, in some cases, unique latency-associated transcription units are expressed.

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  Transfer RNA in Protein Synthesis

  • Dolph L. Hatfield(Author)
  • 2018(Publication Date)
  • CRC Press

    (Publisher)

  tax. The provirus remains latent in the integrated state until stimuli induce viral expression by mechanisms that are not well-characterized. At this point, the cellular machinery carries out viral transcription and translation. With the synthesis of viral RNA and protein, assembled particles exit by budding through the cell membrane, which contributes to the viral envelope.

  Figure 3 shows steps in retroviral replication, in which genomic RNA is converted into a double-stranded DNA form capable of integration into the host genomic DNA. The primer for DNA synthesis is a specific tRNA, which is derived from the host cell and packaged into virions. The nucleotide sequence at the 3′ end of the appropriate primer tRNA hybridizes to a complementary 18–19 residue viral sequence, near the 5′ end of the RNA genome. DNA synthesis using RNA as template is carried out by the viral Reverse Transcriptase. The initial product of elongation of the tRNA is “minus strong stop DNA,” the result of DNA synthesis to the 5′ end of the genome (Figure 3 , step 1). The product molecule (minus viral strand) is then transferred to the 3′ end of genomic RNA, annealing to a terminal repeat therein (step 2). Experiments with heterozygous virus particles indicate that this first strand “jump” is intermolecular.5 The viral Reverse Transcriptase also possesses RNase H activity, which degrades the RNA strand of DNA:RNA hybrids. This RNase H activity may facilitate transfer of the minus strand by removing genomic RNA, thus eliminating the need for the minus strand to melt before subsequent annealing on the 3′ end.

  FIGURE 1. Retrovirus particle. The retroviral particle contains, among other components, the RNA viral genome and Reverse Transcriptase (RT), which are surrounded by a protein core. The core in turn is surrounded by the viral particle, containing lipid membrane from the host cell and viral envelope proteins.3

  FIGURE 2. Organization of the retroviral genome. The genome is indicated in the proviral form, double-stranded DNA integrated into host DNA. The components of the long terminal repeat (LTR) from the 5′ end, are U3, R, and U5, described in Figure 3 . The order of the gag, pol, and env genes are indicated. Details are contained in the text.2

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