Central Dogma

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Central Dogma

• Unidirectional flow of information
• DNA ? RNA ? protein
• In mid 60s started to think differentlya

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Reverse Transcription

• 1963 Howard Temin showed that the replication
  of retroviruses, whose particles contain RNA
  genomes, was inhibited by actinomycin D, an
  antibiotic which binds to DNA
• 1970 Temin and David Baltimore simultaneously
  published the observation that retrovirus
  particles contain an RNA-dependent DNA polymerase
  - reverse transcriptase
• Retrotransposons with striking similarities to
  retrovirus genomes form a substantial part of the
  genomes of all higher organisms, including humans

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Transposition

• Transposable genetic elements - specific
  sequences that are able to move from one position
  in the genome to another - fall into two groups
• simple transposons, which do not undergo reverse
  transcription and are found in prokaryotes (e.g.
  the genome of Enterobacteria phage Mu)
• retrotransposons, which closely resemble
  retrovirus genomes and are bounded by long direct
  repeats (long terminal repeats, LTRs), move by
  transcription, reverse transcription and
  integration in eukaryotes

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Similarities

• Believed responsible for high proportion of
  spontaneous mutations
• Promotes genetic rearrangements in host genome
• deletion, inversions, duplications translocations
• Mechanism generates short duplicates in
  cellular DNA
• Ends have inverted repeats
• Accompanied by replication of the element
• Control own transposition functions
• act in cis (affecting activity on contiguous
  sequence on the same nucleic acid) or in trans
  (encode diffusible products that act on
  regulatory sites in any stretch of nucleic acid
  in cell)

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Bacteriophage Mu

• Complex, tailed particle containing a linear,
  double-stranded DNA genome of about 40 kb
• Temperate bacteriophage whose replication can
  proceed through two pathways
• lysogeny integration of the genome into that of
  the host cell
• lytic replication results in the death of the
  cell
• Integration of the phage genome into that of the
  host bacterium occurs at random sites in the cell
  genome and requires viral and host cell proteins
• linked to replication, make a duplicate copy and
  it has 2 outcomes
• phage is replicated for new virus
• may have second insertion may or may not be
  harmful
• Integrated phage genomes are known as prophage
  and integration is essential for the
  establishment of lysogeny

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Bacteriophage Mu Genome
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Yeast Ty Viruses

• Representative of retrotransposons
• Protein encoded by TyA is capable of forming a
  roughly spherical, 60 nm diameter 'virus-like
  particle' (VLP) similar to gag of HIV also has
  TyB that is homologous to HIV pol
• 5.6 kb RNA transcript can be incorporated into
  these particles, resulting in the formation of
  intracellular structures known as Ty-VLPs
• Unlike true viruses these particles are not
  infectious, but if accidentally taken up by a
  cell, can carry out reverse transcription of
  their RNA to form a double-stranded DNA Ty
  element which can integrate into the host cell
  genome

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Yeast Ty Viruses
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Retroviruses

• The main difference between retrotransposons and
  retroviruses is the presence of an additional
  gene in retroviruses, env, which encodes an
  envelope glycoprotein
• The envelope protein is responsible for receptor
  binding and has allowed retroviruses to propagate
  by infection of other cells

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Retrovirus Genomes

• Retrovirus genomes have four unique features
• Only viruses which are diploid
• Only RNA viruses whose genome is produced by
  cellular transcriptional machinery
• Only viruses whose genome requires a specific
  cellular RNA (tRNA) for replication
• Only ()sense RNA viruses whose genome does not
  serve directly as mRNA immediately after infection

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Reverse Transcription

• During reverse transcription, the two
  single-stranded ()sense RNA molecules which
  comprise the virus genome are converted into a
  double-stranded DNA molecule longer than the RNA
  templates owing to the duplication of direct
  repeat sequences at each end - the long terminal
  repeats (LTRs)

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Reverse Transcription
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Retrovirus Genomes
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Retrovirus Genetics

• Reverse transcription is a highly error-prone
  process
• results in many mutations in retrovirus genomes
  and rapid genetic variation
• Since two RNAs are packaged into each virion and
  used as the template for reverse transcription,
  recombination occurs between the two strands
• can generate progeny viruses different from
  either parent

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Retrovirus Integration

• After reverse transcription is complete, the
  double-stranded DNA migrates into the nucleus
• Mature products of the pol gene form a complex of
  polypeptides which include three distinct
  enzymatic activities
• reverse transcriptase
• RNAse H - involved in reverse transcription
• integrase - catalyses integration of virus DNA
  into the host cell chromatin provirus

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Retrovirus Integration
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DNA in Infected Cell

• After reverse transcription, 3 types of DNA
• linear integrated form
• circular form with 1 LTR
• circular form with 2 LTRs
• No simple genomic target may be numerous
  possible sites of integration
• Provirus is at mercy of cell for replication,
  forms a full length mRNA without terminally
  redundant sequence becomes vRNA, pack 2 in
  virion

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2 Genome Strategies

• One like described for retroviruses package
  RNA into virion
• Two switch RNA and DNA phase of replication
  DNA viral genomes
• use RT as a late step rather than early
• used by hepadnavirus and caulimovirus

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Hepatitis B virus (HBV)

• Virions contain a partially double-stranded
  ('gapped') DNA genome, plus an RNA-dependent DNA
  polymerase (reverse transcriptase)
• Have very small genomes consisting of a ()sense
  strand of 3.0-3.3 kb and a ()sense strand of
  1.7-2.8kb
• On infection of cells, three major genome
  transcripts are produced 3.5, 2.4, and 2.1 kb
  mRNAs
• have same 3 ends but different 5 ends

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HBV Genome

• 4 known genes
• C core protein
• P polymerase
• S 3 polypeptides for surface antigen
• X transactivator of viral transcription

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Repair of Gapped DNA

• Complete the sense strand
• Remove protein primer from sense strand and
  oligribonucleotide primer from sense
• Eliminate terminal redundancy on sense
• Ligate 2 ends
• Unsure of mechanism

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Cauliflower mosaic virus (CaMV)

• Genome consists of a gapped, circular
  double-stranded DNA molecule of about 8 kbp, one
  strand of which contains a single gap, and a
  complementary strand which contains two gaps
• 8 genes encoded in this genome, although not all
  eight products have been detected in infected
  cells
• Gapped genome goes to nucleus and is repaired
• Transcribed to make 2 poly-A tailed transcripts
• 1 long that is translated
• 1 shorter that forms a large inclusion body in
  the cytoplasm that functions in 2nd site of
  replication that is RTed to be packaged in virion

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CaMV Genome
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Reverse Transcription of Virus Genomes
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Evolution of Viruses

• 3 theories
• regressive evolution viruses are degenerate
  life-forms which have lost many functions that
  other organisms possess and have only retained
  the genetic information essential to their
  parasitic way of life?
• cellular origins viruses are subcellular,
  functional assemblies of macromolecules which
  have escaped their origins inside cells?
• independent entities viruses evolved on a
  parallel course to cellular organisms from the
  self-replicating molecules which existed in the
  primitive prebiotic 'RNA world?

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Virus Superfamilies

• Genetic and nucleotide sequence relationships
  between viruses can reveal the origins of
  possible 'superfamilies
• nucleotide sequence is different but functions
  and active sites of proteins are conserved
• have functional and organizational similaries
• There are three orders of related virus families
• Mononegavirales Negative-sense RNA viruses with
  non-segmented genomes Bornaviridae,
  Filoviridae, Paramyxoviridae, Rhabdoviridae
• Caudovirales Tailed bacteriophages
  Myoviridae, Podoviridae, Siphoviridae
• Nidovirales "Nested" viruses - because of their
  pattern of transcription Arteriviridae,
  Coronaviridae

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Possible Virus Superfamilies