Molecular Virology SS2006 Types of Retroviruses Organisation Life Cycle

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Molecular Virology - SS2006Types of
RetrovirusesOrganisationLife Cycle

• Thomas Klimkait
• Institute of medical Microbiology

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Retroviruses

• 1963 Howard Temin Retroviruses contain RNA and
  can be inhibited by Actinomycin D
• Reverse Transcriptase is contained in
  retroviruses (Temin und Baltimore)
• 1981 Gallo discovers HTLV-1, the first human
  pathogenic retrovirus
• 1983 Co-discovery of HIV, the causative agent of
  AIDS is discovered by Montagniers and Gallos
  research groups

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Reverse-transcribing viruses
Retroviruses Alpharetrovirus Avian Leukosis
Virus Betaretrovirus Mouse Mammary tumor
virus Gammaretrovirus Murine leukemia
virus Deltaretrovirus Bovine leukemia
virus Epsilonretrovirus Walley dermal sarcoma
virus Lentivirus HIV-1 Spumavirus Chimp. foamy
virus
Metaviridae Metavirus Saccharomyces c. Ty3
virus Errantivirus Drosophila melanogaster gipsy
virus
Pseudoviridae Pseudovirus Saccharomyces c. Ty1
virus Hemivirus Drosophila melanogaster copia
virus
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Phylogeny
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Classification

• Historically, retroviruses were divided into
  groups based on their EM- morphology
• A-type 'intracisternal particles'.
• Non-enveloped, (non-infectious???)
• immature particles only inside cells,
• (result from endogenous retrovirus-like genetic
  elements?)
• B-type enveloped,
• extracellular particles with a condensed,
  acentric core,
• prominent envelope spikes, e.g. MMTV.

• C-type As B-type, but with a central core
• barely visible spikes
• e.g. mammalian and avian retroviruses (MLV, ALV,
  HTLV, HIV).
• D-type Usually slightly larger (to 120nm)
• spikes less prominent, e.g. MPMV.
• molecular genetic studies replaced morphologic
  differences,
• most comparisons now based on sequence
  conservation.

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Genome

• All genomes consist of two molecules of RNA,
• s/s, ()sense and have 5' cap and 3' poly-(A)
  (equivalent to mRNA).
• size from 8-11kb.
• Retrovirus genomes have 4 unique features
• the only viruses to be truly diploid.
• the only RNA viruses whose genome is produced by
  cellular transcriptional machinery (no
  participation by viral polymerase).
• the only viruses whose genome requires a specific
  cellular RNA (tRNA) for replication.
• the only ()sense RNA viruses whose genome does
  not serve directly as mRNA immediately after
  infection.
• genome molecules physically linked as a dimer by
  hydrogen bonds (co-sediment). In addition, 3rd
  type of nucleic acid present in all particles, a
  specific type of tRNA (usually trp, pro or lys) -
  required for replication
• Gene order in all retroviruses is invariant
• 5' - gag - pol - env - 3'

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Organisation
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Long Terminal Repeat

• U3 unique non-coding region of 200-1,200nt
  forms the 5' end of the provirus after RT
  contains promoter elements for transcription.
• R Region short (18-250nt) sequence
• forms a direct repeat at the both ends of the
  genome, 'terminally redundant'.
• U5 unique, non-coding region of 75-250nt
• is the first part of the genome to be reverse
  transcribed
• forms 3' end of the provirus genome.

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U3 as promoter region
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Special Sequence Features

• LTR
• Primer Binding Site 18nt complementary to the 3'
  end of the specific tRNA primer
• used as start for reverse transcription.
• Leader long (90-500nt) non-translated region
  downstream of the transcription start site
  present at the 5' end of all virus mRNAs.
• Polypurine Tract short (10) run of A/G residues
  responsible for initiating ()strand synthesis
  during RT

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Reverse Transcription to ds
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Integration products
Circles probably form by intracellular ligation.
The linear and 2-LTR circle forms are infectious
(unlike the ()sense vRNA!). Reverse
transcription occurs in the cytoplasm, after
which the provirus DNA migrates into the nucleus
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Integration process
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stepwise Integration

• 1st step, Processing two nucleotides removed
  from the 3' ends of the viral DNA, following a
  conserved dinucleotide, CA.
• 2nd Joining these new 3' ends are jointed to
  host target DNA in a concerted cleavage-ligation
  reaction.
• - Both steps are catalyzed by a multimer of IN,
  minimally a dimer, reaction that can be
  reconstituted in vitro. - requires
  divalent metal ion, Mn2 or Mg2.
• 3rd step, Repair requires DNA synthesis to
  fill in the gaps in host DNA that flank the
  provirus, removal of the overhangs of two
  nucleotides (2(pNpN)) at the 5' ends of the viral
  DNA, and ligation

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• The ends of the LTRs consist of inverted repeats
  of 4-6 bp.
• These are brought together to form a cleavage
  site for IN
• are cleaved to form a staggered cut.
• molecule is inserted into the host cell DNA. The
  net result of the integration process is that
• integrated proviruses contain 1 or 2 less bases
  at the termini
• ends of the integrated LTRs always have the same
  sequence 5' - TG...CA - 3'
• 4-6 bp of host cell DNA flanking the integrated
  provirus are duplicated.

• Model
• a staggered cut (5' overhang) is introduced into
  both the ends of the LTRs and the host cell DNA,
  followed by joining of the cut ends and repair of
  the free 3' ends.
• Once integrated, the provirus is present for the
  lifetime of the cell (consider germ-line
  integration).
• gt There is no specific mechanism for excision of
  the provirus the infected cell cannot be 'cured!

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Integrase Action
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Is integration site-specific?
gt HIV into active chromosomal regions!
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Splicing

• is regulated by the cellular apparatus, which
  interacts with cis-acting sequences present in
  the mRNA.
• proteins encoded by gag, pol and pro genes are
  expressed from a full length genomic RNA (
  vRNA).
• The env protein is expressed from a singly
  spliced mRNA.
• In complex retroviruses, (HTLV, Lentiviruses),
  multiply spliced mRNAs are produced, mark early
  expression.

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Splicing Patterns of Retroviruses
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Assembly (HIV)

• Gag proteins are transported to plasma membrane
• Gag proteins form defined budding structures
• Env proteins follow through the Golgi-pathway
  (glycosylation) are inserted into the Gag-layer
• vRNA follows to lign the buds
• Budding occurs from the membrane
• Gag-Proteins are cleaved gt particle
  maturation

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surface budding (HIV)
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Maturation
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essential Protease (HIV)
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Action of Protease Inhibitors
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HIV Life Cycle