Retroviruses

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Chapter 16

• Retroviruses

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Introduction

• RNA virus that copies genome into DNA during
  replication
• Disputes the Central Dogma make mRNA into DNA
  using reverse transcription
• Found in all classes of vertebrate animals
  fish, amphibians, birds and mammals
• HIV-1 and -2 some cause cancer

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Retrovirus Virion

• Virions contain 2 copies of RNA genome
  described as diploid, forms a dimer
• complementary sequence is called a kissing loop
  complex
• Also have host RNA packaged during assembly
  tRNA bound to RNA at primer binding site near
  the 5 end
• tRNA is specific for each virus
• Nucleocapsid protein (NC) is most abundant
  protein associated with RNA
• Other proteins in smaller amounts have enzyme
  activity

See figure legend for abbreviations
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Retrovirus Virion

• Lattice of capsid protein (CA) is around coated
  RNA with conical, spherical or cylindrical shape
  based on virus
• Layer of matrix proteins (MA) between capsid and
  envelope
• Associated with envelope is transmembrane protein
  (TM) bound to heavily glycosylated surface
  protein (SU)
• interaction is usually non-covalent

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Genome Regions

• 3 major regions in the genome
• gag group specific antigen internal
  structural proteins
• pol polymerase enzymes
• env envelope proteins

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Enzyme Activities in Virion

• RNA-dependent DNA polymerase (RT reverse
  transcriptase)
• DNA-dependent DNA polymerase
• Ribonuclease H (RNase H) removes RNA from a
  RNA-DNA duplex
• Integrase integrates viral genome into host
  genome
• Protease virion matuation

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Attachment and Entry

• Use SU protein with virus attachment site to bind
  cellular receptors causing changes in TM
  conformation exposing hydrophobic fusion sequence
• cell membrane and envelope fuse
• Most use plasma membrane fusion by some use
  endocytosis and then fuse envelope with membrane
  of endosome
• Structure in cytoplasm looses some proteins and
  creates reverse transcription complex

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Reverse Transcription

• Takes place in a complex
• Synthesis of () and (-) DNA begins at 3 OH of
  primer RNA
• (-) DNA used tRNA primer
• () DNA primer is a polypurine tract (PPT) in
  viral genome
• see PPT when RNase H starts at 3 end
• During synthesis each detached from template and
  reattaches at other end of template thru
  base-pairing
• Provirus DNA resulting from reverse
  transcription longer than genome
• Each termini has U3-R-U5 sequence long terminal
  repeat (LTR)
• U3 on one end and U5 on the other

See figure legend for good summary
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Integration of Provirus

• Provirus complexed with protein moves to nucleus
  pre-integration complex
• most retroviruses require cells going into
  mitosis for the breakdown of the nuclear membrane
• productive infection only in dividing cells
• HIV and related viruses can enter intact nuclei,
  no need for cell division for productive
  infection
• Integrase is still attached cuts up the DNA of
  the cell and seals provirus in the gap
• may lead to immediate expression of viral genes
  or little or no expression latent infection
• when this cell divides so does the genomes and
  get daughter cells with viral genome

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Transcription and Genome Replication

• 2 LTRs have identical sequence but functionally
  different 1 acts as initiation site and other
  as termination site
• TF binds upstream LTR and cell RNA pol II starts
  transcription at U3-R junction and moves
  downstream where termination occurs at R-U5
• Transcripts are capped and PolyA tailed with some
  acting as mRNA (may be spliced) and others become
  genome

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Translation and Post-Translational Modifications

• env gene is translated from spliced mRNA in RER
  because glycosylated
• Env proteins move to Golgi cleave to TM and SU
  proteins
• TM/SU stay close and are further glycosylated
  before movement to plasma membrane
• gag and pol genes are translated from genome
  length mRNA into Gag and Gag-Pol polyproteins
• need more Gag proteins so have a mechanism that
  allows ribosomes to terminate after Gag synthesis
  in about 95 ribosomes, others make the
  polyprotein
• Gag and Gag-Pol of most retroviruses are
  myristylated at N

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Murine Leukemia Virus Mechanism

• Mechanism involves reading thru a stop codon near
  end of Gag
• Suppressor tRNA adds amino acids and continues
  translation into pol gag an pol are in same ORF

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Majority of Retroviruses

• Mechanism is ribosomal frame-shifting to get the
  appropriate numbers of Gag and Pol
• gag and pol are in different reading frames that
  differ by -1 nt shift in about 5 of translations
  (used by HIV-1)

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Assembly

• Some retroviruses make immature particles in
  cytoplasm but not assembled at plasma membrane
• N of Gag and Gag-Pol anchor to membrane by
  myristyl group and stabilized by electrostatic
  interactions between MA domain and negative
  charge of phosphate in membrane
• MA domain also binds cytoplasmic tails of TM
  protein in membrane
• NC domain of Gag and Gag-Pol bind polyprotein to
  virus RNA and make genome dimer
• Protein binds 1st to packaging signal near 5 end
  of RNA and tRNA binds to PBS RNA coated with
  many copies of Gag and few copies of Gag-Pol

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Release of Virions

• Immature virion gets envelope by budding from
  cell surface
• Gag and Gag-Pol are arranged radially with N
  facing out and C facing inward
• The late domain of Gag binds host cell factors
  involved in budding process
• During or after budding viral protease cleave Gag
  and Gag-Pol
• Gag cleavage products form matrix, capsid and
  protein component of nucleocapsid
• Pol cleavage products are virion enzymes

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Examples of Retroviruses

• Classified as simple or complex dependent on
  genome
• Simple have 3 retroviral genes gag, pol and env
• some cases have 1 additional gene an oncogene
  because it may cause host cells to become a tumor
  cell
• src gene in Rous sarcoma virus
• Complex have additional genes with the products
  having a variety of function in replication cycle
• HIV-1 and HIV-2 are complex (Chapter 17)

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Representatives of Family Retroviridae

• Many cause disease
• in mammals feline leukemia virus
• in birds Rous sarcoma virus
• in fish walleye dermal sarcoma virus

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Retroviruses as Gene Vectors

• Genetically modified virion to act as gene vector
• Can introduce gene into cell, express to high
  levels after integration into cell genome
• clinical treatment of genetic disorders and
  cancer
• use murine leukemia virus, commonly
• Lentiviruses also used can be delivered to
  non-dividing cells/tissues which is a big
  advantage

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Success Story

• X-linked severe-combined immunodeficiency
  (X-SCID) is successfully treated with retroviral
  vectors
• take stem cells from patient and infect them with
  virus that has missing gene
• virus integrates into genome
• give stem cells back to and provirus expresses
  good genes and develop a normal immune system
• bad side several patients developed cancer

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Endogenous Retroviruses

• Genomes of vertebrates contain retroviral genomes
  endogenous retroviruses (ERV), virus is
  defective
• 100,000 human ERV sequences and find in other
  sequenced organisms
• Some are related to normal retroviral genomes
  exogenous retroviruses
• ERVs in mice similat to sequence in genome of
  mouse mammary tumor virus
• ERVs arise from exogenous retrovirus integrate
  into genome of germline cells survive and
  participate in reproduction
• Can be copied to other areas in the genome give
  rise to related ERV elements

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ERVs (continued)

• Do not normally replicate as they are defective
• Some can replicate when defective ERVs are
  supplied missing gene by another ERV or
  exogenous retrovirus
• Some can replicate in cells other that the one it
  was made in mouse ERV and swine ERV can
  replicate in human cells
• Some have intact genome and can initiate a
  productive infection
• concern in using pig parts in humans for organ
  transplant as may get retroviral infection

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