Replication of Positive-Sense RNA Viruses by yurtgc548

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									Replication of Positive-
 Sense RNA Viruses
Virus Replication
RNA Virus Replication
    Family: Picornaviridae
•   “small”
•   (+)RNA virus
•   Nonenveloped
•   Icosahedral capsid, 30 nm
      Genus: Enterovirus
• “intestine”
• Large group of related viruses:
  –   Poliovirus (poliomyelitis-“gray”, “marrow”)
  –   Echovirus (enteric cytopathic human orphan)
  –   Coxsackievirus
  –   Enterovirus
• Infect URT, GI, CNS, heart, muscle, skin
• pH stable
       Genus: Rhinovirus
•   “nose”
•   Human rhinovirus (>100 serotypes)
•   Common cold viruses
•   Acid pH labile
    Genus: Aphthovirus
• “ulcer”
• Foot-and-mouth disease virus
  (FMDV) of livestock
• Infect cattle, sheep, pigs, goats
    Genus: Cardiovirus
• “heart”
• Encephalomyocarditis virus (EMC) of
  mice
• Infect brain, muscle, heart
     Genus: Heparnavirus
•   “liver”
•   Hepatitis A virus
•   “infectious” hepatitis
•   Transmission via contaminated food
    or water
    Poliovirus: (+)RNA Genome
• 7.7 kb
• Single open
  translational reading
  frame (ORF)
• 5’ end has small viral
  protein (VPg)
• 3’ polyA tail
• Serves directly as
  mRNA (“infectious”)
     Poliovirus: RNA Genome
            Expression
• Attachment and entry by
  receptor-mediated
  endocytosis
• Uncoat and release of
  RNA into cytoplasm
• RNA attaches to cell
  ribosome - recognition by
  Internal ribosome entry
  site (IRES) sequence on
  5’ end of genome RNA
• Viral mRNA translates
  for one large polyprotein
   Poliovirus Polyprotein
• Single polyprotein cleaved by viral
  protease (2A, 3C) into 3 precursor
  proteins (P-1, P-2, P-3)
• Precursor proteins cleaved further
  by viral protease
      Poliovirus Proteins




• P-1: capsid proteins (VP4, VP2, VP3, VP1)
• P-2: protease (2A), regulatory proteins
  (2B, 2C)
• P-3: regulatory protein (3A), VPg,
  protease (3C), RNA polymerase (3D)
      Poliovirus: Transcription of
       Complimentary RNA (RI-1)
• Antigenome copied
  from released (+)RNA
  genome
• Transcription complex:
  – (+)RNA genome
  – VPg serves as primer
    (attaches to 3’ end of
    viral RNA)
  – Host proteins required
  – Viral RNA Replicase
    (viral protein 3D)
       Poliovirus: Replication of
          Genome RNA (RI-2)
• (+)RNA genome copied
  from Antigenome template
• Transcription complex:
  antigenome, VPg, host
  proteins, viral RNA
  replicase
• New (+)RNA serves as:
  – mRNA (more viral proteins)
  – genome RNA (for new virus)
  Poliovirus RNA Synthesis

• Each cell makes ~
  4 x 105 RNA
  molecules
• (-)RNA ~ 10%
• (+)RNA ~ 90%
   Poliovirus: Assembly
• Protomer (VP0, VP1,
  VP3)
• Pentamer (five
  protomers)
• Procapsid (twelve
  pentamers)
• Provirion (sixty
  protomers, “head-full”
  insertion of viral RNA
  genome)
• Virion (cleavage of VP0
  into VP2, VP4)
    Poliovirus: Cell
Cytopathic Effects (CPE)
• Host cell translation stopped shortly (30
  min.) after infection
• Viral protease activates host cell protease
  to cleave a 220 kd cell ribosome protein of
  eIF-4F
• No cap-binding complex of mRNA
• No attachment of host mRNA to ribosome
• No cell protein synthesis, leads to cell
  death
  Poliovirus:
Release By Cell
     Lysis
• Occurs within 5-10
  hours in cell culture
• Each cell releases ~
  25 – 100,000 virus
  particles
• Not all virus
  particles are
  infectious
• Virus spreads to
  other cells
    “The The fight against infantile
 paralysis is a fight to the finish, and
the terms are unconditional surrender.”
     • Franklin D. Roosvelt, 32nd U.S. President
       and polio survivor, 1944
         Family: Flaviviridae
• “yellow”
• (+)RNA, 10 kb
  – Single ORF
  – 5’ cap, 3’ polyA tail
  – Translates for single
    polyprotein, cleaved into
    structual (capsid, M, E) and
    non-structual (NS) proteins
• Enveloped
• Icosahedral, 40-50 nm
• Replicate in arthropods,
  animals, humans
          Genus: Flavivirus
• Former “Group B Arboviruses”
  (animal to human transmission
  by arthropod bite)
• Yellow fever virus (monkeys)
• West Nile virus (birds)
• St. Louis encephalitis virus
  (birds)
• Hepatitis C virus
  (transmission by blood and
  body secretions)
     Family: Togaviridae
•   “coat”
•   (+)RNA virus
•   Enveloped
•   Icosahedral capsid, 60-70 nm
•   Replicate in arthropods, animals,
    humans
    Genus: Alphavirus
• Former “Group A Arboviruses”
• Sindbis virus (bird, monkey, rare mild
  disease in human)
• Equine encephalitis virus (horse, bird,
  rodent)
        Genus: Rubivirus
•   “red”
•   Rubella virus
•   “3 day measles”
•   Transmission by direct contact, aerosols
•   Mild skin rash disease, but infected moms
    may transmit to fetus (congenital defects,
    death)
  Sindbis Virus: (+)RNA Genome
• Genome (11 kb), two
  ORF, 5’ cap, 3’ polyA
  tail
• Genome RNA
  translates ORF-1
• Synthesis of a
  subgenomic mRNA
 (4 kb) to translate
  ORF-2
    Sindbis Virus: “Early” Gene
            Expression
• Attachment, endocytosis,
  uncoat and release of
  (+)RNA into cytoplasm
• RNA binds to ribosome
  and translates ORF-1 for
  a polyprotein
• Polyprotein cleaved into
  four nonstructural
  proteins
 Sindbis Virus:
Transcription of
 Complementry
  RNA (RI-1)

• Antigenome copied
  from released
  viral Genome RNA
• By “early” viral
  replication
  enzymes
 Sindbis Virus:
 Replication of
 Genome RNA
    (RI-2)
• Genome (+)RNA (49s)
  copied from
  Antigenome
• By viral replication
  enzymes
• New RNA serves as:
  – mRNA
  – Genome RNA
   Sindbis Virus:
    “Late” Gene
    Expression
• Viral RNA replicase
  binds to Interior
  replicase start site of
  antigenome
• Synthesis of a
  subgenomic mRNA
  (26s), 5’ Cap, 3’ PolyA
• Translation of ORF-2
  for structural (capsid,
  envelope) proteins
    Sindbis Virus RNA
• Temporal regulation of viral RNA
  synthesis
• 1–3 hours: both (+)RNA and (-)RNA
• >3 hours: mainly (+)RNA
• 10x more subgenomic mRNA (26s)
  than genomic mRNA (49s)
   Sindbis Virus: Assembly and
       Release By Budding
• Envelope proteins
  glycosylated in
  ER/Golgi,
  transported and
  inserted into plasma
  membrane
• Capsid forms in
  cytoplasm
• Bud through the
  virus modified plasma
  membrane, picks up
  envelope, and
  released from cell
        Family:
      Coronavirus
• Envelope, large petal
  spikes,
 80-120 nm
• Flexible helical
  nucelocapsid,
 10-20 nm
• (+)RNA, 30 kb, 5’ cap, 3’
  polyA, five ORFs
• Synthesis of subgenomic
  mRNAs
    Genus: Coronavirus
• Human coronavirus (common cold,
  LRTI, gastroenteritis)
• SARS-CoV (severe acute respiratory
  syndrome, fatal pneumonia)
• Also infections of mammals and birds
            Reading
• Chapter 14:
  – Replication of Positive-Sense RNA
    Viruses
  – Case Study: enteroviruses
   Discussion Questions
• 1. Why is the RNA genome of poliovirus by
  itself “infectious” when transfected into a
  host cell?
• 2. How does (+)RNA viruses replicate
  their genome via two replicative
  intermediates? Why is this necessary?
• 3. Is the replication strategy of Sindbis
  virus similar/different from poliovirus?
MICR 401 FIRST EXAM
•   Tuesday, Oct. 18, 2011
•   History through Positive-sense RNA Virus
•   Lecture and Reading
•   Objective questions (MC, T/F, ID)
•   Short essay questions (similar to Class
    Discussion Questions and Basic Virology
    Questions for Chapter)

								
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