BioMI 409

1
BioMI 409 Genome Replication and Expression III
RNA Viruses and Retroviruses 9/25/03
2
One cant believe impossible things, said
Alice. I dare say you havent had much
practice, said the Queen. Why sometimes Ive
believed as many as six impossible things before
breakfast. Lewis Carroll Alice in Wonderland
3
Sindbis A Togavirus
External surface
Internal NCP
Togaviruses are the simplest enveloped viruses

• No Matrix protein

• Spikes are heterodimers of glycoproteins E1E2
• Icosahedral capsid made from single protein C

From Flint et al. Principles of Virology (2000),
ASM Press
4
Sindbis Alphavirus sgRNA Strategy
RNA virus, but generates a subgenomic RNA for
capsid and envelope proteins
RNA structure at 3 end of genome may control
RNA initiation
(subgenomic promoter)
sgRNA Common strategy in RNA plant
viruses Temporal control of RNA virus gene
expression
From Flint et al. Principles of Virology (2000),
ASM Press
5
Sindbis P1234 Polyprotein is Cleaved into 3
Different Multisubunit Polymerase Forms
P1234 is sequentially cleaved (nsP2viral
protease) to produce
nsP1 P23 nsP4 polymerase for full length
(49S) RNA and RNA synthesis
nsP1nsP2nsP3nsP4 polymerase for subgenomic
(26S) and genomic RNA synthesis
From Flint et al. Principles of Virology (2000),
ASM Press
6
Sindbis Recognition Specificities of Different
P1234 Cleavage Products
P123 nsP4 site in RNA to initiate full length
RNA synthesis
nsP1 P23 nsP4 site in RNA to initiate full
length RNA synthesis
Still recognizes RNA initiation site for full
length RNA synthesis
nsP1nsP2nsP3nsP4 site in RNA for subgenomic
()RNA synthesis as well as full-length RNA
(genome) synthesis
Cleavages likely produce conformational changes
that alter template specificity
From Flint et al. Principles of Virology (2000),
ASM Press
7
Reovirus dsRNA Virus Strategy
Subviral particles in cytoplasm are sites of RNA
synthesis
core
Extruded into cytoplasm through channels in
5-fold axes
Remain in cytoplasm translated
Packaged into new subviral particles templates
for RNA synthesis to produce new dsRNAs
From Flint et al. Principles of Virology (2000),
ASM Press
8
Identifying the VSV Virion-Associated RdRp
LP methyl transferase (cap),
RNPs templates
polyA, elongation, protein kinase
9
Identifying Different Virion-Associated
Polymerase Activities
Pelleted Nucleoprotein-Genome Complexes from
Compare with reaction intermediates found in
infected cells
10
Classification of Retroviruses
Pathogenicity
Oncoviruses - tumor-inducing (sarcoma, leukemia
viruses)
Lentiviruses - slow virus diseases (HIV)
Spumaviruses - foamy diseases (vacuolization,
persistent infection

without overt disease)
Morphology of virus nucleocapsid core (type A,
B, C, D)
Cell receptors utilized
Endogenous (inherited through germline as
integrated provirus) or exogenous
Presence/absence of oncogene
Nucleotide sequence, genome structure
11
All Retroviruses are Similar in Virion Structure,
Genome Organization, Mode of Replication
from Fields et al., (1996) Fundamental Virology,
3rd edition
12
Unique features of Retrovirus Genome
"" sense RNA genome that does NOT function first
as mRNA early in infection
13
Retrovirus Genome (Diploid)

• Ranges from 7-10 kb in size (1 copy)

• Diploid 2 copies/virion
• (non-denaturing gradients vs. gels)

• Important in high recombination rate

From Flint et al. Principles of Virology (2000),
ASM Press
14
Retrovirus Genome
RT polymerase RNase H
Genome class of mobile genetic elements
(retrotransposons)
15
Retrovirus Genome Structure
Variation among different retroviruses
Additional Proteins

• Presence/Absence of oncogene (simple
  retroviruses)

• Transactivators that regulate viral gene
  expression
• (complex delta, epsilon, lenti, spuma)

16
Overview of Retrovirus Multiplication
From Flint et al. Principles of Virology (2000),
ASM Press
17
Retrovirus Replication
18
The Amazing RT Reaction Initiation

• RT elongates DNA copy from tRNA primer to 5-end
  of viral genome

• RNase H activity (part of RT) digests 5-end of
  viral genome RNA

• Newly synthesized R (DNA copy) now available to
  basepair
• with 3-end of viral genome (first template
  exchange)

From Flint et al. Principles of Virology (2000),
ASM Press
19
The Amazing RT Reaction First Template Exchange
From Flint et al. Principles of Virology (2000),
ASM Press
20
The Amazing RT Reaction Second Template Exchange

• Template circular DNA with overlapping 5-ends
  bordering U3-R-U5

• RT (helicase activity?) displaces annealed DNA
  strand and continues
• synthesis to ends

From Flint et al. Principles of Virology (2000),
ASM Press
21
Features of Retrovirus Integration (The Telltale
Changes)
Experimentally use flanking sequences to identify
preintegration site
Integration is site specific for the viral genome
(ends), but at random sites in the host genome
From Flint et al. Principles of Virology (2000),
ASM Press
22
Steps in Retroviral DNA Integration
Gapped intermediate is produced
Repair synthesis and ligation (host enzymes?)
From Flint et al. Principles of Virology (2000),
ASM Press
23
Further Reading
1. Handout/WEB 2 graphics on UV mapping a RNA
genome
2. Reading for Retroviruses Flint et al. (2000)
Chapter 7
Focus on Retro mechanisms of replication and
integration
Do not get hung up on structural models of
proteins
Look over Hepdnavirus replication strategy (just
to get idea). Relevant information on WEB.
3. Reading for DNA virus replication
DiMaio Coen (2001) Chapter 6 in Fundamental
Virology (good overview)
Flint et al. Chapter 9 Again, DO NOT get hung up
on structural models