rna synthetic strategies of dsrna viruses€¦ · reovirus rotavirus bluetongue virus rice dwarf...
TRANSCRIPT
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RNA Synthetic Strategies of
dsRNA Viruses
Sarah McDonald Associate Professor at Wake Forest University
September 24, 2018
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Some dsRNA Viral Families
• Totiviridae
Yeast LA virus
• Cystoviridae
Phi6 bacteriophage
• Reoviridae
Reovirus
Rotavirus
Bluetongue virus
Rice dwarf virus
• Birnaviridae
Infectious bursal
disease virus
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Two Stages of RNA Synthesis
dsRNA
genome
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Two Stages of RNA Synthesis
dsRNA
genome Transcription
1
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Two Stages of RNA Synthesis
dsRNA
genome Transcription
1 +RNA
(protein synthesis
template)
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Two Stages of RNA Synthesis
Genome Replication
(minus-strand synthesis)
Transcription
1
2
dsRNA
genome
+RNA
(protein synthesis
template)
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Two Stages of RNA Synthesis
Genome Replication
(minus-strand synthesis)
Transcription
1
2Characteristic Features:
dsRNA
genome
+RNA
(protein synthesis
template)
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Two Stages of RNA Synthesis
Genome Replication
(minus-strand synthesis)
Transcription
1
2
• Catalyzed by a viral
RNA-dependent RNA
polymerase (RdRp)
Characteristic Features:
dsRNA
genome
+RNA
(protein synthesis
template)
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Two Stages of RNA Synthesis
Genome Replication
(minus-strand synthesis)
Transcription
1
2
• Catalyzed by a viral
RNA-dependent RNA
polymerase (RdRp)
• Particle-associated for
Toti, Cysto and Reo;
not for Birna.
Characteristic Features:
dsRNA
genome
+RNA
(protein synthesis
template)
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RNA Synthesis for Totiviridae
LA Virion (T=1)
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RNA Synthesis for Totiviridae
• The LA virus RdRp is expressed as fusion to the capsid
protein, which is similar to Reoviridae core shell.
LA Virion (T=1)
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RNA Synthesis for Totiviridae
• The LA virus RdRp is expressed as fusion to the capsid
protein, which is similar to Reoviridae core shell.
• Thus, the RdRp is tethered within the particle interior
where it functions during transcription and replication.
LA Virion (T=1)
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RNA Synthesis for Totiviridae
LA Virion (T=1)
• The LA virus RdRp is expressed as fusion to the capsid
protein, which is similar to Reoviridae core shell.
• Thus, the RdRp is tethered within the particle interior
where it functions during transcription and replication.
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RNA Synthesis for Totiviridae
LA Virion (T=1)
• The LA virus RdRp is expressed as fusion to the capsid
protein, which is similar to Reoviridae core shell.
• Thus, the RdRp is tethered within the particle interior
where it functions during transcription and replication.
Similar
particle
type
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RNA Synthesis for Cystoviridae
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RNA Synthesis for Cystoviridae
• The Phi6 RdRp (P2) is incorporated into the virion
during particle assembly.
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RNA Synthesis for Cystoviridae
• The Phi6 RdRp (P2) is incorporated into the virion
during particle assembly.
• Several copies of P2 are bound to P1 inner capsid.
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RNA Synthesis for Cystoviridae
• The Phi6 RdRp (P2) is incorporated into the virion
during particle assembly.
• Several copies of P2 are bound to P1 inner capsid.
transcriptiongenome
replication
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RNA Synthesis for Cystoviridae
• The Phi6 RdRp (P2) is incorporated into the virion
during particle assembly.
• Several copies of P2 are bound to P1 inner capsid.
Similar
particle
type
transcriptiongenome
replication
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RNA Synthesis for Reoviridae
Virion (T=13)
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RNA Synthesis for Reoviridae
Virion (T=13)
• The rotavirus RdRp (VP1) is incorporated into the
virion during genome packaging/particle assembly.
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RNA Synthesis for Reoviridae
Virion (T=13)
• The rotavirus RdRp (VP1) is incorporated into the
virion during genome packaging/particle assembly.
• Several copies of VP1 are bound to VP2 shell.
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RNA Synthesis for Reoviridae
Virion (T=13)
• The rotavirus RdRp (VP1) is incorporated into the
virion during genome packaging/particle assembly.
• Several copies of VP1 are bound to VP2 shell.
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RNA Synthesis for Reoviridae
Virion (T=13)
• The rotavirus RdRp (VP1) is incorporated into the
virion during genome packaging/particle assembly.
• Several copies of VP1 are bound to VP2 shell.
Different
particle
types
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RNA Synthesis in Birnaviridae
Virion (T=13)
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RNA Synthesis in Birnaviridae
Virion (T=13) • The birnavirus RdRp (VP1)
is attached to the 5’ end of
the viral dsRNA genome
segments.
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RNA Synthesis in Birnaviridae
Virion (T=13) • The birnavirus RdRp (VP1)
is attached to the 5’ end of
the viral dsRNA genome
segments.
• VP3 is a dsRNA-binding
polypeptide that is thought
to stimulate VP1 activity.
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RNA Synthesis in Birnaviridae
Virion (T=13) • The birnavirus RdRp (VP1)
is attached to the 5’ end of
the viral dsRNA genome
segments.
• VP3 is a dsRNA-binding
polypeptide that is thought
to stimulate VP1 activity.
• However, unlike other
dsRNA viruses, VP1 does
NOT require a core shell
protein to function as an
RdRp.
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Several RdRp Structures Available
• Totiviridae
Yeast LA virus
• Cystoviridae
Phi6 bacteriophage
• Reoviridae
Reovirus
Rotavirus
Bluetongue virus
Rice dwarf virus
• Birnaviridae
Infectious bursal
disease virus
![Page 30: RNA Synthetic Strategies of dsRNA Viruses€¦ · Reovirus Rotavirus Bluetongue virus Rice dwarf virus • Birnaviridae Infectious bursal disease virus • Totiviridae Yeast LA virus](https://reader033.vdocuments.us/reader033/viewer/2022051910/5fff0cd92c0bee6ea330207a/html5/thumbnails/30.jpg)
• Totiviridae
Yeast LA virus
• Cystoviridae
Phi6 bacteriophage
• Reoviridae
Reovirus
Rotavirus
Bluetongue virus
Rice dwarf virus
• Birnaviridae
Infectious bursal
disease virus
Several RdRp Structures Available
![Page 31: RNA Synthetic Strategies of dsRNA Viruses€¦ · Reovirus Rotavirus Bluetongue virus Rice dwarf virus • Birnaviridae Infectious bursal disease virus • Totiviridae Yeast LA virus](https://reader033.vdocuments.us/reader033/viewer/2022051910/5fff0cd92c0bee6ea330207a/html5/thumbnails/31.jpg)
Rotavirus RdRp Structure
Lu et al., 2008
• Globular, cage-like enzyme; comprised of 3 domains
• 4 tunnels permeate catalytic center
active
site
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Rotavirus v. Reovirus RdRps
McDonald et al.,
2009 (review)
Tao et al.,
2002
Lu et al.,
2008
(active) (inactive)
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Rotavirus v. Reovirus RdRps
McDonald et al.,
2009 (review)
Tao et al.,
2002
Lu et al.,
2008
+RNA
binding
(overshot)
(active) (inactive)
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Rotavirus v. Reovirus RdRps
C-terminal
plug block
dsRNA exit
McDonald et al.,
2009 (review)
Tao et al.,
2002
Lu et al.,
2008
+RNA
binding
(overshot)
(active) (inactive)
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Rotavirus v. Reovirus RdRps
C-terminal
plug block
dsRNA exit
retracted
priming
loop
McDonald et al.,
2009 (review)
Tao et al.,
2002
Lu et al.,
2008
+RNA
binding
(overshot)
(active) (inactive)
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Rotavirus RdRp Structures
Lu et al., 2008
• Globular, cage-like enzyme; comprised of 3 domains
• 4 tunnels permeate catalytic center
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Rotavirus RdRp During Transcription
Estrozi et al., 2013
• Reoviridae RdRps are oriented such that their +RNA
exit tunnels abut the core shell.
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Rotavirus RdRp During Transcription
Estrozi et al., 2013
• Reoviridae RdRps are oriented such that their +RNA
exit tunnels abut the core shell.
• Ideal for core shell dependence of RdRp and ease of
transcript exit out of the core.
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RdRp Structures Available
• Totiviridae
Yeast LA virus
• Cystoviridae
Phi6 bacteriophage
• Reoviridae
Reovirus
Rotavirus
Bluetongue virus
Rice dwarf virus
• Birnaviridae
Infectious bursal
disease virus
![Page 40: RNA Synthetic Strategies of dsRNA Viruses€¦ · Reovirus Rotavirus Bluetongue virus Rice dwarf virus • Birnaviridae Infectious bursal disease virus • Totiviridae Yeast LA virus](https://reader033.vdocuments.us/reader033/viewer/2022051910/5fff0cd92c0bee6ea330207a/html5/thumbnails/40.jpg)
Phi6 RdRp Structure
Butcher et al., 2006
Red=fingers; Green=palm;
Blue=thumb
Yellow=priming domain
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Phi6 RdRp Structure
• Similar to the
Reoviridae
polymerase domain
Butcher et al., 2006
Red=fingers; Green=palm;
Blue=thumb
Yellow=priming domain
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Phi6 RdRp Structure
• Similar to the
Reoviridae
polymerase domain
• Extended C-terminal
elaboration is
involved in priming
(initiation of RNA
synthesis).
Butcher et al., 2006
Red=fingers; Green=palm;
Blue=thumb
Yellow=priming domain
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Phi6 RdRp Structure
• Similar to the
Reoviridae
polymerase domain
• Extended C-terminal
elaboration is
involved in priming
(initiation of RNA
synthesis).
• Only 3 tunnels
(single product exit).
Butcher et al., 2006
Red=fingers; Green=palm;
Blue=thumb
Yellow=priming domain
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Rotavirus v. Phi6 RdRp Structure
McDonald et al., 2009 (review)
• Mechanism of
RNA synthesis
differs between
Cysto and Reo.
![Page 45: RNA Synthetic Strategies of dsRNA Viruses€¦ · Reovirus Rotavirus Bluetongue virus Rice dwarf virus • Birnaviridae Infectious bursal disease virus • Totiviridae Yeast LA virus](https://reader033.vdocuments.us/reader033/viewer/2022051910/5fff0cd92c0bee6ea330207a/html5/thumbnails/45.jpg)
Rotavirus v. Phi6 RdRp Structure
McDonald et al., 2009 (review)
• Mechanism of
RNA synthesis
differs between
Cysto and Reo.
• For Cysto, the
parent +RNA is
split off dsRNA
immediately; only
dsRNAs are made.
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Rotavirus v. Phi6 RdRp Structure
McDonald et al., 2009 (review)
• Mechanism of
RNA synthesis
differs between
Cysto and Reo.
• For Cysto, the
parent +RNA is
split off dsRNA
immediately; only
dsRNAs are made.
• For Reo, nascent
+RNA is made
(parent is kept
tethered via 5’ cap)
during
transcription.
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Rotavirus v. Phi6 RdRp Structure
McDonald et al., 2009 (review)
• Mechanism of
RNA synthesis
differs between
Cysto and Reo.
• For Cysto, the
parent +RNA is
split off dsRNA
immediately; only
dsRNAs are made.
• For Reo, nascent
+RNA is made
(parent is kept
tethered via 5’ cap)
during
transcription.
active
site
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Catalytic Motifs A-F
• Conserved motifs (A–F) are formed by the palm and fingers
subdomains.
• Motifs A and C contain aspartate residues that anchor Mg2+ ions (crucial
for two-metal ion mediated phosphodiester bond formation).
McDonald et
al., 2009
(review)
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RdRp Structures Available
• Totiviridae
Yeast LA virus
• Cystoviridae
Phi6 bacteriophage
• Reoviridae
Reovirus
Rotavirus
Bluetongue virus
Rice dwarf virus
• Birnaviridae
Infectious bursal
disease virus
![Page 50: RNA Synthetic Strategies of dsRNA Viruses€¦ · Reovirus Rotavirus Bluetongue virus Rice dwarf virus • Birnaviridae Infectious bursal disease virus • Totiviridae Yeast LA virus](https://reader033.vdocuments.us/reader033/viewer/2022051910/5fff0cd92c0bee6ea330207a/html5/thumbnails/50.jpg)
Birnavirus RdRp Structure
• Birnavirus RdRp has 3 domains (similar to Reo);
only 3 tunnels (similar to Cysto)
• Birnavirus motifs are arranged in the permuted order of
C-A-B-D-E
• The essential “GDD” sequence of motif C is “AND”
Pan et al., 2007
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Birnavirus RdRp Structure
Motif C
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Take-Home Messages
• dsRNA viruses perform two types of RNA
synthesis during their replication cycles:-Transcription (+RNA synthesis)
-Genome replication (dsRNA synthesis)
• Viral RdRps are located within the particle interior;
Totiviridae, Cystoviridae, and Reoviridae members
catalyze RNA synthesis in association with
particle.
• Birnaviridae RdRps do not perform particle-
associated RNA synthesis.
• Structures of RdRps inform an understanding of
RNA synthesis mechanisms.