Upload
others
View
2
Download
0
Embed Size (px)
Citation preview
RNA Synthetic Strategies of
dsRNA Viruses
Sarah McDonald Associate Professor at Wake Forest University
September 24, 2018
Some dsRNA Viral Families
• Totiviridae
Yeast LA virus
• Cystoviridae
Phi6 bacteriophage
• Reoviridae
Reovirus
Rotavirus
Bluetongue virus
Rice dwarf virus
• Birnaviridae
Infectious bursal
disease virus
Two Stages of RNA Synthesis
dsRNA
genome
Two Stages of RNA Synthesis
dsRNA
genome Transcription
1
Two Stages of RNA Synthesis
dsRNA
genome Transcription
1 +RNA
(protein synthesis
template)
Two Stages of RNA Synthesis
Genome Replication
(minus-strand synthesis)
Transcription
1
2
dsRNA
genome
+RNA
(protein synthesis
template)
Two Stages of RNA Synthesis
Genome Replication
(minus-strand synthesis)
Transcription
1
2Characteristic Features:
dsRNA
genome
+RNA
(protein synthesis
template)
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)
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)
RNA Synthesis for Totiviridae
LA Virion (T=1)
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)
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)
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.
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
RNA Synthesis for Cystoviridae
RNA Synthesis for Cystoviridae
• The Phi6 RdRp (P2) is incorporated into the virion
during particle assembly.
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.
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
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
RNA Synthesis for Reoviridae
Virion (T=13)
RNA Synthesis for Reoviridae
Virion (T=13)
• The rotavirus RdRp (VP1) is incorporated into the
virion during genome packaging/particle assembly.
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.
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.
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
RNA Synthesis in Birnaviridae
Virion (T=13)
RNA Synthesis in Birnaviridae
Virion (T=13) • The birnavirus RdRp (VP1)
is attached to the 5’ end of
the viral dsRNA genome
segments.
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.
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.
Several RdRp Structures Available
• Totiviridae
Yeast LA virus
• Cystoviridae
Phi6 bacteriophage
• Reoviridae
Reovirus
Rotavirus
Bluetongue virus
Rice dwarf virus
• Birnaviridae
Infectious bursal
disease virus
• Totiviridae
Yeast LA virus
• Cystoviridae
Phi6 bacteriophage
• Reoviridae
Reovirus
Rotavirus
Bluetongue virus
Rice dwarf virus
• Birnaviridae
Infectious bursal
disease virus
Several RdRp Structures Available
Rotavirus RdRp Structure
Lu et al., 2008
• Globular, cage-like enzyme; comprised of 3 domains
• 4 tunnels permeate catalytic center
active
site
Rotavirus v. Reovirus RdRps
McDonald et al.,
2009 (review)
Tao et al.,
2002
Lu et al.,
2008
(active) (inactive)
Rotavirus v. Reovirus RdRps
McDonald et al.,
2009 (review)
Tao et al.,
2002
Lu et al.,
2008
+RNA
binding
(overshot)
(active) (inactive)
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)
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)
Rotavirus RdRp Structures
Lu et al., 2008
• Globular, cage-like enzyme; comprised of 3 domains
• 4 tunnels permeate catalytic center
Rotavirus RdRp During Transcription
Estrozi et al., 2013
• Reoviridae RdRps are oriented such that their +RNA
exit tunnels abut the core shell.
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.
RdRp Structures Available
• Totiviridae
Yeast LA virus
• Cystoviridae
Phi6 bacteriophage
• Reoviridae
Reovirus
Rotavirus
Bluetongue virus
Rice dwarf virus
• Birnaviridae
Infectious bursal
disease virus
Phi6 RdRp Structure
Butcher et al., 2006
Red=fingers; Green=palm;
Blue=thumb
Yellow=priming domain
Phi6 RdRp Structure
• Similar to the
Reoviridae
polymerase domain
Butcher et al., 2006
Red=fingers; Green=palm;
Blue=thumb
Yellow=priming domain
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
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
Rotavirus v. Phi6 RdRp Structure
McDonald et al., 2009 (review)
• Mechanism of
RNA synthesis
differs between
Cysto and Reo.
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.
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.
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
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)
RdRp Structures Available
• Totiviridae
Yeast LA virus
• Cystoviridae
Phi6 bacteriophage
• Reoviridae
Reovirus
Rotavirus
Bluetongue virus
Rice dwarf virus
• Birnaviridae
Infectious bursal
disease virus
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
Birnavirus RdRp Structure
Motif C
Pan et al., 2007
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.