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.