<figure><img src="image_1.png" /></figure>

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# Viroids & the RNA World: from genomic scale (RNA) to atomic scale (ribozyme)

Fabrice Leclerc

<figure><img src="image_2.png" /></figure>

## To cite this version:

Fabrice Leclerc. Viroids & the RNA World: from genomic scale (RNA) to atomic scale (ribozyme). Master. BGA Biochimie et Génétique des ARN, Paris, France. 2020. hal-03313809

HAL Id: hal-03313809

https://hal.science/hal-03313809

Submitted on 6 Aug 2021

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# Viroids & the RNA World

Fabrice Leclerc

Institute for Integrative Biology of the
Cell (I2BC)

CNRS-CEA-Univ. Paris Saclay
fabrice.leclerc@i2bc.paris-saclay.fr

<figure><img src="image_3.png" /></figure>
HHRz
79-NT

Viroids
&

the RNA World

from genomic scale (RNA)

to
atomic scale (ribozyme)

249-NT

Viroid (ASBVd)

<figure><img src="image_4.png" /></figure>

<figure><img src="image_5.png" /></figure>

<figure><img src="image_6.png" /></figure>

<figure><img src="image_7.png" /></figure>
# Introduction

* evolution (RNA world) & genomics

* RNA biology & RNomics
  (molecular and cellular functions)

* structural biology & structural
  bioinformatics (structural basis for
  functions)

* enzymology & computational enzymology
  (catalysis)
# Prebiotic & RNA Worlds

<figure><img src="image_8.png" /></figure>

Martin *et al.*, Life, 2015.

Attwater *et al.*, Nat. Chem., 2013. Horning & Joyce, PNAS, 2016.
# Virus World & RNA World

## viroids

« The ancient Virus World and evolution of cells »

<figure><img src="image_9.png" /></figure>
# Viroids: « survivors from the RNA World »

## functions

* error-prone replication

* replication fidelity

* replication

* genome assembly

* ribosome-free

* replication

* small size

* high GC

* circRNA

* periodicity

* no protein-coding

* ribozyme

## features

<figure><img src="image_10.png" /></figure>
# Viroids: Families

<table><thead><tr><th>FAMILY</th><th>SUBFAMILY</th><th>GENUS</th><th>SPECIES</th></tr></thead><tbody><tr><td rowspan="5">Pospiviroidae</td><td rowspan="2">Pospiviroinae</td><td>Pospiviroids</td><td>Potato spindle tuber (PSTVd)<br>Mexican papita (MPVd)<br>Tomato planta macho (TPMVd)<br>Chrysanthemum stunt (CSVd)<br>Citrus exocortis (CEVd)<br>Tomato apical stunt (TASVd)<br>Iresine 1 (IrVd 1)<br>Columnea latent (CLVd)</td></tr><tr><td>Hostuviroids</td><td>Hop stunt (HSVd)</td></tr><tr><td rowspan="2">Apscaviroinae</td><td rowspan="2">Apscaviroids</td><td>Coconut cadang-cadang (CCCVd)<br>Coconuttin angaja (CTIVd)<br>Hop latent (HLVd)<br>Citrus IV (CVd-IV)</td></tr><tr><td>Apple scar skin (ASSVd)<br>Citrus III (CVd-III)<br>Apple dimple fruid (ADFVd)<br>Grapevine yellow speckle 1 (GYSVd 1)<br>Grapevine yellow speckle 2 (GYSVd 2)<br>Citrus bent leaf (CBLVd)<br>Pear blister canker (PBCVd)<br>Australian grapevine (AGVd)</td></tr><tr><td>Coleviroinae</td><td>Coleviroids</td><td>Coleus blumei 1 (CbVd 1)<br>Coleus blumei 2 (CbVd 2)<br>Coleus blumei 3 (CbVd 3)</td></tr><tr><td rowspan="2">Avsunviroidae</td><td rowspan="2">Flores et al.,<br>Arch. Virol.,<br>1998.</td><td>Avsunviroids</td><td>Avocado sunblotch (ASBVd)</td></tr><tr><td>Pelamoviroids</td><td>Peach latent mosaic (PLMVd)<br>Chrysanthemum chlorotic mottle (CChMVd)</td></tr></tbody></table>

PSTVd

<figure><img src="image_11.png" /></figure>

ASBVd

<figure><img src="image_12.png" /></figure>
# Viroids: Plant Parasites

<figure><img src="image_13.png" /></figure>

Ding & Itaya, Mol Plant Microbe Interact, 2007.
# Viroids: Replication

<figure><img src="image_14.png" /></figure>

<figure><img src="image_15.png" /></figure>

Symmetric pathway (family *Avsunviroidae*)

Rz: hammerhead ribozyme
# Viroids: 2D Structures

<figure><img src="image_16.png" /><figcaption>(+) PSTVd</figcaption></figure>

Pospiviroids

<figure><img src="image_17.png" /><figcaption>(+) PLMVd</figcaption></figure>

Avsunviroids

<footnote n="10">10 Ding & Itaya, Mol Plant Microbe Interact, 2007.</footnote>
# Viroids: Pospiviroids

<figure><img src="image_18.png" /></figure>
# Viroids: Avsunviroids

<figure><img src="image_19.png" /><figcaption>## B Avsunviroids</figcaption></figure>

<figure><img src="image_20.png" /><figcaption>## C</figcaption></figure>

<figure><img src="image_21.png" /></figure>
# Viroids & RNA silencing

<figure><img src="image_22.png" /></figure>
ASBVd: 2D Structures

<figure><img src="image_23.png" /></figure>

Ding & Itaya, Mol Plant Microbe Interact, 2007.
# ASBVd(-): 2D Structures

<figure><img src="image_24.png" /><figcaption>## Hammerhead (HHR)</figcaption></figure>

Leclerc *et al.*, Sci. Rep., 2016.

<figure><img src="image_25.png" /></figure>

<figure><img src="image_26.png" /></figure>

Flores *et al.*, Adv. Virus Res., 2000.
# HHR motifs in life forms

<figure><img src="image_27.png" /></figure>

<figure><img src="image_28.png" /></figure>

<figure><img src="image_29.png" /></figure>

yeast

Plasmid transcription

<figure><img src="image_30.png" /></figure>

<figure><img src="image_31.png" /></figure>

<figure><img src="image_32.png" /></figure>

RNA dependant
replication

<figure><img src="image_33.png" /></figure>

<figure><img src="image_34.png" /></figure>

<figure><img src="image_35.png" /></figure>

Delan-Forino *et al*.,
J. Virol., 2011.
Perreault *et al*.,
PLoS Comput. Biol., 2011
Gupta & Swati,
Interdiscip. Sci., 2016.
# A prototype for RNA Catalysis

## “Minimum” Hammerhead Ribozyme

<figure><img src="image_36.png" /></figure>
# Self-Cleaving / Self-Splicing

<figure><img src="image_37.png" /><figcaption>ribozyme</figcaption></figure>

<figure><img src="image_38.png" /><figcaption>SN2(P) reaction
("in-line")</figcaption></figure>

<figure><img src="image_39.png" /><figcaption>Asp424</figcaption></figure>

<figure><img src="image_40.png" /></figure>

<figure><img src="image_41.png" /><figcaption>RNase A</figcaption></figure>

<caption>3'-5'-exonuclease</caption>
# Catalytic Metal Ions: One/ Two

<figure><img src="image_42.png" /></figure>

X-ray (active)

<figure><img src="image_43.png" /></figure>

41-NT

<figure><img src="image_44.png" /></figure>

experimental: Pontius *et al.*, 1997; Lott *et al.*, 1998

theoretical: Boero *et al.*, JCTC, 2005

theoretical: Leclerc & Karplus, JPC, 2006
# Contribution of Metals to Catalysis

<figure><img src="image_45.png" /></figure>

$$ \Delta G^{\text{exp}} = 20.1 \text{ kcal/mol} $$

Lopez *et al.*, 2006

Torres *et al.*, 2003

Leclerc & Karplus, 2006
HHR: reaction mechanism

<figure><img src="image_46.png" /><figcaption>Reaction Path Following: B3LYP/6-31+G(d,p)//HF/3-21+G(d)</figcaption></figure>
Folding Metal Ions Models

<figure><img src="image_47.png" /></figure>

X-ray (active)

<figure><img src="image_48.png" /></figure>

<figure><img src="image_49.png" /></figure>

experimental:
Peracchi *et al.*, 1997
Metal Binding Sites in the
Hammerhead Ribozyme ?

<figure><img src="image_50.png" /><figcaption>Chartrand *et al.*, RNA, 1997</figcaption></figure>

<figure><img src="image_51.png" /><figcaption>Hansen *et al.*, RNA, 2008</figcaption></figure>
Minimum/Full-Length
HHR

<figure><img src="image_52.png" /></figure>

41-NT

<figure><img src="image_53.png" /></figure>

50-NT

<figure><img src="image_54.png" /></figure>

56-NT

<figure><img src="image_55.png" /></figure>

50-NT

<figure><img src="image_56.png" /></figure>

<figure><img src="image_57.png" /></figure>

<figure><img src="image_58.png" /></figure>

<figure><img src="image_59.png" /></figure>
Variations in HHR motifs

<figure><img src="image_60.png" /></figure>

Type II
Hammerhead
Consensus

<figure><img src="image_61.png" /></figure>

R = purine; Y = pyrimidine

<figure><img src="image_62.png" /></figure>

variable-length hairpin

nucleotide
identity

N 97%

● 97%

○ 75%

N 90%

● 90%

○ 50%

N 75%

<figure><img src="image_63.png" /></figure>

marine
metagenome

<figure><img src="image_64.png" /></figure>

*Drosophila*
*pseudoobscura*

<figure><img src="image_65.png" /></figure>

*Yarrowia*
*lipolytica*

<figure><img src="image_66.png" /></figure>

mouse gut
metagenome
# Loop-Loop interactions in HHR folding

<figure><img src="image_67.png" /></figure>

Penedo *et al.*, RNA, 2004.
Loop-Loop interactions in
HHR catalysis

<figure><img src="image_68.png" /></figure>

<figure><img src="image_69.png" /></figure>

<figure><img src="image_70.png" /></figure>

<figure><img src="image_71.png" /></figure>
# Minimum HHR, Tertiary Contact and Catalysis

<figure><img src="image_72.png" /><figcaption>(b)</figcaption></figure>

<figure><img src="image_73.png" /></figure>

<figure><img src="image_74.png" /><figcaption>Stem I</figcaption></figure>

<figure><img src="image_75.png" /></figure>
HHR morphing

Highly active
form

<figure><img src="image_76.png" /></figure>

O'Rourke *et al.*, JMB, 2015
A Nucleobase
Catalyst

<figure><img src="image_77.png" /></figure>

experimental: Chi *et al.*, PLoS Biol., 2008

theoretical: Lee *et al.*, JACS, 2008

<figure><img src="image_78.png" /></figure>

X-ray (active)
<figure><img src="image_79.png" /></figure>

<figure><img src="image_80.png" /></figure>
# Metal Catalysts in the Hammerhead Ribozymes ?

<figure><img src="image_81.png" /></figure>

Osborne *et al.*, Biochem., 2009

<figure><img src="image_82.png" /></figure>
# Cooperative Models in Self-Cleaving ?

<figure><img src="image_83.png" /></figure>
Cooperative Models in
Self-Cleaving ?

<figure><img src="image_84.png" /></figure>

<figure><img src="image_85.png" /></figure>
Metal Ions back in the
Hammerhead Catalysis

<figure><img src="image_86.png" /></figure>

<figure><img src="image_87.png" /></figure>
# HHR: Active Conformation and Metal Ions

<caption>A</caption>

66-NT

<figure><img src="image_88.png" /><figcaption>B</figcaption></figure>

<figure><img src="image_89.png" /></figure>

<figure><img src="image_90.png" /></figure>
HHR: Metal Ions and
Catalysis

<figure><img src="image_91.png" /><figcaption>B</figcaption></figure>

<figure><img src="image_92.png" /><figcaption>C</figcaption></figure>

<figure><img src="image_93.png" /></figure>
# ASBVd(-)/HHR(-): SANS

## Study

ASBVd(-)

<figure><img src="image_94.png" /></figure>

HHR(-)

<figure><img src="image_95.png" /></figure>
HHR(-) dimerization/
catalysis

$E_a \sim 8\text{kcal/mol}$

$E_a \sim 20\text{kcal/mol}$

$E_a \sim 6\text{kcal/mol}$

$E_a \sim 30\text{kcal/mol}$

<figure><img src="image_96.png" /></figure>

$1/T (\text{K}^{-1})$
# HHR(-): 2D structures

<figure><img src="image_97.png" /></figure>
3D Modeling of HHR(-)

<figure><img src="image_98.png" /></figure>

<figure><img src="image_99.png" /></figure>

<figure><img src="image_100.png" /></figure>

loops HI-HII

<figure><img src="image_101.png" /></figure>

<figure><img src="image_102.png" /></figure>

<figure><img src="image_103.png" /></figure>

<figure><img src="image_104.png" /></figure>

<figure><img src="image_105.png" /></figure>

<figure><img src="image_106.png" /></figure>

<figure><img src="image_107.png" /></figure>
HHR folding

<figure><img src="image_108.png" /></figure>

Penedo *et al.*, RNA, 2004.

$R_g(I)/(F) \sim 1.1$

$ASBVd: R_g(I)/(F) \sim 1.2$
HHR morphing

Highly active
form

<figure><img src="image_109.png" /></figure>

O'Rourke *et al.*, JMB, 2015

$R_g(I)/(F) \sim 1.1$

ASBVd: $R_g(I)/(F) \sim 1.2$
Modeling/SANS

$$
\begin{align*}
\mathrm{R}_{\mathrm{g}}^{\mathrm{exp}} &= 31\AA \\
\mathrm{R}_{\mathrm{g}}^{*\mathrm{calc}} &= 31 (26)\AA
\end{align*}
$$

<figure><img src="image_110.png" /></figure>

$$
\begin{align*}
d^{\text{exp}} &= 96.0\AA \\
d^{\text{calc}} &= 96.7\AA
\end{align*}
$$
# HHR (-): dimerization

<figure><img src="image_111.png" /><figcaption>## A</figcaption></figure>

<figure><img src="image_112.png" /><figcaption>## B</figcaption></figure>

<figure><img src="image_113.png" /></figure>

<figure><img src="image_114.png" /></figure>
# HHR (-): dimerization
B

<figure><img src="image_115.png" /></figure>

E<sub>int</sub>(10°C) = -9.3 kcal/mol

E<sub>dimer</sub>(10°C) = -47.1 kcal/mol

E<sub>int</sub>(25°C) = -8.5 kcal/mol

E<sub>dimer</sub>(25°C) = -33.6 kcal/mol

E<sub>int</sub>(45°C) = -5.8 kcal/mol

E<sub>dimer</sub>(45°C) = -15.7 kcal/mol

<figure><img src="image_116.png" /></figure>

monomer 1 HI

HII

HIII

monomer 2

<figure><img src="image_117.png" /></figure>

E<sub>dimer</sub>(10°C) = -53.7 kcal/mol

E<sub>dimer</sub>(25°C) = -38.9 kcal/mol

E<sub>dimer</sub>(45°C) = -19.2 kcal/mol

<figure><img src="image_118.png" /></figure>
HHR(-) dimer

<figure><img src="image_119.png" /><figcaption>A</figcaption></figure>

<figure><img src="image_120.png" /><figcaption>B</figcaption></figure>

<figure><img src="image_121.png" /><figcaption>C</figcaption></figure>

<figure><img src="image_122.png" /></figure>

<figure><img src="image_123.png" /></figure>

<figure><img src="image_124.png" /></figure>
HHR(-): monomer 1

<figure><img src="image_125.png" /></figure>

<figure><img src="image_126.png" /></figure>

<figure><img src="image_127.png" /></figure>
HHR(-): monomer 2

<figure><img src="image_128.png" /></figure>
HHR(-): monomer seeds

<figure><img src="image_129.png" /></figure>
HHR(-) monomers

<figure><img src="image_130.png" /></figure>

<figure><img src="image_131.png" /></figure>

<figure><img src="image_132.png" /></figure>

<figure><img src="image_133.png" /></figure>

<figure><img src="image_134.png" /></figure>

<figure><img src="image_135.png" /></figure>

<figure><img src="image_136.png" /></figure>

<figure><img src="image_137.png" /></figure>

<figure><img src="image_138.png" /></figure>

<figure><img src="image_139.png" /></figure>
HHR: Monomer & Dimer

A

<figure><img src="image_140.png" /></figure>

B

<figure><img src="image_141.png" /></figure>

C

<figure><img src="image_142.png" /></figure>

PDB ID:
# Rg & self-association

<figure><img src="image_143.png" /></figure>
# MD setup for HHR dimer

* ~300 000 atoms

* K⁺ & Cl⁻ (0.15M)

* T = (283K, 313K)

* NAMD

<figure><img src="image_144.png" /></figure>
Dynamics of HHR(-) dimer

<figure><img src="image_145.png" /></figure>
<figure><img src="image_146.png" /><figcaption>HHR dimer: MD trajectory</figcaption></figure>
ASBVd(-): self-association

<figure><img src="image_147.png" /></figure>

monomer 1
<figure><img src="image_148.png" /><figcaption># ASBVd(-): self-association</figcaption></figure>
<figure><img src="image_149.png" /><figcaption>ASBVd(-): self-association</figcaption></figure>
# What did we learn ?

* for RNA and ribozymes: « too short » may be bad

* don't forget about dynamics

* SANS & modeling approaches to infer self-association modes

* theoretical approaches to explore reaction mechanisms and pathways
# Viroids: Plant Parasites

**Genus Pospiviroids:**
PSTVd (potato spindle tuber)

<figure><img src="image_150.png" /></figure>

**Genus Coleviroids:**
CbVd 1 (coleus blumei 1)

<figure><img src="image_151.png" /></figure>

**Genus Hostuviroids:**
HSVd (hop stunt)

<figure><img src="image_152.png" /></figure>

**Genus Avsunviroids:**
ASBVd (avocado sunblotch)

<figure><img src="image_153.png" /></figure>

**Genus Cocadviroids:**
CCCVd (coconut cadang-cadang)

<figure><img src="image_154.png" /></figure>

**Genus Pelamoviroids:**
PLMVd (peach latent mosaic)

<figure><img src="image_155.png" /></figure>

**Genus Apscaviroids:**
ASSVd (apple scar skin)

<figure><img src="image_156.png" /></figure>
# Viroid & HDV: Hepatitis D

<figure><img src="image_157.png" /></figure>
# Acknowledgments

* Zdenek Chval (University of South Bohemia, CK)

* Daniela Chvalová (University of South Bohemia, CK)

* Xavier Lopez (Euskal Herriko Unibertsitatea, SP)

* Annick Dejaegere (ESBS Strasbourg)

* Darrin M. York (Rutgers University, USA)

* Martin Karplus (Harvard University, USA)

* Giuseppe Zaccai (IBS, Grenoble)

* Jacques Vergne (MNHN, Paris)

* Anne Martel (ILL, Grenoble)

* Martina Rihova (Institute of Physics, Prague, CK)

* Marie-Christine Maurel (MNHN, Paris)

* William G. Scott (UCSC, Santa-Cruz, USA)

<figure><img src="image_158.png" /></figure>
Elements

An International Magazine of Mineralogy, Geochemistry, and Petrology

December 2016
Volume 12, Number 6

ISSN 1811-5209

# Origins of Life: Transition from Geochemistry to Biogeochemistry

NITA SAHAI and HUSSEIN KADDOUR, Guest Editors

Transition from Geochemistry to Biogeochemistry

Staging Life: Early Ocean

Incubating Life: Prebiotic Sources

Foundation Stones to Life

Metal Catalysts

Protometabolism and Early Protocells

Fabrice Leclerc, Ph.D.

I2BC /Dept. de Biologie des Génomes,
« Séquence Structure Fonction des ARN »

SSFA (D. Gautheret)

fabrice.leclerc@u-psud.fr
# Assembling the RNA World

a

**Prebiotic chemical synthesis**

* How can nucleotides be synthesized in appreciable quantities?

* How can nucleotides be polymerized into strands long enough to be functional?

b

**Finding functional ribozymes**

* How small is the fraction of random sequences that can act as functional ribozymes, and how long do functional sequences have to be?

* Are there ribozymes that can make accurate copies of themselves by any mechanism?

c

**Evolutionary theory and modelling**

* Is it easier to begin life with an autocatalytic set or a single polymerase?

* What is the error threshold for cooperative trans-acting replicators in a spatially structured model?

d

**Molecular cooperation**

* Can larger cooperative sets be assembled?

* Can an autocatalytic set be designed that uses single nucleotides as input?

g

**RNA replication inside cells**

* Did surface-based replication precede protocells?

* How were growth and division of protocells controlled by the genetic material inside?

f

**Early Earth environment**

* What were the temperature, pressure, salinity and other physical conditions in which replication got going?

* What other molecules were relevant to replication (for example, lipids, mineral catalysts and peptides)?

e

**Energy and metabolism**

* What was the source of energy and raw materials for RNA-based organisms?

* Can we find a set of ribozymes that will catalyze all the metabolic reactions involved in their own synthesis?
# Viroids: Phylogeny

## REPLICATION

## ASYMMETRIC PATHWAY

## SYMMETRIC PATHWAY

## FAMILY

*Pospiviroidae*

*Avsunviroidae*

## SUBFAMILY

*Pospiviroinae*

*Apscaviroinae*

*Coleviroinae*

## GENUS

*Pospiviroids*

*Hostuviroids*

*Cocadviroids*

*Apscaviroids*

*Coleviroids*

*Avsunviroids*

*Pelamoviroids*

## SPECIES

*Potato spindle tuber (PSTVd)*

*Mexican papita (MPVd)*

*Tomato planta macho (TPMVd)*

*Chrysanthemum stunt (CSVd)*

*Citrus exocortis (CEVd)*

*Tomato apical stunt (TASVd)*

*Iresine 1 (IrVd 1)*

*Columnea latent (CLVd)*

*Hop stunt (HSVd)*

*Coconut cadang-cadang (CCCVd)*

*Coconuttin angaja (CTiVd)*

*Hop latent (HLVd)*

*Citrus IV (CVd-IV)*

*Apple scar skin (ASSVd)*

*Citrus III (CVd-III)*

*Apple dimple fluid (ADFVd)*

*Grapevine yellow speckle 1 (GYSVd 1)*

*Grapevine yellow speckle 2 (GYSVd 2)*

*Citrus bent leaf (CBLVd)*

*Pear blister canker (PBCVd)*

*Australian grapevine (AGVd)*

*Coleus blumei 1 (CbVd 1)*

*Coleus blumei 2 (CbVd 2)*

*Coleus blumei 3 (CbVd 3)*

*Avocado sunblotch (ASBVd)*

*Peach latent mosaic (PLMVd)*

*Chrysanthemum chlorotic mottle (CChMVd)*
Modeling RNA-RNA
Interactions

<figure><img src="image_159.png" /></figure>
HHR motifs

<figure><img src="image_160.png" /><figcaption>Type I</figcaption></figure>

Prokaryotes
Plants
Metazoans

<figure><img src="image_161.png" /><figcaption>Type II</figcaption></figure>

Prokaryotes

<figure><img src="image_162.png" /><figcaption>Type III</figcaption></figure>

Prokaryotes
Plants
<figure><img src="image_163.png" /><figcaption># HHR (-): symmetric dimerization</figcaption></figure>
# ASBVd(-): symmetric self-association

<figure><img src="image_164.png" /></figure>
ASBVd(-): symmetric
self-association

C

<figure><img src="image_165.png" /></figure>

<figure><img src="image_166.png" /></figure>

monomer 1

monomer 2
ASBVd(-): symmetric
self-association

<figure><img src="image_167.png" /></figure>

T = 45C
# HHR design & engineering

<figure><img src="image_168.png" /></figure>
Glms ribozyme

<figure><img src="image_169.png" /></figure>

B

<figure><img src="image_170.png" /></figure>
# Viroid Pathogenicity & Localization

<figure><img src="image_171.png" /></figure>
Viroid& RNA silencing

<figure><img src="image_172.png" /></figure>

TRENDS in Plant Science
Viroid disease symptoms

<figure><img src="image_173.png" /><figcaption>Potato spindle tuber viroid</figcaption></figure>

<figure><img src="image_174.png" /><figcaption>Apple viroid disease</figcaption></figure>

<figure><img src="image_175.png" /><figcaption>chrysanthemum
stunt viroid</figcaption></figure>

<figure><img src="image_176.png" /><figcaption>Avocado
Sunblotch Viroid</figcaption></figure>

<figure><img src="image_177.png" /></figure>

<figure><img src="image_178.png" /><figcaption>Coconut cadang-cadang viroid</figcaption></figure>
<figure><img src="image_179.png" /><figcaption>HSVd</figcaption></figure>

<figure><img src="image_180.png" /><figcaption>CCCVd</figcaption></figure>

<figure><img src="image_181.png" /><figcaption>ASSVd</figcaption></figure>

<figure><img src="image_182.png" /><figcaption>CbVd1</figcaption></figure>

<figure><img src="image_183.png" /><figcaption>B.</figcaption></figure>

<figure><img src="image_184.png" /><figcaption>PSTVd</figcaption></figure>

<figure><img src="image_185.png" /></figure>
<figure><img src="image_186.png" /><figcaption>B. Minus strand</figcaption></figure>

<figure><img src="image_187.png" /><figcaption>C. Plus strand</figcaption></figure>

<figure><img src="image_188.png" /></figure>