Acta virologica 66: 206 – 215, 2022

doi: 10.4149/av_2022_302

A novel RNA virus, Thesium chinense closterovirus 1, identified byhigh-throughput RNA-sequencing of the parasitic plant Thesium chinense
Chaerim Shin1, Dongjin Choi1, Ken Shirasu2,3, Yasunori Ichihashi4, Yoonsoo Hahn1*
Department of Life Science, Chung-Ang University, Seoul 06974, South Korea; 2RIKEN Center for Sustainable Resource Science,
Yokohama, Kanagawa 230-0045, Japan; 3Graduate School of Science, The University of Tokyo, Bunkyo, Tokyo 113-0033, Japan;
4
RIKEN BioResource Research Center, 3-1-1 Koyadai, Tsukuba, Ibaraki 305-0074, Japan

1

Received March 15, 2022; accepted May 20, 2022
Summary. – The genome sequence of a closterovirus (genus Closterovirus, family Closteroviridae), tentatively named Thesium chinense closterovirus 1 (TcCV1), was identified by performing high-throughput
RNA-sequencing of the haustoria and root tissues of Thesium chinense, a parasitic plant. The TcCV1
genome was predicted to encode nine proteins, eight of which have orthologs in previously identified
closteroviruses. The TcCV1 RNA-dependent RNA polymerase (RdRp) and heat shock protein 70 homolog(Hsp70h) showed 27.8–68.2% and 23.8–55.1% amino acid identity, respectively, to orthologous proteins ofknown closteroviruses. The putative +1 ribosomal frameshifting site required for producing RdRp was
identified as GUUUAGC with UAG stop codon and the skipped nucleotide U. Phylogenetic trees based on
RdRp and Hsp70h show that TcCV1 is a novel member of the genus Closterovirus, forming a subclade witha group of known closteroviruses, including mint virus 1 and carnation necrotic fleck virus. The genome
sequence of TcCV1 may be useful for studying the genome evolution of closteroviruses.
Keywords: Thesium chinense closterovirus 1; Closterovirus; Closteroviridae; Thesium chinense

Introduction
Closteroviridae is a family of RNA viruses that infectplants, leading to considerable yield losses in commer­cially
important plants (beet, carrot, citrus, grape, rasp­berry,strawberry, tobacco, and tomato) (Agranovsky et al., 1994;
Karasev et al., 1995; Tzanetakis et al., 2007; Tzanetakis andMartin, 2007; Menzel et al., 2009; Martelli et al., 2012; Wanget al., 2016; Fiallo-Olive and Navas-Castillo, 2019; Martelli,
2019; Liu et al., 2021). The family Closteroviridae comprises
Corresponding author. E-mail: hahny@cau.ac.kr; phone: +82-2820-5812.
Abbreviations: CP = coat protein; CPm = minor coat protein;
CNFV = carnation necrotic fleck virus; CTV = citrus tristezavirus; ER = endoplasmic reticulum; Hsp70h = heat shockprotein 70 homolog; MV1 = mint virus 1; ORF = open reading
frame; RdRp = RNA-dependent RNA polymerase; RF = ribosomal frameshifting; RSS = RNA silencing suppressor; TcCV1 =Thesium chinense closterovirus 1*

of four known genera (Ampelovirus, Closterovirus,
Crinivirus, and Velarivirus) and a group of unassigned
species (Agranovsky, 2016; Martelli, 2019; Fuchs et al.,2020).
Members of the family Closteroviridae have long filamentous virions and positive-sense, single-stranded RNA
genomes that are 13–19 kilobases (kb) long (Martelli, 2019;
Fuchs et al., 2020). They have either monopartite (Ampelovirus, Closterovirus, Velarivirus, and the unassigned
group) or di/tripartite (Crinivirus) genomes, which bothcontain open reading frames (ORFs) for seven conserved
proteins and additional proteins. For example, beet yellows virus (BYV; species Beet yellows virus; genus Closterovirus) encodes nine proteins: ORF1a polyprotein, ORF1ab
fusion protein, 6 kilodalton (kDa) protein (p6), heat shockprotein 70 homolog (Hsp70h), 64 kDa protein (p64; also
known as heat shock protein 90 homolog or Hsp90h),
minor coat protein (CPm), major coat protein (CP), 20
kDa protein (p20), and 21 kDa protein (p21) (Agranovsky etal., 1994; Dolja, 2003). The first seven proteins are shared

SHIN, CH. et al.: Novel closterovirus from Thesium chinense
among most Closteroviridae family members, with di­fferent molecular masses according to species (Martelli,2019). Additional ORFs are present in many viruses thatare unique to a species or group of species (Agranovsky,
2016; Fuchs et al., 2020; Park et al., 2021; Song et al., 2021).
ORF1a polyproteins and ORF1ab fusion proteins aretranslated directly from viral genomic RNA, whereas
other proteins are produced using subgenomic RNAs
(Dolja, 2003; Agranovsky, 2016; Fuchs et al., 2020; Song etal., 2021). The ORF1a polyprotein, which contains a papain-like leader protease, RNA methyltransferase, Zemlya,
and RNA helicase domain, is proteolytically processed
into at least four mature peptides. ORF1b contains an
RNA-dependent RNA polymerase (RdRp) domain that is
produced as an ORF1ab fusion protein via +1 ribosomal
frameshifting (RF), which occurs at a low rate near theend of ORF1a. The 7-nucleotide (nt) long consensus sequences of GUUUAGC, GUUUAAC, and GUUCGGC, where
the stop codons (UAG and UAA) and a rare arginine codon(CGG) are in bold font and nucleotides skipped during
the +1 RF are underlined, have been proposed to cause
the +1 RF during the translation of ORF1a (Karasev et al.,
1995; Agranovsky, 2016; Atkins et al., 2016; Zheng et al.,2018; Park et al., 2021). ORF1a polyproteins and ORF1ab
fusion proteins are involved in viral genome replicationand the transcription of subgenomic RNAs (Dolja et al.,2006; Song et al., 2021).
Five proteins (p6, Hsp70h, p64, CPm, and CP) or theiranalogous counterparts are involved in the intercellular
movement of viruses and assembly of virions (Dolja et al.,2006; Song et al., 2021). The p6 protein is a small integral
membrane protein that mediates cell-to-cell movementof the virus and is associated with the endoplasmic reticulum (ER) (Alzhanova et al., 2000). Hsp70h, which may
be derived from the cellular molecular chaperone HSP70
via horizontal gene transfer, is involved in viral genome
replication, virion assembly, and cell-to-cell movement(Peremyslov et al., 2004). CP is the major capsid proteinthat forms a long helical body of filamentous virions andencapsidates approximately 95% of the viral genomic
RNA (Dolja et al., 2006). CPm forms a terminal structure
at one end of the virion together with Hsp70h and p64(Dolja, 2003).
Extra ORFs encoding small proteins are located in the 3ʹ
region or between the conserved ORFs. One of these extra
ORFs is the RNA silencing suppressor (RSS) (Dolja et al.,2006; Song et al., 2021). Double-stranded RNA molecules
generated during viral genome replication trigger plant
defense responses that utilize RNA-silencing mechanisms(Waterhouse et al., 2001). Viral RSS proteins interfere withand suppress the host defense system (Wang et al., 2012;
Flores et al., 2013). Proteins with RSS function have been
identified in some Closteroviridae members, including

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BYV p21, citrus tristeza virus (CTV) p23, and grapevine
leafroll-associated virus 2 (GLRaV2) p24 (Song et al., 2021).
Transcriptome data from plant tissues often containsequences originating from RNA viruses that symptomatically or asymptomatically infect the host plant (Heet al., 2015; Susi et al., 2019; Liu et al., 2021; QuintanilhaPeixoto et al., 2021). Novel viral genome sequences can be
identified by sequence comparisons of assembled planttranscriptome contigs and known viral protein sequences(Orilio et al., 2018; Bejerman et al., 2020, 2021; Choi et al.,2021; Goh et al., 2021; Shin et al., 2021). In this study, the genome sequence of a novel positive-sense, single-stranded
RNA virus belonging to the genus Closterovirus (family
Closteroviridae) was identified using the transcriptome
data obtained from the haustoria and roots of the parasitic
plant Thesium chinense (Ichihashi et al., 2018).

Materials and Methods
Transcriptome data. Transcriptome data were previously
obtained from the haustoria and roots of T. chinense (Ichihashi
et al., 2018). The sequence data of 14 sequencing runs fromseven T. chinense individuals are available in the Sequence
Read Archive (SRA) of the National Center for Biotechnology
Information (NCBI) under the SRA Project Acc. No. SRP114897.
High-quality reads were collected by filtering the raw reads
using sickle (version 1.33; https://github.com/najoshi/sickle),
with the parameter “-q 30 -l 55,” and assembled into contigs
using the SPAdes Genome Assembler (version 3.15.3; http://
cab.spbu.ru/software/spades), with the parameter “--rnaviral”(Bushmanova et al., 2019).
Identification of viral genome contigs. The assembled
T. chinense transcriptome contigs were compared with known
viral RdRp domain sequences using the DIAMOND program
to identify the putative viral genome contigs. Known viral
RdRp sequences were extracted from 20 Pfam families (release
35.0; https://pfam.xfam.org; Pfam Acc. Nos. PF00602, PF00603,
PF00604, PF00680, PF00946, PF00972, PF00978, PF00998,
PF02123, PF03431, PF04196, PF04197, PF05273, PF05788, PF05919,
PF06317, PF06478, PF07925, PF08467, and PF12426).
Putative ORFs of the viral genome contigs were predicted using the getorf program of the EMBOSS package (version 6.6.0.0;
http://emboss.sourceforge.net), with the parameter “-reverse
N -find 1 -minsize 150.” The molecular weights of the predicted
viral proteins were calculated using the pepstats program in
the EMBOSS package. Functional domains in the viral proteins
were predicted using the Pfam database sequence search toolwith an E-value cutoff of 0.001. The transmembrane domains
were predicted using TMHMM (version 2.0; https://services.
healthtech.dtu.dk/service.php?TMHMM-2.0).
Phylogenetic analysis. NCBI BLAST (https://blast.ncbi.nlm.
nih.gov/Blast.cgi) was used to collect known viral genome

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and protein sequences. Pairwise identities of the viral protein
sequences were calculated using the needle program in the
EMBOSS package. Multiple sequence alignments were visualized using BOXSHADE (version 3.31; https://launchpad.net/
ubuntu/focal/+package/boxshade). Sequence logo representation was created using WebLogo (version 3; http://weblogo.
threeplusone.com).
Multiple alignments of viral protein sequences were performed using MAFFT (version 7.475; https://mafft.cbrc.jp/alignment/software), with the parameter “--auto” (Nakamura
et al., 2018). Poorly aligned regions from the multiple sequencealignments were removed using trimAl (version 1.4.rev22;
http://trimal.cgenomics.org), with the parameter “-automated1”(Capella-Gutierrez et al., 2009). Maximum likelihood phylogenetic trees were inferred using IQ-TREE (version 2.1.3; http://
www.iqtree.org), with the parameter “-B 1000” (Minh et al.,2020), and visualized using MEGA software (version 10.2.4;
https://www.megasoftware.net) (Kumar et al., 2018).

Results and Discussion
Genome sequence of Thesium chinense closterovirus 1(TcCV1)
T. chinense RNA-sequencing reads previously obtained
from the haustoria and roots of seven individual plants
were assembled into transcriptome contigs (Ichihashi etal., 2018). Sequence similarity searches of the transcriptome contigs against the known viral RdRp sequences re-vealed two putative viral genome contigs with the lengthsof 15293 nt and 15289 nt. These contigs, named C1 and C2,
respectively, shared 94.0% nucleotide identity with eachother, indicating that they are two variants of the same virus. The C1 and C2 contigs showed the highest amino acid(aa) sequence identity with the RdRp domain sequenceof mint virus 1 (MV1), which was previously identified inmint (Mentha spp.) (Tzanetakis et al., 2005). MV1 is a member of the genus Closterovirus, supporting the notion that
the C1 and C2 contigs are genome sequences of viruses
closely related to the genus Closterovirus.
Sequence similarity searches of the NCBI protein
database using C1 and C2 contigs as queries indicatedthat they had the highest sequence similarities (app­roximately 60–70% aa identity) with viruses of thegenus Closterovirus, including MV1, Rehmannia virus
1 (ReV-1), tobacco virus 1 (TV1), carnation necrotic fleckvirus (CNFV), carrot closterovirus 1 (CtCV1), and carrot
yellow leaf virus (CYLV) (Karasev et al., 1994; Tzanetakis
et al., 2005; Menzel et al., 2009; Adams et al., 2014; Wanget al., 2016; Kwon et al., 2018). Therefore, contigs C1 andC2 were considered to be genome sequences of a putative novel closterovirus (the genus Closterovirus; thefamily Closteroviridae) and named Thesium chinense
closterovirus 1 (TcCV1). The TcCV1 genome sequenceswere deposited in NCBI under Acc. Nos. OM801605 (C1)and OM801606 (C2). The C1 and C2 contig sequences
share 94.0% nucleotide identity with each other andencode an identical set of proteins exhibiting 94.1–99.5%
aa identities with their respective orthologs. The longer

Fig. 1
Genomic structure of Thesium chinense closterovirus 1 (TcCV1)
Schematic representation of the TcCV1 genome (C1 contig). The predicted open reading frames (ORFs) are shown as boxes with ORF numbers and encoded protein names. The coordinates of the ORFs are listed in the box at the bottom left. The predicted functional domains
are indicated by the blue lines below the ORFs. Coordinates and Pfam names (with Acc. Nos.) are presented in the box at the bottom right.
Nucleotide sequences surrounding the stop codon (red) of ORF1a are shown above the genome, with the corresponding amino acid sequence of the ORF1ab fusion protein at the top. The +1 ribosomal frameshifting by skipping the uridine (U) residue at genome position
7677 produces the ORF1ab fusion protein.

SHIN, CH. et al.: Novel closterovirus from Thesium chinense
contig, C1, was chosen to represent the TcCV1 genome
sequence for further analysis.
The TcCV1 genome sequence (the C1 contig with 15293
nt length) was predicted to encode nine ORFs: ORF1a,
ORF1b, and ORF2 to ORF8 (Fig. 1). The ORF1a polyprotein
is 2486 aa long and was predicted to have a papain-likeleader protease, RNA methylase, Zemlya, and RNA helicase
domain. The Zemlya domain is a recently described functional domain identified only in the genus Closterovirus(Gushchin et al., 2017). No homologs of the Zemlya domain
have been found in proteins of other genera of the family
Closteroviridae, other viruses, or cellular organisms. The
Zemlya domain was proposed to mediate remodeling of
ER membranes to form viral replication factories during
closterovirus infection (Gushchin et al., 2017). The presence of the Zemlya domain further supports the inclusionof TcCV1 in the genus Closterovirus.
TcCV1 ORF1b encodes an RdRp domain that is produced
at a low rate, presumably via the +1 RF during translationof ORF1a (Atkins et al., 2016; Martelli, 2019). A tentative +1
RF sequence predicted in the TcCV1 genome is GUUUAGC
with the UAG stop codon of ORF1a and the skipped U nucleotide. The +1 RF may occur by skipping the U residue,
the first nucleotide of the stop codon, at the nt position
7677 (C1 contig), which causes the translational machinery
to continue to translate ORF1b. The predicted ORF1ab fusion protein is 2957 aa long.
TcCV1 ORF2 encodes a small protein with a length of
65 aa and a calculated molecular weight of 7 kDa, which
is hereafter referred to as p7. TcCV1 p7 showed sequencesimilarities with conserved p6-like proteins of other closteroviruses. Closterovirus p6-like proteins are associatedwith the ER membrane and are involved in cell-to-cell
movement of the virus (Alzhanova et al., 2000). TcCV1 p7
was predicted to have a transmembrane domain in the
N-terminal half (aa positions 7–29), indicating that it may
have the same function as other p6-like proteins.
TcCV1 ORF3 encodes a 602 aa protein that contains
a viral Hsp70 domain and shows sequence similarity with
Hsp70h proteins of members of the family Closteroviridae.
The viral Hsp70h protein may be transferred to an ancestral Closteroviridae virus from the cellular molecularchaperon HSP70 (Satyanarayana et al., 2000; Dolja et al.,2006). Hsp70h plays multiple roles during the viral life
cycle including genome replication, virion formation, andcell-to-cell movement (Peremyslov et al., 2004).
The ORF4 sequence of TcCV1 encodes a 549 aa proteinwith a calculated molecular mass of 62 kDa. This protein,
named p62, showed sequence similarities with p64-likeproteins of approximately 60 kDa that are seen in members of the family Closteroviridae. TcCV1 p62 and orthologous p64-like proteins share a viral Hsp90h domain andare also known as Hsp90h proteins (Song et al., 2021).

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ORF5 and ORF6 of TcCV1 encode 222 aa and 203 aa proteins that are orthologous to closterovirus CPm and CP,
respectively. CPm and CP share a closterovirus-specificcoat protein domain and may have originated from the
duplication of an ancestral gene in the common ancestor of the family Closteroviridae (Dolja et al., 2006). CP
encapsidates most of the viral genomic RNA, whereas
CPm together with Hsp70h and p64-like proteins form theterminal structure at one end of the virion (Dolja, 2003;
Dolja et al., 2006).
ORF7 predicted in the TcCV1 genome sequence may
encode a 159 aa protein, named p18, with a calculated
molecular weight of 18 kDa. However, no homologous
protein sequences were detected in the current NCBI
sequence databases or in the genomes of known closteroviruses. Therefore, the hypothetical protein p18 is
specific to TcCV1. ORFs encoding proteins with unknown
functions are commonly found in many closteroviruses
and are specific to a virus or shared only among closely
related viruses (Agranovsky, 2016; Park et al., 2021; Songet al., 2021).
ORF8, the last ORF of TcCV1, encodes a 23 kDa (204 aa)
protein named p23. TcCV1 p23 exhibits sequence similarities with p21-like proteins found in many closteroviruses,
including BYV p21, CTV p23, and GLRaV2 p24. TcCV1 p23
and its orthologous proteins have an RSS domain that isessential for interference with and suppression of thehost RNA-silencing mechanism (Dolja, 2003; Wang et al.,2012; Flores et al., 2013).
Analysis of the +1 RF sequences of closterovirus
genomes
The closterovirus RdRp proteins encoded by ORF1b are
produced via the +1 RF during the translation of ORF1a(Atkins et al., 2016; Martelli, 2019; Park et al., 2021). The +1
RF was first proposed to occur at the ORF1a stop codon orat a rare codon preceded by two uridines (UU) (Agranovsky
et al., 1994; Karasev et al., 1995). The most frequently observed motif is GUU_stop_C (underscores mark codon
boundaries of ORF1a and ‘stop’ indicates a stop codon),whereas CTV has a rare arginine codon (CGG) instead ofa stop codon (Karasev et al., 1995; Park et al., 2021).
To determine whether there were additional conserved
sequences at the +1 RF sites, the amino acid and nucleotide
sequences of 22 closteroviruses were comparatively analyzed (Fig. 2). First, multiple alignments of the C-termini
of ORF1a polyprotein sequences and N-termini of ORF1b
protein sequences were conducted (Fig. 2a). For ORF1b,
the N-terminus was extended to the first sense codon.
The +1 RF must occur within the overlapping region of
ORF1a and the extended ORF1b, which is a +1 reading
frame relative to ORF1a.

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The positions of the ORF1a stop codons were conservedin 19 of 22 viruses. Three viruses, CTV, Elephantopus scaber
closterovirus (ESCV), and rose leaf rosette-associated virus(RLRaV), have an extended C-terminus of approximately
20 aa compared to the other nineteen viruses. A highly
conserved arginine-valine (RV) dipeptide was observedat the end of ORF1a in all examined viruses except CNFV,
which has an arginine-alanine dipeptide, suggesting thatthe dipeptide sequence is important and must be includedin the ORF1ab fusion protein. Therefore, it is highly likelythat the +1 RF may occur after the RV dipeptide position in
all members of the genus Closterovirus, regardless of thepresence of a stop codon after the RV dipeptide.

The extended N-termini of ORF1b ORFs of 22 closteroviruses varied in length and did not contain any notably conserved sequences (Fig. 2a). However, the ORF1b sequence
after the proposed +1 RF site displayed very high sequenceconservation, which further supported the notion that all
known closteroviruses may use the same +1 RF site for the
production of the ORF1ab fusion protein. When the TcCV1
ORF1ab fusion protein sequence deduced using the proposed +1 RF site was analyzed, the RdRp domain was predicted to be at aa positions 2457–2904, which spans acrossthe ORF1ab boundary at position 2486–2487. The RdRp
domains of all ORF1ab fusion proteins predicted fromknown closteroviruses span their predicted +1 RF sites.

(a)

(b)

Fig. 2
Sequence comparisons of closterovirus +1 ribosomal frameshifting (RF) sites(a) Multiple alignments of ORF1a C-terminal (top) and ORF1b N-terminal (bottom) sequences of 22 closteroviruses are shown. ORF1b
N-terminal sequences were extended up to the first in-frame stop codon. Stop codons are shown as red Xs. Amino acid residues that are
identical or similar in ≥ 50% of the viruses are highlighted in black or gray, respectively. Asterisks above or below the alignments indicate
residues that are identical in all viruses. Predicted RdRp domain is marked by blue lines. The dotted blue line denotes the +1 RF that
produces ORF1ab fusion proteins. (b) Nucleotide sequences at the proposed +1 RF sites of 22 closteroviruses are presented. Stop codonsof ORF1a are marked by the three dots (note that CTV, ESCV, and RLRaV have a CGG codon at this position). Nucleotide residues that arepresumably skipped by +1 RF are shaded in gray and marked as position 0. The corresponding TcCV1 ORF1ab fusion protein sequence isshown at the top. Sequence logo representation of the alignment is presented at the bottom.

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Table 1. Sequence identities of the RdRp and Hsp70h proteins of TcCV1 and related viruses
No.

Genus

1

Closterovirus

2

Virus

Acronym

Genomea

RdRpb

Hsp70hb

Mint virus 1

MV1

NC_006944

68.2% (322/472)

50.1% (305/609)

Rehmannia virus 1

ReV-1

NC_040572

66.2% (312/471)

50.0% (307/614)

3

Tobacco virus 1

TV1

NC_027712

65.9% (313/475)

49.5% (304/614)

4

Carnation necrotic fleck virus

CNFV

NC_038419

64.8% (307/474)

45.1% (274/607)

5

Carrot closterovirus 1

CtCV1

KF533697

65.1% (308/473)

52.8% (328/621)

6

Carrot yellow leaf virus

CYLV

NC_013007

62.5% (295/472)

55.1% (340/617)

7

Citrus tristeza virus

CTV

NC_001661

54.5% (274/503)

41.0% (254/619)

8

Elephantopus scaber closterovirus

ESCV

MN814306

55.7% (268/481)

42.3% (262/620)

9Rose leaf rosette-associated virus

RLRaV

NC_024906

52.1% (256/491)

41.0% (250/610)

10Strawberry chlorotic fleck-associated virus

SCFaV

NC_008366

57.7% (277/480)

39.2% (240/612)

11

Raspberry leaf mottle virus

RLMoV

NC_008585

55.7% (264/474)

42.4% (263/620)

12

Fig virus a

FiVA

MN817232

44.1% (241/546)

33.7% (211/626)

13

Fig virus b

FiVB

MN817233

45.5% (245/538)

32.0% (196/613)

14

Arracacha virus 1

ArrV-1

NC_040570

56.0% (265/473)

39.8% (243/611)

15

Grapevine leafroll-associated virus 2

GLRaV2

NC_007448

56.1% (267/476)

42.4% (257/606)

16

Beet yellow stunt virus

BYSY

NC_043106

54.8% (259/473)

45.9% (283/616)

17

Soybean leaf crinkle mottle virus

SLCMV

LC601607

56.4% (266/472)

47.0% (287/610)

18

Beet yellows virus

BYV

NC_001598

56.4% (268/475)

46.0% (281/611)

19

Carnation yellow fleck virus

CYFV

NC_022978

55.6% (262/471)

46.1% (281/609)

20Blackcurrant closterovirus 1

BCCV-1

NC_040834

51.5% (250/485)

38.8% (242/623)

21

Blackcurrant leafroll-associated virus 1

BcLRaV-1

NC_040722

52.0% (251/483)

38.8% (242/624)

22

Unassigned

23

Persimmon virus b

PeVB

NC_025967

33.9% (177/522)

30.3% (197/651)

Actinidia virus 1

AcV1

NC_035453

34.5% (183/530)

29.8% (197/662)

24

Olive leaf yellowing-associated virus

OLYaV

MT809205

40.0% (202/505)

29.7% (195/657)

25

Blueberry virus a

BVA

NC_018519

39.5% (218/552)

26.4% (179/678)

Little cherry virus 2

LChV2

NC_005065

32.5% (156/480)

26.5% (169/638)

27

26

Grapevine leafroll-associated virus 1

GLRaV1

NC_016509

34.2% (162/474)

28.3% (182/643)

28

Grapevine leafroll-associated virus 4

GLRaV4

NC_016416

32.5% (158/486)

25.8% (164/635)

Ampelovirus

29
30

Velarivirus

Plum bark necrosis stem pitting-associated virus

PBNSPaV

NC_009992

31.2% (148/474)

27.0% (172/638)

Areca palm velarivirus 1

ArPV1

NC_027121

28.0% (144/515)

23.8% (161/677)

31

Little cherry virus 1

LCV1

NC_001836

30.0% (145/484)

25.7% (177/690)

32

Cordyline virus 1

CV1

NC_038421

27.9% (135/484)

25.5% (164/642)

33

Cordyline virus 3

CV3

NC_043107

29.0% (144/497)

27.0% (170/630)

Blackberry yellow vein-associated virus

BYVaV

NC_006962,
NC_006963

29.5% (146/495)

27.9% (180/645)

35

Cucurbit yellow stunting disorder virus

CYSDV

NC_004809,
NC_004810

27.8% (133/479)

27.1% (170/628)

36

Sweet potato chlorotic stunt virus

SPCSV

NC_004123,
NC_004124

30.0% (146/486)

27.0% (174/644)

37

Lettuce infectious yellows virus

LIYV

NC_003617,
NC_003618

29.0% (141/487)

27.5% (176/641)

34

Crinivirusa
NCBI Acc. Nos. for genome sequences (note that criniviruses have two genomic segments); bamino acid sequence identities to the TcCV1
RdRp and Hsp70h proteins in the format of “percent identity (identical residues/aligned length)”.

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Fig. 3Phylogenetic relationships of TcCV1 and related viruses
Maximum likelihood phylogenetic trees were inferred based on the RdRp (a) and Hsp70h (b) protein sequences. TcCV1 forms a subclade
named the “CNFV-subclade” (light green box) with six previously known viruses within the genus Closterovirus (green background).
Bootstrap branch support values of 95% or higher, calculated from 1000 replicates, are shown at the nodes. See Table 1 for detailed information of these viruses.

Nucleotide sequences surrounding the putative +1 RFsite collected from 22 closteroviruses were analyzed (Fig.
2b). Four different sequences were observed at the 7 nt long
+1 RF site: GUUUAGC (16 viruses), GUUCGGC (3 viruses),GUUUAAC (2 viruses), and GCUUAGC (1 virus), where theskipped residues are underlined. In addition to the 7 nt
positions, three positions were found to be conserved.
When the skipped residue position was set to 0, the C, G,
and U nucleotides at positions −6, −5, and +8, respectively,
were identical in all 22 closteroviruses. The CG dinucleotide
at positions −6 and −5 are the first two nucleotides of the
CGN (where N represents A, C, G, or U) codon for arginine,
which is conserved in all ORF1ab fusion proteins. The
U nucleotide at position +8 is the second nucleotide of thecodons for valine (GUN) or isoleucine (AUH; H represents
A, C, or U), which are similar amino acids found at thisposition in all ORF1ab fusion proteins. Therefore, it is notclear whether these sequences are conserved in the +1 RFsite or encoded amino acid.
Phylogenic position of TcCV1 among closteroviruses
The phylogenetic position of TcCV1 within the genus
Closterovirus was inferred using the RdRp (ORF1b) andHsp70h (ORF3) sequences. A total of 37 known viruses ofthe family Closteroviridae (21 from the genus Closterovirus
and 4 representatives from each of the three other genera
and the unassigned group) were collected (Table 1). The
RdRp and Hsp70h protein sequences of the known viruses
were compared with those of TcCV1. The TcCV1 RdRp andHsp70h showed 27.8–68.2% and 23.8–55.1% aa identity, respectively, to the orthologous proteins of known viruses.
Multiple alignments were separately generated fromthe RdRp and Hsp70h protein sequences and filtered toSHIN, CH. et al.: Novel closterovirus from Thesium chinense
remove phylogenetically uninformative sites. Maximum
likelihood phylogenetic trees constructed with 1000
bootstrap replicates for both RdRp and Hsp70h showedthat TcCV1 is a novel member of the genus Closterovirus(family Closteroviridae) (Fig. 3). In both trees, TcCV1
formed a strong subclade (bootstrap support values of
100% and 99%, respectively) tentatively referred here as
the “CNFV-subclade” after CNFV, which was identified
first in the group. The “CNFV-subclade” includes TcCV1,
MV1, ReV-1, TV1, CNFV, CtCV1, and CYLV. Branchingpatterns of the “CNFV-subclade” members showed strong
concordance between the RdRp and Hsp70h phylogenetic
trees, indicating that the evolutionary history of thisgroup can be unambiguously resolved. However, otherclosteroviruses showed largely discordant branchingpatterns between RdRp and Hsp70h phylogenetic
trees, suggesting the possibility of complex genomic
evolutionary processes involving genomic recombination
or lineage-specific evolutionary rates.

Conclusion
The genome sequence of a novel closterovirus (genus
Closterovirus), TcCV1, was identified in transcriptome dataobtained from the haustoria and root tissues of T. chinense.
The TcCV1 genome encodes nine proteins, eight of whichhave orthologs in closely related closteroviruses. Comparisons of amino acid and nucleotide sequences of the putative +1 RF sites required for ORF1ab fusion protein production implied that the +1 RF occurs at a conserved locationin all closteroviruses. Phylogenetic analyses showed thatTcCV1 is a novel closterovirus that forms a strong subcladewith a group of previously known closteroviruses. The
genome sequence of TcCV1 may be useful for studying theevolution of closterovirus genome organization.
Acknowledgment. This work was supported by National
Research Foundation of Korea (NRF) grants funded by theGovernment of Korea (grant numbers 2018R1A5A1025077 and2020R1A2C1013403).

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