New Insights About ORF1 Coding Regions Support the Proposition of a New Genus Comprising Arthropod Viruses in the Family Totivir

Home , Leishmaniavirus, Totiviridae, Totivirus

Virus Research 211 (2016) 159–164

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Virus Research

j ournal homepage: www.elsevier.com/locate/virusres

Short communication

New insights about ORF1 coding regions support the proposition of a

new genus comprising arthropod viruses in the family Totiviridae

a,b,1 a,1 b

Márcia Danielle A. Dantas , Gustavo Henrique O. Cavalcante , Raffael A.C. Oliveira ,

a,b,∗

Daniel C.F. Lanza

Laboratório de Biologia Molecular Aplicada, Departamento de Bioquímica, Universidade Federal do Rio Grande do Norte, Natal, RN, Brazil

Programa de Pós-Graduac¸ ão em Bioquímica, Universidade Federal do Rio Grande do Norte, Natal, RN, Brazil

r t i c l e i n f o a b s t r a c t

Article history: Analyzing the positions of 2A-like polypeptide cleavage sites in all available genomes of arthropod

Received 14 July 2015

totiviruses we propose the limits of all ORF1 coding sequences and observed that two proteins pre-

Received in revised form 6 October 2015

viously predicted in infectious myonecrosis virus genome are unique in the arthropod totiviruses group.

Accepted 14 October 2015

A putative protein cleavage site upstream the major capsid protein was also identiﬁed only in these

Available online 20 October 2015

genomes. In addition, protein models generated using ab initio and threading approaches revealed con-

served structures possibly related to formation of viral protrusions and RNA packaging, clarifying the

Keywords:

mechanisms involved in the extracellular transmission. These data appoints that the group formed by

Infectious myonecrosis virus

arthropod totiviruses are sufﬁcient distinctive to be clustered in new genus belonging to the Totiviridae

Giardia lamblia virus

family, in agreement with previous phylogenetic analysis.

Drosophila melanogaster virus

Double strand RNA viruses

The family Totiviridae is composed by icosahedral virions of and Victorivirus infect exclusively fungi (King et al., 2012; ICTV,

∼

40 nm in diameter, that normally encapsidate a monossegmented 2014).

double-stranded RNA (dsRNA) genome. This family comprises sev- The ﬁrst virus belonging to the Totiviridae family related to

eral viruses of major medical, veterinary or agricultural importance infect an arthropod was the IMNV, isolated from penaeid shrimps

such as Trichomonas vaginalis virus 1, Infectious myonecrosis virus in 2005 (Tang et al., 2005; Poulos et al., 2006). Five years later

(IMNV) and Helminthosporium victoriae virus 190S respectively, Drosophila melanogaster totivirus (DmV) and Armigeres subalba-

with genomes typically organized in two overlapping open read- tus totivirus (AsV) were isolated from Drosophila melanogaster cell

ing frames (ORFs), encoding a major capsid protein (MCP) and a lines and Armigeres subalbatus mosquitoes respectively (Wu et al.,

RNA dependent RNA polymerase (RdRp) (Wickner et al., 2005). 2010; Zhai et al., 2010). Most recently, two new isolates that cluster

Genomes with more than two ORFs occurs only in Leishmania RNA in the monophyletic group formed by IMNV, AsV and DmV were

viruses, that shows an additional small ORF1 encoding a putative described. Omono river virus (ORV) and Tianjin totivirus (TianV)

protein, and Piscine myocarditis virus, containing a downstream were isolated from Culex mosquitoes and bat guano respectively

ORF3 whose deduced product presents a predicted chemokine- and should belong to the same species since they share an amino

superfamily motif (Scheffter et al., 1995; Haugland et al., 2011). To acid sequence identity higher than 50% (Wickner et al., 2005; Isawa

date, the Totiviridae family comprises ﬁve genera recognized by the et al., 2011; Yang et al., 2012).

International Committee on Taxonomy of Viruses (ICTV). The gen- The arthropod totiviruses are the last studied and has not

era Giardiavirus, Leishmaniavirus and Trichomonasvirus comprise an exact phylogenetic classiﬁcation. Our recent results concern-

viruses that infect protozoa and the members of the genera Totivirus ing RdRp phylogeny revealed that the ﬁve arthropod totiviruses

already described are grouped in a monophyletic group closest

related to the Giardiavirus clade (Oliveira et al., 2014). In the

present work we presented further evidence of their peculiar

∗ ORF1 organization and predictions of unique motifs and polypep-

Corresponding author at: Laboratório de Biologia Molecular Aplicada—LAPLIC,

Departamento de Bioquímica, Centro de Biociências, Universidade Federal do Rio tides, corroborating the singularity of the arthropod totiviruses

Grande do Norte, Natal, RN CEP: 59072-970, Brazil. Fax: +55 84 32153415. group within Totiviridae family. From this point of the text the

E-mail addresses: [email protected], [email protected]

group formed by arthropod totiviruses will be referred as artivirus

(D.C.F. Lanza).

1 (arthropod totiviruses), as proposed by Zhai et al. (2010).

Equal contributors.

http://dx.doi.org/10.1016/j.virusres.2015.10.020

160 M.D.A. Dantas et al. / Virus Research 211 (2016) 159–164

Fig. 1. Schematic representation of arthropod totiviruses and giardiaviruses genomes. (A) Amino acid sequences of 2A-like motifs identiﬁed in the ORF1 polypeptide from

all artiviruses genomes. (B) Schematic representations comparing full genomes of artiviruses and its closest related viruses of the giardiavirus clade. The coding regions

of RNA binding protein (RBP), Small protein 1 (SP1), Small protein 2 (SP2), Major capsid protein (MCP) and RNA dependent RNA polymerase (RdRp) are represented by

rectangles with different colors. The positions of the initial nucleotide referenced by the complete genome, and the size of each predicted polypeptide encoded by ORF1, are

represented by the numbers below and above the bars respectively. The dotted rectangle represents the conserved site located at the extreme C-Terminal of SP2. Phylogenetic

groups were reconstructed based on RNA-dependent RNA polymerases sequences as previous described (Oliveira et al., 2014). The numbers in branch nodes indicate posterior

probabilities. Abbreviations: ORV—Omono river virus; TianV—Tianjin totivirus; DmV—Drosophila melanogaster totivirus; AsV—Armigeres subalbatus virus; IMNV—Infectious

myonecrosis virus; GCV—Giardia canis virus; GLV—Giardia lamblia virus; PMCV—Piscine myocarditis virus.

A screening covering all Totiviridae species (Supplementary release factors 1 and 3. This mechanism is common in some viruses

Table 1), using the Basic Local Alignment Search Tool (BLAST) belonging to families Picornaviridae, Dicistroviridae, Tetraviridae

and a careful visual inspection, revealed that 2A-like motifs are and Reoviridae (Palmenberg et al., 1992; Donnelly et al., 2001;

unique in artivirus, occurring in all viruses of this group. 2A- Doronina et al., 2008; Luke et al., 2014). Considering the locations

like sequences and their respective positions in each polypeptidic of 2A-like motifs, we deduced the polyprotein cleavage products

sequence encoded by ORF1 are presented in Fig. 1A and B, respec- encoded by ORF1 for all artivirus genomes. The set of amino acid

tively. 2A-like motifs (-DxExNPG P-) are an interesting solution to sequences corresponding to each coding region was aligned using

release nascent peptides from a polyprotein, using the eukaryotic MUSCLE (Edgar, 2004) and T-Coffee (Notredame et al., 2000) with

M.D.A. Dantas et al. / Virus Research 211 (2016) 159–164 161

Fig. 2. Identities between the polypeptides encoded by arthropod totiviruses. (A–B) Alignments of the full polypeptide sequences of RBP and SP2. Sequences were deduced

from the ﬁve complete genomes available in GenBank, aligned using MUSCLE and T-Coffee and edited using Jalview v.2.8. The RBP of Drosophila melanogaster virus was

not detected, thus this sequence was removed from the alignment. DSRM domains and the 11-amino acid conserved sites are highlighted within the black rectangles at RBP

and SP2 sequences respectively. (C) Identity matrix of the predicted polypeptides. The darker boxes in heat maps designate highly identities between the two compared

sequences.

default parameters, and manually adjusted using the interface The alignments revealed that, in addition to coding regions

Jalview v.2.8 (Waterhouse et al., 2009). Genome schemes obtained of RdRp and MCP, the coding regions for a RNA binding protein

were arranged in according to our previous phylogenetic analysis (RBP) and Small protein 2 (SP2), previously predicted in IMNV

(Oliveira et al., 2014) in Fig. 1B. ORF1 (Nibert, 2007; Dantas et al., 2015), have correspondents in

162 M.D.A. Dantas et al. / Virus Research 211 (2016) 159–164

Fig. 3. DSRM and SP2 structural models. The models were calculated using the software I-TASSER that combines ab initio and threading approaches. The reliability scores of

each model are shown in Supplementary Table 2. (A) DSRM tertiary structures for each of the four sequences analysed. The conserved structure formed by two ␣-helices and

three ␤-sheets, typical of dsRNA-binding motifs, is clear in all predicted structures. (B) SP2 predicted structures for all arthropod totiviruses. In all cases the tertiary structure

is rich in alpha helix regions and ressembles a ﬁber shape.

all other artivirus genomes (Fig. 2A and B and Supplementary Fig. to determine the location of DSRM domain in the unique DmV avail-

1). Comparing the full sequence alignments, the higher amino acid able sequence. The DmV genome sequence was obtained by deep

identities were observed for RdRp (36.24% to 62.92%) and the mini- sequencing and assembly of total small RNAs (vdSAR) and the gaps

mal for the RNA binding protein (RBP) (11.23% to 28.40%). Curiously, were ﬁlled using 5 and 3 RACE (Wu et al., 2010). Although this

the SP2 proteins exhibit considerably identity between the differ- method has proven to be effective to characterize unknown viruses

ent species, ranging from 15.13% to 35.07%, comparable in some in insect cells, we speculate that this approach may not be efﬁcient

cases with the identity between MCPs (28.47% to 62.30%) (Fig. 2C). to cover the entire 5 extremity of DmV genome.

Using the BLAST algorithm, that consider only the regions of local The most interesting results of this work are related to SP2.

similarity inside the sequences, were observed regions with sig- Besides this protein occurs in all artiviruses, the alignment of SP2

niﬁcant similarity even in SP2 and RBP alignments, that showed predicted amino acid sequences revealed a great conservation,

regions with similarity higher than 40% (data not show). The high- mainly in its second half and extreme C-terminal region (Fig. 2B).

est values of similarity are evidences that amino acids with similar The last eleven amino acids of SP2 (highlighted in the black rectan-

physicochemical characteristics have been maintained in some gle of Fig. 2B) shared high identity (ranging from 58.33 to 91.67%),

regions, indicating some conservation in the protein structures. and it seems to be a putative cleavage site where an unclear prote-

The RBP coding regions were conﬁrmed by the presence of a olytic mechanism occurs to release SP2 from the ORF1 polypeptide.

double-stranded RNA-binding domain (DSRM) (highlighted within Secondary structure predictions and 3D models of SP2 were calcu-

the black rectangle in Fig. 2A) and are located upstream SP2 in all lated using Foldindex (Prilusky et al., 2005), COILS (http://embnet.

genomes (Fig. 1B). Despite the great sequence diversity of RBPs, vital-it.ch/software/COILS form.html) and I-TASSER revealing very

their respective DSRM 3D models generated using the software I- similar secondary structure patterns (Supplementary Fig. 2) and

TASSER (Zhang, 2008; Roy et al., 2010) achieved excellent reliability protein shapes (Fig. 3B), indicating a conserved function of SP2 in

scores evaluated using MOLPROBITY (Chen et al., 2010) and SWISS- every artivirus species. In all artiviruses, SP2 have predominantly

MODEL (Arnold et al., 2006) (Supplementary Table 2) revealing a an unfolded or “dynamic” structure resembling a ﬁber, with high

conserved structure formed by two ␣-helices and three ␤-sheets, alpha helix content and a conserved and structured C-terminal

typical of dsRNA-binding motifs (Fig. 3A). The presence of this region (Fig. 3B and Supplementary Fig. 2). The structured region

domain exclusively in the artivirus group within the Totiviridae may acts as a foot, anchoring the viral protrusions in MCP and

family, is a indication that RBP plays typical, speciﬁc, and primordial forming the pore complex. As described by Tang et al. (2008),

roles on viral RNA packaging. DSRM domains are widely recurrent the outer surface of IMNV capsid is formed by pentagon-shaped

in other RNA binding proteins such as Drosophila staufen pro- regions, and each pentagon has a small central knob surrounded

tein (Bycroft et al., 1995), dsRNA dependent protein kinase (PKR) by two sets of ﬁve mesas. The lower density and resolution of

(Nanduri et al., 1998), adenosine deaminase that act on RNA (ADAR) the IMNV protrusions might be explained by the lateral ﬂexibil-

(Steﬂ et al., 2006) and RNA helicase A (Nagata et al., 2012; Li et al., ity of SP2 complexes and the mesas could be formed by SP2 units,

2014) always acting as a dsRNA binding factor and/or in RNA pack- interacting with a respective unit of the MCP pentamer. Viral pro-

aging associated processes. We did not have sufﬁcient information trusions arranged as multimeric proteins that restrict the mobility

M.D.A. Dantas et al. / Virus Research 211 (2016) 159–164 163

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