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Hel
CavVNDiVHanaVMénoVNséVMoumoV
4207 4280ICSYKSQLQN FISLQQQKIL SDNVNLSTID SAQGDEFDIV ILCLSQINNF TLNPNRFNVA ISRAKSVLFI TVPP.......... .......... .E........ .......... .......... .......... .......... .............. .......... .......... .......... .......... .......... .......... .............H .AT..N...I PEF.K..... .......... .......... .......... .......... .............. .A....L.VI PEY....... .......... .......... .......... .......... .............. .T.......I P.F.K..... .......... .......... .......... V......... ....
VIV
CavVNDiVHanaVMénoVNséVMoumoV
4117YHFRCHPTIF QYFKDLYYAD KDMECATSIA DRIIRPLNPI NTVQVSEPTF RNQGVILNQD EADKVLEILV LVNQTLALHS SYEYQPTIAI.......S.. .......... .......... .......... .......... .......... .......... .......... .................M.. .......... .H........ .......... .....G.... .......... .......... .......... T.........D......H.. .....Q.... ...V...KAE ..L....... ...H.GP..Y .......... .T....D..I ..T...T..A .Q....SL..H......S.. .F........ ...V...AAS .......H.. ..I..EG.SY ......R.EN .TE...D... ..S.V.S..A .FN..............L.. .F..N..... ...V.S.QAI .......H.. ...PIQG..Y ......R.EH .TE...D... ....I.S..T .FDFK.....
IV
CavVNDiVHanaVMénoVNséVMoumoV
4027RICVTTIQSF STVQHVKDID LVILDEFSLT SDNYLLTGLA HLKPSTRVLF SGDPRQLSGV DEVRKPLQSR FHTLINYYTE TYPREVHVLK.......... .......... .......... .......... .......... .......... ..I....... .......... .................... .A....V-V. V......... ......S... ..RL...... .......... .VI..L.H.. .......... ...L..L..T.......... ......R... .......... .........S .......... .......... ..T.....P. ......F... ...N......C......... P.....-.V. .......... .........S ...L...... ....K..... ..I....LP. ........A. ...K.LL...C......... P.....-.V. .......... .........S ...L...... ...T...... ..I..T..P. ........A. ...N..L...
II III
CavVNDiVHanaVMénoVNséVMoumoV
3937RFKIMFGGPG TGKSHTLSIL INHLHEKGLR ILVYTPSHQS ANALLYKIAN LIKRRTIQNP GLVRIITDGM KEEIKPHPYI TYRTNMLDKD.......... .......... .......... .......... .......... .......... .......... .......... .................... .......AT. .....D..F. V......... .......... MM...G.... .......... .D.L...... ....H.I.T........... .......... V.....Q.FK ..I....... .....F.... IM...H...S .......E.. .D......F. ...AS..E............ ....Y..... ......S.FK ..I....... .......... VMRK.N.... R......E.. .D.L...... .P..H...Q........... .......... V....AS.FK M.I....... .........G .M...S.H.. .......... .D.L...... .P..H.....
I
ZCavVNDiVHanaVMénoVNséVMoumoV
3601NKGRLITYNC YVCGENAYLT CATCERAFCN SADTNHGSHM EQHLQYSGHT CLYLNCKTVK CHHCFTTDIN LLYTTGRD-H YCEAHKPKNA...K...... .......... .......... .......... .......... .......... .Q....M... ........-. ...S............S... ....D..... .......... NT.......I .......... .......... .Q..Y.S... .......E-. F..I.........Q...... ........I. .....KS... .K..I....I .........D ........I. .R...AI... K...S.K.MY F..T....H....K...... .......... .......... .S.SI....L .......E.N ......R... .S..Y..... V...S.KN-V F............K.VA... .......... .......... .S.PL..... .........D ..F....... .R..Y.Y... V.......-V F..Q.R....
3689* * * * * * * * * * *++++ +
3CL
CavVNDiVHanaVMénoVNséV
1565DTTTGKSNIW TSYKLQHPSE IMITLNNEIN LPNPANYDFE TTKVVYQHPL RNVCATLETL QHLTNKTNAK LPYDSRLLSD FNITAEQYNQ.......... .......... .......... ....T..... .......... .......... ........V. ....P..... ........A..IK....... .......... .......... ....E..... .SS....... ..IS...... .Y........ IA........ ........A.NC...TP... ....I..... .....D.G.. ..Q.ED...Y .A.IC..... .D.R...Q.. .Y...QG.T. ....PQ..A. ...........KLQ...... ....M..... .......... ..E.VD...N .N..I.H... ...M...... .Y...S...R M...K..... ........E.
CavVNDiVHanaVMénoVNséV
1385
NKSAASNPSI SHIVLEMPVA INPLIKYTTR TSVSSLRGAV VNGYIYIQRH LFGSKKQEFE ACYNNGKGLL NCKNLERSKY DIDSAELIGT.......... ......L... .........K .......... .......... .......... .......... .....D.... .................... A.N...L..T .......... .......... .......... ...N...V.. .......... .....D.... ..............L..... PY.A.PL..S .....T.DS- --......S. .......L.. ....NQKD.D SH.A.....N K......... ...N................ P.K..GL..T .....T.MSN KYA.....S. .......... ....N.ND.L V........E K......... ..........
! !
pro
CavVNDiVHanaVMénoVNséV
1655 1688YGYYIDYNNF VNNFNRYTTT TIGTKSFETC IKYG...N...... I......... .......... ....H...V..... .....H.... .......... ....H......K.. IQ..IK..N. L.NSR...MS ......L.V...Q. .K...Q.... L....T..S. ....
CavVNDiVHanaVMénoVNséV
1475LIRIPLHDKH SIPHISIHPD PLSYNGPVTL YLSRYDTELN KDVLCVHTGF MSEGHHDIKT VFGDCGGMLF DPKGRLLGLH CAGSDDVVFM.........Q .......... .......... .......... .......... .......... .......... .......... ................V..S .V.N.N...A ..T....... .......... .......... I......... .......... .......... ................V.PK .V.DVK...K ....T..... ......S.TA .......... IA......R. .......... ..R.Q..... ....A..S.L..K...V.RN .VVD.EL..E ..T....... ......S.QG .......... ........R. .......... .T........ ....E.....
& ! &
OMTAALAKAPLDW DHLTLEIPGY NTRKQHSS-H MTTKALGILH ILQDSMLYTN RKTLNPNLPV ILPGSASYLG DTVLANEMSK TLKQTKFVHI...S...HN. .......... ........-. .......... .......... .......... .......... ........A. .......I..ST.S...PT. N.....L..C .--RK.V.-. T....S...Y .......... .......... .I..A..QF. ....T..I.. N..R...INVSN.Q...SN. N.TQ.L.... E...ISPPG. .LN......S .........H .T...K...I .H..A.GFN. ..I....FR. Y...S.IIN..H.NL..HT. ..Q....... S..INNPC-. .Q...I.V.R .....I...P .....AKY.. .......NY. E.I....FRR H...S..I...H.N...Q.. ..Q....... T..TNNPC-. .Q.....V.. ..K......S .L...AAR.I .......HY. E......FK. Y..LS..I..
CavVNDiVHanaVMénoVNséVMoumoV
4848
X I
#
DPRLKIDNNT THHRKTLMEM LDIGYTTELI ISDIHDNNN- PWIPELMEYT LKYLIDTGTL IMKITSRGAT EAVLQQLEHM AKNFTYVRVC.......... .......... .......... .......K.- .......T.. ....V..... .......... .D......DL S..............L..HN ..Y..P.K.. .......... .........N T..H..I... ....IE.... .......A.. .DT.EY...L S..............R-.DD ..YKLL.KD. .PK.FN.... .....--SDV E.....I... N...QQS... .......M.. .KAI....TL SE.............L.... ..FK...KD. .P...P.... .....N.DDP T..T..ID.. H...L..... .......... STA.E...NL SRI................. ..FK...N.. ..V..S.... .......T.K N..A..I..S T...LES... .......... .EA.AL.QAY S.......T.
CavVNDiVHanaVMénoVNséVMoumoV
4937
IV VI
# #
NLNAVTFSSE LWIVFANKRK PPVQGWTSHE LRAELRKHWY SMTRSIIQPL MRARQSVFRY SPK.......... .......... .......... .......... .......... ..S....... .........P..V ......D... ...L...... ..N....... ..A.N..... T...PCI... ......V..C... .......N.. ...N.....D ..N....... ...HN.LH.T I.S.LD.... ....V....C... ......D... .T.K.....D V.S....... ...QN.KN.I L.Y.LDI... ....I....C... .......... ...N....YD ..T...M... ...QN.RTTV L.P.IGL.K. ...
CavVNDiVHanaVMénoVNséVMoumoV
5026 5088
VIII
#
NMTSFVLSHLEKL AYEPQANLKA FTSMDYRLKN FNPEMCKLRR ELQKVWYEKY IDTK--KTHC NMGCGKEPLQ QALHNIDVLQ GKSNPQNNMN........N. .......... .......... .......... .......SQ. TN.N--.... .......... .......... ..................N. ..D....... .......... .......... ....T..QQ. .A.N--.... .........K H......I.. ..T........V.QPQ..SI ..DN-...N. ........R. .D........ D...H..A.. SE.S--I... ......Q..K .......IN. ..N........L.QP.F... ...QG...R. .L......R. ..I....... ...IE...R. TSINTSR... .......K.S N......IK. ..-.LH.D...L.Q.EY.N. ...Q....S. .L......R. .......... ...DA..TQ. LTASKP.I.. ......QL.K R......IKL .N-K.Y....
CavVNDiVHanaVMénoVNséVMoumoV
4614
I
THTCDSEEHI YFDSHWYKDG GFKKPSYIFS DINKEHYYKL GTTGLCLYLN SKYAKYVHEY RTVSGNDVFK S-LYSPYCDL GRKPHQAVIE.......... ........A. ..T....... .......... .......... .......... .......... .-........ .......E.......A.... .......... ..S....... ........N. .....V.... ......I... .K........ .-..N.F... ..E....A....L..AQ... ........T. ..S....... .......... .A........ ..H...L.AF QPI....... TEF.NSQ.TT N.T..HLS....I.S.D... ........IN ..S....... .M......N. .......... ......C.V. QE.T.T.... .-...SS.E. EL...K.I.T....NA.... ........EN ..Q....... .M......N. .S........ ..H...C.K. .EA..D.... .-...LE.DK DQK..THA.Q
CavVNDiVHanaVMénoVNséVMoumoV
4702 *
PSCSIPDCII TS-NIGERFQ TLVCNVHQDQ MELISKIAQA TKYGYQFIYT GKILLNNH.......... ..-....... .......K.. .DI....S.. .......... ..T............... ..-..D.K.. .....I.K.. ..I....S.. .......V.. ..T.....DNT.M.A... Q.-SSN.D.H .FI.DG.Y.. .QI..D.SK. ........K. .PTT.S..DCT.T....N S.-TL.SQ.. .I..SL.YG. .DI....S.. .Q...R.V.. .S-E...KDCT.T..... ..NTSD.N.. V...SK.Y.. .DI....S.. .....R.... KANE....
CavVNDiVHanaVMénoVNséVMoumoV
4791 4847* * *
ExoN
GIRNDTTVDL RPLLNFCVDN MHVKPVIVTW SGASDHCFLR AHTLYPDIST VCNITTRCTS QPIYASPQGR HTYYLCQYHA HQLKDHINIT.......... K......... .......... .........K ........A. .....I.... .......... .......... ......V.....S...I... K......... V......... ..V......K .Y........ .......... .......... .......... ............K.NV...I KH.....I.. .......I.. A.DN.....K .N....PAAQ I....P.... T.....A.TC .......F.. QTF.EQLT....SDN.I..V KHM....I.. V......... A.T....... .N.....V.Q T...AS..L. .......... .......... .KF.EQL........I.... KHM....I.. T......... A..T.....K .N......TQ I......... ......R... NI........ ..H.EQL...
CavVNDiVHanaVMénoVNséVMoumoV
4447* * * *
I
DCICFDAEFL NPRDNLQEPV MLSYGFSSKY GKRRIAGIPV RYIKDKFNRI IPHKYNYKDN NKPLTSTYSC DWMKKQHPDQ YKHLLTSVLQ.......... .....V.... .......... .......... .......... V.Q....... ........T. ...R....E. ........M..Y........ .....V..H. .......... .......T.. ........K. V.I..P...K H.......V. E.....K.E. .QY..N.IT..Y.F..S... ....DPR... ......T.A. .N...S.T.M ..VYS...QL T.V....... ........IA T.L.AKA.E. .NY.RDTIT..Y.Y..S... ..K..PP... .........H .....S.T.M .....NY... T.I...F..K D.......VS S....DL.T. F...NNA.CK.YVY..T... ..I..R..C. .V.......N .N.....T.M ..V....DKL V...F.F..K ........IT G.L.A.L.Q. F.Q.N.A..N
CavVNDiVHanaVMénoVNséVMoumoV
4357 & &
HFVNLEIIDL KVDRNQYTNE RTLRVYHNDY LKLTLDLDN- VASNSLTDCH TRYCRTIHAP PTPHDPLDDA IMTQCIYQ.......... ........D. .......... .........- .......... ......V... A......... .................. ..EY..F.S. .I......N. .........- .......... K.....V..S I......... .................. .AIN.KQ... K...LT..NE ....IE.SQD IK....N... AY..N.T.R. I......... L.....FK.......... S..K.KH.D. .N...T..NE ...K.EISKS I...K.Q... DF..N...T. I......... ......F........L.. .AEF.VH.G. K.IS.T..EE ...KFEIHED ...CK...V. DF..N.V.T. I......... ...R.VF.
CavVNDiVHanaVMénoVNséVMoumoV
4537 4613
II III
& & § §
Figure S1: Multiple sequence alignments of putative mesonivirus replicative
domains. Partial pp1a/pp1ab sequences were aligned using ClustalX 2.0 (6) as
implemented in Geneious (5). Amino acid numbers relate to positions in CavV
pp1a/pp1ab. Conserved sequence motifs in the helicase (Hel) (3, 4), 3'-to-5'
exoribonuclease (ExoN) (7, 10), guanine-N7 methyltransferase (1, 8) and ribose-2'-O
methyltransferase domains (2, 10) are indicated by black bars and roman numbers.
Other conserved residues are indicated as follows: !, Cys, His and Asp residues
predicted to form the catalytic triad of the mesonivirus 3CLpro (12, 13); &, residues
predicted to participate in the S1 specificity pocket of the mesonivirus 3CLpro (13); *,
conserved Cys/His residues in the helicase-, ExoN- and NMTase-associated zinc-
binding domains (8-11, 13) that are predicted to be involved in zinc ion coordination;
+, other conserved Cys and His residues in the mesonivirus helicase-associated
zinc-binding domain; &, conserved acidic residues (Asp, Glu) predicted to be
involved in metal ion coordination by nidoviral (and cellular) exoribonucleases of the
DEDD superfamily (7, 10); #, conserved residues predicted to form the catalytic
tetrad of mesonivirus (and other viral and cellular) ribose-2'-O methyltransferases (2,
10).
gp64 ? gp116
229TILS 1128LAPR
N
1MNRR
C
sgsg
Protein
mRNA2 mRNA3
Genome 2 3 5’ An-3’
5’5’ An-3’
An-3’
Okavirus (YHV)
Alphamesonivirus (CavV)
2b 2a 5’ An-3’
5’ An-3’
? S
224STRID
S1 S2
224STRID 431WDSS
N
1PATVS
B
sg
Genome
Protein
mRNA2
N Arterivirus 5’ E 1a 1b
GP5
M
GP3
GP4 GP2
An-3’
N Okavirus An-3’1a
1b 5’
gp116 3
? gp64
4?
N Bafinivirus An-3’1a 1b M
5’ S
1b
N Torovirus An-3’1a
1b M 5’
HE S
1b
N Alphacoronavirus 5’ 1a 1b
An-3’ M S E
2b Alphamesonivirus An-3’1a
1b 5’ 2a
3a 3b
4? N
S1
M?
S2 ?
A
Figure S2: Nidovirus genome organization. (A) Schematic view of the genome
organization of viruses representing all currently established nidovirus (sub)families.
Mesonivirus (B) and ronivirus (C) genome organization in the glycoprotein and
nucleocapsid protein coding regions. Open reading frames (ORFs) are shown by
boxes, sgRNAs are indiated by lines, and proteins are shown by rounded boxes.
Leader sequences are symbolized by red and 5’ cap structrures by black boxes. N-
terminal protein sequences and genome positions are indicated. 3' poly (A) tails are
indicted by An. The following genomes are shown: Human coronavirus NL63 (HCoV-
NL63, NC_005831, genus Alphacoronavirus, subfamily Coronavirinae, family
Coronaviridae); Bovine torovirus (BToV) strain Breda (NC_007447, genus Torovirus,
subfamily Torovirinae, family Coronaviridae), White bream virus (WBV, NC_008516,
genus Bafinivirus, subfamily Torovirinae, family Coronaviridae), Equine arteritis virus
(EAV) strain Bucyrus (NC_002532, genus Arterivirus, family Arteriviridae), Cavally
virus (NC_015668, genus Alphamesonivirus, family Mesoniviridae), and Gill-
associated virus (GAV, NC_010306, genus Okavirus, family Roniviridae). Proteins
and corresponding ORFs encoding these proteins are indicated as follows: E,
envelope protein; gp and GP, glycoprotein; HE, hemagglutinin-esterase protein; M,
membrane protein, N, nucleocapsid protein; S, spike protein.
Table S1: Primers used to generate DIG labeled northern blot probes.
Virus Primer name Primer sequence Probe length
CavV CavV-5’-2-F
CavV-5’-106-R
5’-CTAATGAAAATTTTGTTTTCTCAC
5’-ACTGATGTGGCTAGGTTTGGTGAT
105 nt
CavV-3’-19551-F
CavV-3’-20092-R
5’-AAGTTCGACCACTGAATGAGAC
5’-CGCCATACTACTAAGCCCTAAC
542 nt
HanaV HanaV-5’-1-F
HanaV-5’-108-R
5’-ACTAAAGAAAATTCAGTT
5’-GCAACTGATGTGGCTAGGTTTGGTG
108 nt
HanaV-3’-19655-F
HanaV-3’-20062-R
5’-TGCATTAATCTACCACTATAATCTCGCC
5’-TGCCAAGCTCCATACTACTAAGCC
408 nt
MénoV MénoV-5’-1-F
MénoV-5’-121-R
5’-ACTTTGATATCTTTTGATAATCGCC
5’-CAGAGGGTCTCAAACAAACAAAGTTGC
121 nt
MénoV-3’-19717-F
MénoV-3’-19971-R
5’-CACTTTCCGAGGCACGACACAACC
5’-TGCCAATCTGATAACTACTGAGCCCT
255 nt
NséV NséV-5’-1-F
NséV-5’-111-R
5’-ACTAAAGAATAATTTGTATTCAACC
5’-AGCAATAGACGTGGCTAGTTAAATTTCTAGAG
111 nt
NséV-3’-19790-F
NséV-3’-20060-R
5’-TGCAGCATGAACTCGACCACCA
5’-GCGCCAAACTACTAAGTCCTAACCG
271 nt
References
1. Chen Y, Cai H, Pan J, Xiang N, Tien P, Ahola T, and Guo D. 2009. Functional screen reveals SARS coronavirus nonstructural protein nsp14 as a novel cap N7 methyltransferase. PNAS 106:3484-3489.
2. Decroly E, Imbert I, Coutard B, Bouvet M, Selisko B, Alvarez K, Gorbalenya AE, Snijder EJ, and Canard B. 2008. Coronavirus nonstructural protein 16 is a cap-0 binding enzyme possessing (nucleoside-2'O)-methyltransferase activity. J Virol 82:8071-8084.
3. Gorbalenya AE, and Koonin EV. 1993. Helicases: amino acid sequence comparisons and structure-function relationships. Curr Opin Struct Biol 3:419-429.
4. Ivanov KA, Thiel V, Dobbe JC, van der Meer Y, Snijder EJ, and Ziebuhr J. 2004. Multiple enzymatic activities associated with severe acute respiratory syndrome coronavirus helicase. J Virol 78:5619-5632.
5. Kearse M, Moir R, Wilson A, Stones-Havas S, Cheung M, Sturrock S, Buxton S, Cooper A, Markowitz S, Duran C, Thierer T, Ashton B, Meintjes P, and Drummond A. 2012. Geneious Basic: an integrated and extendable desktop software platform for the organization and analysis of sequence data. Bioinformatics 28:1647-1649.
6. Larkin MA, Blackshields G, Brown NP, Chenna R, McGettigan PA, McWilliam H, Valentin F, Wallace IM, Wilm A, Lopez R, Thompson JD, Gibson TJ, and Higgins DG. 2007. Clustal W and Clustal X version 2.0. Bioinformatics 23:2947-2948.
7. Minskaia E, Hertzig T, Gorbalenya AE, Campanacci V, Cambillau C, Canard B, and Ziebuhr J. 2006. Discovery of an RNA virus 3'->5' exoribonuclease that is critically involved in coronavirus RNA synthesis. PNAS 103:5108-5113.
8. Nga PT, Parquet Mdel C, Lauber C, Parida M, Nabeshima T, Yu F, Thuy NT, Inoue S, Ito T, Okamoto K, Ichinose A, Snijder EJ, Morita K, and Gorbalenya AE. 2011. Discovery of the first insect nidovirus, a missing evolutionary link in the emergence of the largest RNA virus genomes. PLoS Path 7:e1002215.
9. Seybert A, Posthuma CC, van Dinten LC, Snijder EJ, Gorbalenya AE, and Ziebuhr J. 2005. A complex zinc finger controls the enzymatic activities of nidovirus helicases. J Virol 79:696-704.
10. Snijder EJ, Bredenbeek PJ, Dobbe JC, Thiel V, Ziebuhr J, Poon LL, Guan Y, Rozanov M, Spaan WJ, and Gorbalenya AE. 2003. Unique and conserved features of genome and proteome of SARS-coronavirus, an early split-off from the coronavirus group 2 lineage. J Mol Biol 331:991-1004.
11. van Dinten LC, van Tol H, Gorbalenya AE, and Snijder EJ. 2000. The predicted metal-binding region of the arterivirus helicase protein is involved in subgenomic mRNA synthesis, genome replication, and virion biogenesis. J Virol 74:5213-5223.
12. Ziebuhr J, Snijder EJ, and Gorbalenya AE. 2000. Virus-encoded proteinases and proteolytic processing in the Nidovirales. J Gen Virol 81:853-879.
13. Zirkel F, Kurth A, Quan PL, Briese T, Ellerbrok H, Pauli G, Leendertz FH, Lipkin WI, Ziebuhr J, Drosten C, and Junglen S. 2011. An insect nidovirus emerging from a primary tropical rainforest. mBio 2:e00077-00011.