COBRE Y SUS ALEACIONES

Assembly of sequences from 22 random cDNA clones produced a contiguous consensus cDNA sequence of 2797 nt (Fig. 4.15). The nucleotide composition of this sequence was A = 31.1%, U = 28.0%, G = 19.9% and C = 21.0%. Sequence analysis revealed a single large AUG initiated ORF (ORF 1) on the positive strand when both standard and fungal mitochondrial codons were used to define ORFs (Fig. 4.16). Analysis of nucleotides within the ORF 1 region identified a prevalence for A or U (59.49%) at the third or wobble position of codons. The 5´ and 3´ UTR regions were, respectively, at least 192 and 124 nt long, although sequencing of these regions was not completed in this study. Mitochondrial codon usage identified four additional ORFs, one (ORF 2) on the positive strand and three on the negative strand (ORF 3, ORF 4 and ORF 5) (Fig. 4.16). Additional information regarding these ORFs is provided in Appendix 4.6. As a BLASTP search with polypeptides putatively encoded by these four ORFs did not identify any significant sequence similarity (E values) with previously reported protein domains or motifs, ORFs encoding these putative polypeptides were considered less likely to be expressed inside host cells. Hence, these putative polypeptides were not considered for further analyses in this study.

ORF 1 was 2481 nt in length and was located between nucleotide positions 193 and 2673 (Fig. 4.16). This putative ORF encoded a polypeptide of 826 aa (Fig. 4.17) with a molecular weight of 92.94 kDa. A BLASTP search revealed that this putative polypeptide shared aa sequence similarity with polyproteins containing a putative RdRp region in

mycoviruses in the genus Mitovirus. A CD-search further showed that the conserved RdRp

region of ORF 1 belonged to the family pfam05919, which is a member of the superfamily cl05469, comprising RdRps from several members of the genus Mitovirus. The conserved RdRp domain spanned between aa positions 284 and 632 (E value, 1.74 X 10-78). A Pfam

search found this conserved domain spanned aa positions 220 and 752 (E value, 2.1 X 10-94).

Nevertheless, the putative RdRP domain contained six conserved motifs (I-VI), including the highly conserved GDD motif, typical of Mitovirus RdRp (Fig. 4.18). These results indicated that this dsRNA was a distinct member of the genus Mitovirus in the family Narnaviridae. The proposed name for this dsRNA is Rhizoctonia mitovirus 1 RS002 (RMV-1-RS002; Genbank accession No. KC792591).

166

Figure 4.15 Schematic representation of a series of 22 overlapping cDNA sequences assembled into a partial genome of RMV-1-RS002 from

RS002. cDNA sequences from individual clones are shown by parallel gray boxes. The left bar (0-13) shows the number of independent cDNA sequences used to construct the consensus sequence throughout the genome. Assembly was created using Geneious v5.5.

167

Figure 4.16 Organisation of putative ORFs in the partial genome of RMV-1-RS002. Nucleotide numbers in brackets beside individual ORFs indicate their corresponding lengths. Different reading frames on the positive (1, 2 and 3) and negative (-1, -2 and -3) strands are shown on the left.

Figure 4.17 Schematic representation of the conserved RNA-dependent RNA polymerase (RdRp) domain in the putative polypeptide encoded by ORF 1 of RMV-1-RS002. The shaded box indicates the position of the conserved mitovirus-specific RdRp domain. Amino acid positions (1 to 826) are given above the polyprotein sequence.

168

RMV-1-RS002 (310) KVRPVAIIDYFTQELLSPFHDLVAGILR--SIPQDGTFNQNAIASKVKEFTATAGNS---LFSFDLTAATDRLPVILQRRIISHIIKI---

TeMV (291) KSRIIAIGDYYSQCLLSPFMVTLRSCLE--CIPQDYTYNQEAGFSKVLDFTR-LGKT---CYSLDLSKATDRLPLALQERVMGIILGD---

TaMV (241) KVRVFAMVDCITQWFLHPLHKYLFSVLR--TTKEDATFDQEKGINLVRLALSKKLDK--SVFSFDLSAATDRLPMDIQMVILNSLTPFWLAEKAVRHGVK OMV-1b (262) KVRVFAMADCITQWVLHPLHQYLFSILKQ-ISIVDATFDQEEGVKTLS-AKIKAGKR--VVFSLDLSAATDRLPLTIQAQILNHIVP---

OMV-1a (256) KVRIFAMVDAVTQWLLKPLHEAIFKLLN--IFAFDGTFDQIGQLEKFMDRMKDKAP---YYYSFDLSAATDRLPLSIQILILKHLVS---

BMV-1 (295) KVRVFAMVDIWTQSILNPLHKKIFSIIR--ELPTDGTFDQLKPLDRLHELSTQ---DRFSFDLSAATDRLPLTLQKDILTLLVS---

OMV-3b (295) KVRVFAMVDIWTQSILNPLHKKIFSIIR--ELPTDGTFDQLKPLDRLHELSTQ---DRFSFDLSAATDRLPLTLQKDILTLLVS---

ShMV (273) KARVFAMADSITQSVMAPLNSWVFSKLK--GLPMDGTFNQQAPLNRLVQLYKEGLLHDVEFYSYDLSSATDRLPMAFQKQIISVLFG---

OMV-3a-OnuLd (272) KARVFAMADSITQSVMAPLNSWVFSKLK--DLPMDGTFNQQAPLNRLVQLYQDGLLHDVEFYSYDLSSATDRLPMAFQKQIISVLFG---

RVM2 (347) KIRLFALMDSITQSVMSPLHDYMFAILR--NIPNDGTFDQEASIARSQEKAVTAGK----AFSYDLTAATDRLPVILTAFILSTIVG---I CcMV2a (95) KARIVASTNSWIQCSLFGLHNKIFSILR--SIPQDGTFDQNKPFDLLLESL-QPGYM---LYGFDLSAATDRLPIAFQKDILNHLG---

CMV-1-cpNB631 (339) KARIVAITNSWIQTAFYSLHLHVFKLLK--NIDQDGTFDQERPFKLLIKWLNEPTQK---FYGFDLTAATDRLPIDLQVDILNIIFK---

OMV-5-OnuLd (253) KMRVIAMVDYHSQFVLKKIHNSLFNKLK--LIKSDRTFTQDPIFTT----PTMGHR----FWSMDLSAATDRFPIDLQERLLSYLYGS---

SsMV1/KL-1 (211) KMRPIAMVDYYSQLVLKPIHDGILKKLR--TLPCDRTFTQDPFNNWG---KTMGHK----FWSLDLTSATDRFPISLQERVIAHLLGD---

OMV-4-OnuLd (238) KERVIAMVDYTTQFALRPIHNILLNNLS--KLPCDRTFTQDPFHKWN---DDHKER----YHSLDLSAATDRFPIFLQQKLISLIFND---

TMV-1 (205) KRRIIAMVDYHSQLVLRSIHDGLLNKLR--NLPQDRTYNQDPNNAW----EENKEC----FHSLDLSSATDRFPVKLQSRLLTEMYSD---

GMV-S1 (243) KVRIVAMLDYTTQLFLRPIHNDLFKLLK--KLPQDRTFTQNPLNDW----EDNEHS----FWSIDLTAATDRFPISLQRRLLLYIYSD---

GMV-S2 (243) KVRIVAMLDYTTQLFLRPIHNDLFKLLK--KLPQDRTFTQNPLNDW----ENNEHS----FWSIDLTAATDRFPISLQRRLLLYIYSD---

OMV-6-OnuLd (250) KERVIAIFDYGSQMVLKPIADVLFDLLR--NIPSDRTFTQSPFFTHTD--LDNKSK----FWSIDLSSATDRFPIVFQKRVLQKILG---

CcMV1a (295) KSRPFAIFDYWSQTVLSPLHDWAYATLR--SIPQDCTFNQAEGLSKVT--ARPSQKY---FYSYDLEAATDRFPIQFQKKVLSLIFN---

SsMV2/KL-1 (246) KSRPFAIVDYITQSALTPLHDRLYRVLG--SIPQDCTFDQNKGFKDLL--YGGGP---YYSFDLTSATDRFPIFVQEMVLAWLTS---

HMV-1 (266) KSRIIGEMNFWAQCALKPLHDKEMKALR--SIRQDLTFYQGIGPQVLK--LHPGSK----YYSFDLKSATDRFPVELQEKVIQAFYG---

BcDRV (295) KVRVFAMVDIWTQSILNPLHKKIFSIIR--ELPTDGTFDQLKPLDRLHELSTQ---DRFSFDLSAATDRLPLTLQKDILTLLVS---

CcMV2c (95) KARIVASTNSWIQCSLFGLHNKIFSILR--SIPQDGTFDQNKPFDLLLESL-QPGYM---LYGFDLSAATDRLPIAFQKDILNHLG---

CcMV2b (95) KARIVASTNSWIQCSLFGLHNKIFSILR--SIPQDGTFDQNKPFDLLLESL-QPGYM---LYGFDLSAATDRLPIAFQKDILNHLG---

ScNV-20S (300) KVRVVSAMETHELVLGHAARRRLFKGLRRERRLRDTLKGDFEATTKAFVG---CAGTVISSDMKSASDLIPLSVASAIVDGLEAS---GRLLP-

ScNV-23S (331) KARIVTTHSASRVTFGHQFRRYLLQGIRRHPALVDVIGGDHRRAVETMDGDFGLLRPDGRLLSADLTSASDRIPHDLVKAILRGIFSDP---DRRPPG

RMV-1-RS002 (393) DRFALLWQKVLTFRDFSLG---NGHS---VRYAVGQPMGAKSSFPMLGLTHHIIVQIAA-LRVGF---STMFKDYVILGDDIMIANEKVATQYRR

TeMV (373) SELARLWSSLLSSREFVTQ---TGHK---VRYEVGQPQGFKSSFHSLAMTHHVIVRLAA-LKAG---EMNFTDYVILGDDIVLTNEKVVKDYMI

TaMV (337) GGLGDAWADLLVDRDYYLPR--WSGYARDS-KVRYAVGQPMGALSSWAMLALTHHMIVQFAAASVGVT---G-WFKEYMVLGDDIVIYNSEVAKAYST

OMV-1b (345) -KLGDHWANLLVNRDYSVPN--HTTLPVNPGTVRYGAGQPMGAYSSWAMLALTHHFIVQYCAFKVYHT---NSFFKDYLILGDDLLLLDAKVAKQYLQ

OMV-1a (338) VPFSEAWGRLLTGRTYHLFY--KK----TSYEVQYQVGQPMGALSSWGMLALTHHLVVQYSAYLAFGK---VNLFKDYIVLGDDIVIGNHEVSIRYHY

BMV-1 (374) PSFAEAWGTALVGRPYKWKF--GT----TEDELMYSVGQPMGALSSWGMLALTHHTIVQVAA-SRAGY---KDLFLDYALLGDDICIANKAVADNYLL

OMV-3b (374) PSFAEAWGTALVGRPYKLKF--GT----TEDELMYSVGQPMGALSSWGMLALTHHTIVQVAA-SRAGY---KDLFLDYALLGDDICIANKAVADNYLL

ShMV (358) SDFADDWATLLVGRDWYLK---DIPYRYSVGQPMGALSSWAMLALSHHVIVQIAA-MRVG---KLSFTNYALLGDDIVIADKAVATSYHM

OMV-3a-OnuLd (357) SKFAKDWATLLVGRDWYLK---DIPYRYSVGQPMGALSSWAMLALSHHVIVQIAA-MRVG---KLPFTNYALLGDDIVIADKAVATSYHM

RVM2 (429) RTFGGLWRSILVKRPFGFNSNVAEKLKVSDGPYFYEVGQPMGALSSWPGLALTHHWIVQVAAFRVTNS---KSWNTEYEILGDDIVIFNELIAQEYLN

CcMV2a (176) -YPGGPWRRLLG-IKYNSP---CGF---ISYAVGQPMGAYSSFAMLALTHHVLVQVAA-QKAGF---SDRFTDYCILGDDIVIANSLVAEAYKS

CMV-1-cpNB631 (421) NSPGSSWRSLLR-IKYKSP---QGF---LTYAVGQPMGAYSSFAMLALTHHVIVQVAA-LNSGF---TTRFTDYCILGDDIVIAHDTVASEYLK

OMV-5-OnuLd (331) EISS-AWKQLLIDRTYKTP---EGDE---LHYKVGQPMGAYSSWAAFTLTHHLVVFYSA-RMAG---IKDFTNYILLGDDIVINNDKVAKYYIR

SsMV1/KL-1 (290) ESKARAWRNILVDRDYKLP---SGGW---TRYSVGQPMGAYSSWTTFTLTHHLVVHYAA-RLCG---IVDFDRYILLGDDIVINHDKVARRYIS

OMV-4-OnuLd (317) YEFGKNWRNLLVDRNYDY----QGIS---YRYSVGQPMGAYTSWAAFTLTHHLVVHWAA-ELAG---LKNFKDYIILGDDIVIKNNKVAQIYIN

TMV-1 (283) PSFGENWMNLLLNRDYLLPE--EGLSG---ERLRYAVGQPMGAYSSWAAFTLSHHLVVAWCT-YKSKK---VIRSSQYIILGDDIVIKDNDIARKYIG

GMV-S1 (321) PEIANSWQNLLVHREYAR----NGLN---PIKYSVGQPMGAYSSWPAFTLSHHLVVHWCA-HLCN---INKFKDYIILGDDIVIHNDNIAKKYIE

GMV-S2 (321) PEIANSWQNLLVHREYAR----NGLN---PIKYSVGQPMGAYSSWPAFTLSHHLVVHWCA-HLCH---INKFKDYIILGDDIVIHNDKVAKKYIE

OMV-6-OnuLd (329) KQMTDSWERIMIGSKFLAP---DGDT---VSYNCGQPMGAQSSWPMFTLAHHVIVRVAA-NRCG---LSNFDKYIILGDDIVINNDNVALKYME

CcMV1a (375) TTYAQAWAEIMTQEPFRVKG--LSDP---LRWGAGQPLGAKSSWAIFTLCHHLVVHIAA-VRTN---SD-PYYVILGDDIVLRGSRLATVYKR

SsMV2/KL-1 (324) EQYASAWVQAMVGIPFSTPN--GPE---VEFKCGQPLGAKSSWAMFTLSHHFVVQYCA-MVLN---IDNPRYKILGDDIVICDHALAAKYLE

HMV-1 (345) EDFARSWRSLITDQAFAY-G--ESE---VRYGCGQPIGAYSSWATFTLCHHMIVQMLC-HRYR---APRSHYIILGDDIVIAHDKVAEGYCE

BcDRV (374) PSFAEAWGTALVGRPYKWKF--GT----TEDELMYSVGQPMGALSSWGMLALTHHTIVQVAA-SRAGY---KDLFLDYALLGDDICIANKAVADNYLL

CcMV2c (175) -YPGGPWRRLLG-IKYNSP---CGF---ISYAVGQPMGAYSSFAMLALTHHVLVQVAA-QKAGF---SDRFTDYCILGDDIVIANSLVAEAYKS

CcMV2b (175) -YPGGPWRRLLG-IKYNSP---CGF---ISYAVGQPMGAYSSFAMLALTHHVLVQVAA-QKAGF---SDRFTDYCILGDDIVIANSLVAEAYKS

ScNV-20S (387) VEIAG-LRACTGPQHLVYPD--GSE---ITTRRGILMGLPTTWAILNLMHLWCWDSADRQYRLEGHPFRATVRSDCRVCGDDLIGVGPDSLLRSYD

ScNV-23S (426) TSLADVFDLVLGPYHLHYPD--GSE---VTVRQGILMGLPTTWPLLCLIHLFWVELSDWAPARPNHSRGFVLGESFRICGDDLIAWWRPERIALYN

RMV-1-RS002 (478) IMES-LGLAISQHKSIISTNS-TTSAQIAEICRR

TeMV (457) IMEL-LGVKISLEKSLFHKDY-SS---MAEFCKR

TaMV (428) LMGT-LGVGISDTKSLTSKIG---VFEFAKR

OMV-1b (437) VMNQ-LDVGVNLAKSLISVRG---YGEFAKQ

OMV-1a (427) LMTEILKVKINPTKGIMSPH---SLEFAKR

BMV-1 (462) IMRD-LGVEINLSKSLISSTG---VVEFAKR

OnuMV3b (462) IMHD-LGVEINLSKSLISSTG---VVEFAKR

ShMV (441) IMTQILGVEINLSKSLVSSN---SFELAKR

OMV-3a-OnuLd (440) IMTQILGVEINLSKSLVSNN---SFEFAKR

RVM2 (524) IMAV-IGCEINLNKSISSRCR---PVFEFAKR

CcMV2a (257) LIFD-LGLEISESKSVISG---TFTEFAKK

CMV-1-cpNB631 (504) LMET-LGLSISSGKSVISS---EFTEFAKK

OMV-5-OnuLd (414) TMKR-LGVELSMNKTHVSK---NTYEFAKR

SsMV1/KL-1 (374) IMNK-LGVDISVAKTHVSK---NTYEFAKR

OMV-4-OnuLd (400) LMTK-WGVDISLSKTHVSY---DTYEFAKR

TMV-1 (372) QMSK-LGVAISMQKTHVSK---DTYEFAKR

GMV-S1 (405) IMGK-LGVGLSNSKTHVSK---DTYEFAKR

GMV-S2 (405) IMGK-LGVGLSDSKTHVSK---DTYEFAKR

OMV-6-OnuLd (413) IMND-FKVEISRNKTHVSN---DTYEFAKR

CcMV1a (458) IMSE-LGVSISETKSHVSK---DTFEFAKM

SsMV2/KL-1 (407) VMSQ-LGVEISSVKTHVSE---NLFEFAKR

HMV-1 (427) IMRA-LSVDISDLKTHVSK---DSYEIAKR

BcDRV (462) IMRD-LGVEINLSKSLISSTG---VVEFAKR

CcMV2c (257) LIFD-LGLEISESKSVISG---TFTEFAKK

CcMV2b (257) LIFD-LGLEISESKSVISG---TFTEFAKK

ScNV-20S (477) RNLGLVGMILSPGKHFRSNRRGVFLERLLEFQTR

ScNV-23S (517) QIAVDCGAQFSAGKHLESKTWGIFTEKVFTVKPV

Motif I Motif II

Motif III Motif IV

Motif V Motif VI

Figure 4.18 Multiple alignments of amino acid sequences from the RNA-dependent RNA polymerase-like domains within the putative polypeptides encoded by RMV-1-RS002 and members of the genera Mitovirus and Narnavirus. ClustalW was used to align amino acid sequences. Conserved amino acid residues within alignments are indicated by black shading with white lettering. The numbers of amino acid residues for corresponding sequences are given in brackets on the left side of their respective sequences. The positions of motifs are indicated by roman numerals above corresponding sequence positions (I-VI).

169 Comparison of the aa sequences of the RdRp domains from RMV-1-RS002 and other

Mitovirus members (Table 4.5) showed that the RMV-1-RS002 domain shared greatest

sequence identity (49%) with that from Tuber excavatum mitovirus (TeMV) (Appendix 4.7). When the full length putative polypeptide of RMV-1-RS002 was compared with the same regions of other mitoviruses, it also showed greatest sequence identity (23%) with TeMV (Appendix 4.8).

A phylogenetic tree was inferred from the conserved aa sequence motifs of the

putative RdRp region from RMV-1-RS002 and other members of the genus Mitovirus, using

the Maximum Likelihood method (Fig. 4.19). All Mitovirus members formed a distinct clade,

which was separated from a second clade consisting of two Narnavirus species in the family

Narnaviridae. As expected, the RdRp region of RMV-1-RS002 grouped (with bootstrap

support of 81%) with the corresponding region of TeMV.

To examine the potential of the RMV-1-RS002 genome to form secondary structures at both its ends, the sequences of the partial 3´ and 5´ UTR of the positive strand were folded using the MFOLD software. This showed that the partial 5´ UTR sequence of RMV-1- RS002 had potential to form at least three stem-loop structures with a ΔG value of -35.10 kcal/mol, amongst which the long stem-loop structure had a ΔG value of -14.40 kcal/mol (Fig. 4.20A). Furthermore, a stable stem-loop structure with a ΔG value of -14.30 kcal/mol was also predicted within the partial 3´ UTR of RMV-1-RS002 (Fig. 4.20B).

170

Table 4.5 Mitoviruses and narnaviruses used in this study, with respective acronyms and details of their genomes and Genbank accession numbers. Accepted (ICTV) virus names are in italics

Virus Name Acronym Host

Genome length 5´ UTR length (nt) 3´ UTR length (nt) AU content (%) Polypeptide length (aa)