* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
Download Complete nucleotide sequence of Colorado tick fever virus
Survey
Document related concepts
Human cytomegalovirus wikipedia , lookup
Hepatitis C wikipedia , lookup
2015–16 Zika virus epidemic wikipedia , lookup
Middle East respiratory syndrome wikipedia , lookup
Ebola virus disease wikipedia , lookup
West Nile fever wikipedia , lookup
Hepatitis B wikipedia , lookup
Marburg virus disease wikipedia , lookup
Influenza A virus wikipedia , lookup
Herpes simplex virus wikipedia , lookup
Transcript
Journal of General Virology (1997), 78, 2895–2899. Printed in Great Britain . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .SHORT . COMMUNICATION Complete nucleotide sequence of Colorado tick fever virus segments M6, S1 and S2 Houssam Attoui, Philippe De Micco and Xavier de Lamballerie Laboratoire de Virologie Mole! culaire, Tropicale et Transfusionnelle, Faculte! de Me! decine de Marseille, Universite! de la Me! diterrane! e, 27 Boulevard Jean Moulin, 13385 Marseille Cedex 5, France The nucleotide sequences of the tenth (M6), eleventh (S1) and twelfth (S2) dsRNA genomic segments of the Florio strain (N-7180) of Colorado tick fever virus were determined and found to be 675, 998 and 1884 bp, respectively, in length. A nonanucleotide motif and a hexanucleotide motif were found to be highly conserved in the 5« and 3« non-coding regions (NCRs), respectively, of the three segments. The first and last three nucleotides of each segment were of inverted complementarity, and segment-specific inverted terminal repeats were detected in the NCRs of the three segments. These findings suggest the occurrence of intracellular panhandle structures for the RNA transcripts. A readthrough phenomenon is suspected in segment M6. The environment surrounding the opal codon (position 1052–1054) of segment M6 conforms to that of leaky opal codons described in the literature. Viruses classified as members of the family Reoviridae have been isolated from a wide range of hosts including vertebrates, invertebrates and plants (Joklik, 1983). All of them exhibit a multisegmented double-stranded (ds) RNA genomic pattern. Phylogenetic studies based on sequence analysis of RNA polymerases suggest that these viruses have a polyphyletic origin (Koonin, 1992). They are classified into nine genera (Murphy et al., 1995) namely Aquareovirus, Coltivirus, Cypovirus, Fijivirus, Orbivirus, Orthoreovirus, Oryzavirus, Phytoreovirus and Rotavirus. Genus Coltivirus, the type species of which is Colorado tick fever (CTF) virus, encompasses those viruses isolated from humans, animals and insects exhibiting a 12 dsRNA segmented genomic pattern. CTF virus was characterized by L. Florio and co-workers 50 years ago as being the Author for correspondence : Xavier de Lamballerie. Fax 33 4 91 18 95 98. e-mail virophdm!lac.gulliver.fr The GenBank accession numbers of the sequences reported in this paper are : U53227, U72694 and AF000720. aetiological agent of a severe tick-borne disease, CTF, which is usually self-limiting and appears following the bite of the infected adult Rocky Mountain wood tick, Dermacentor andersoni (Florio et al., 1946, 1950). Antigenic variants of CTF virus have been reported (Karabatsos et al., 1987). Other members of the genus include Eyach virus, two Eyach antigenic variants (Ar}T577 and Ar}T578), and other probable members isolated in Indonesia and China (Murphy et al., 1995). The 12 dsRNA segments of CTF virus have been arranged into three classes, the L1–4 (long) class, the M1–6 (medium) class and the S1–2 (short) class. The total genome comprises approximately 28 000 nucleotides (Brown et al., 1993). In this paper we report the sequence and genetic organization of the M6, S1 and S2 genomic segments of CTF virus. The Florio strain (N-7180) of CTF virus was purchased from the ATCC. BHK-21 cells were grown in suspension in SEagle’s minimum essential medium (S-EMEM) with 10 % foetal bovine serum (FBS), 100 IU}ml penicillin G, 100 µg}ml kanamycin and 100 µg}ml streptomycin at 37 °C under 5 % CO . Virus infectivity was titrated as described previously # (Deig & Watkins, 1964) and recorded as p.f.u.}ml. Cells adjusted to 10' cells}ml were infected with CTF virus (10 p.f.u. per cell) and incubated until gross cytopathic effect was detected (5 days p.i.). Infected cells were allowed to settle overnight at 4 °C, centrifuged at 800 g and treated with Freon 113 as described elsewhere (McCrae, 1985). The virus was purified on a discontinuous sucrose gradient consisting of onethird 66 % (w}w) sucrose and two-thirds 40 % (w}v) sucrose in 0±2 M Tris–HCl pH 7±8 (Mertens et al., 1987) and centrifuged using an SW40 rotor at 27 000 r.p.m. at 4 °C for 2 h. The viral band was recovered, diluted in 0±2 M Tris–HCl buffer pH 7±8, recentrifuged at 100 000 g to remove the sucrose, and resuspended in EMEM. DsRNA was extracted from infected cells and purified virus using a guanidinium thiocyanate-derived procedure (RNA NOW, Biogentex). Extracted RNA from infected cells was precipitated overnight in 2 M LiCl at 4 °C and centrifuged at 18 000 g for 5 min in order to precipitate high molecular mass RNA transcripts (Ramig et al., 1977). The supernatant was precipitated at ®20 ° for 2 h with 1±5 vol. isopropanol and 0±5 vol. 7±5 M ammonium acetate, washed with 70 % ethanol 0001-4944 # 1997 SGM CIJF Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Sun, 18 Jun 2017 07:19:49 H. Attoui, P. De Micco and X. de Lamballerie (a) (b) (c) Fig. 1. Northern blot hybridization of CTF virus genome using DIG-labelled PCR probes derived from segments M6, S1 and S2 and PCR amplification of full-length segments. (a) Electrophoretic separation of CTF dsRNAs (V) on a 1±5 % agarose gel, stained with ethidium bromide. Size markers (M) are indicated in nucleotides. (b) Chemiluminescence detection of hybridization products after the RNA in (a) was transferred to a nylon membrane. Lanes M and V show DIG-labelled molecular mass marker (labelled in bp ; prepared from pBR328 plasmid cleaved with BglIpBR328 cleaved with HinfI), and complete CTF virus genome, respectively. Hybridization signals were detected only with M6, S1 and S2 segments. (c) Amplification of CTF virus dsRNA segment S2, S1 and M6 templates using specific primer sets S2COMPS/S2COMPA, S1COMPS/S1COMPA and M6COMPS/M6COMPA. Lane M shows size marker. Lanes 1, 2 and 3, respectively, show full-length segments S2, S1 and M6. and vacuum dried. The RNA was further purified by treatment with RNase-free DNase I, (Boehringer Mannheim). DsRNA segments were fractionated by electrophoresis on a 10 % acrylamide slab gel (Laemmli, 1970). RNA bands were excised and recovered from the acrylamide by shaking overnight in 2 ml TNE buffer. The RNA was precipitated by adding 10 vols butanol and recovered using an RNaid kit (BIO 101). The cloning process of viral segments was based on the single primer amplification method (Lambden et al., 1992) with some modifications. The 3« ddATP blocked primer A1 (PO -5« % CCACGTGCCAGATGCTCTGGA-ddA 3«) (2±5 mM) was ligated to both 3« ends of viral dsRNA (150 ng) using 10 U of T4 RNA ligase (Boehringer Mannheim). Ligation efficiency was monitored by chemiluminescent detection of ligated 3«DIG-11-ddUTP blocked oligonucleotides (data not shown). The ligation product was gel purified and cDNA was synthesized using RNase H− reverse transcriptase. dsRNA was denatured at 99 °C for 1 min in 15 % dimethyl sulfoxide (DMSO). Reverse transcription was carried out at 42 °C for 1 h in a final volume of 20 µl containing 7 % DMSO, 50 mM Tris–HCl pH 8±3, 75 mM KCl, 3 mM MgCl , 10 mM DTT, # 0±2 mM each dNTP, 200 ng primer A1 tailed segment S2, 0±6 mM primer A2 (PO -5« GGTGCACGGTCTACGAGACCT % 3«) complementary to primer A1, 20 U of RNase inhibitor (Boehringer Mannheim) and 200 U of MMuLV Superscript II (Gibco BRL) ; the reaction mixture was heated to 70 °C for 15 min, and the RNA template was digested with 1 unit of RNase H (Boehringer Mannheim) at 37 °C for 45 min. Full-length cDNA was amplified by PCR using 2±5 U of Taq polymerase (Boehringer Mannheim) and 1 µM primer A2 under standard conditions. PCR products were gel purified and ligated into pGEM-T vector (Promega). According to our results, there was no need either for purification of the cDNA or for annealing at 65 °C for 16 h as previously described CIJG (Lambden et al., 1992) to obtain full-length segments. Moreover, the repair step of the annealed partial cDNA was achieved within the PCR reaction mixture by Taq polymerase. These modifications resulted in a shorter and simpler protocol. The identity of the amplified product was confirmed by hybridization of DIG-dUTP labelled full-length PCR products with complete CTF virus genome by Northern blot analysis (Dyall-Smith et al., 1983). Before transfer to Hybond-N nylon membrane (Amersham), CTF virus genomic segments were fractionated on a 1±5 % (w}v) agarose gel which was further treated with 50 mM NaOH for 45 min, and with 250 mM sodium acetate pH 5±2 for 15 min. Hybridization products were revealed by chemiluminescence using the Boehringer Mannheim DIG detection kit (Fig. 1 a, b). Recombinant plasmids containing full-length PCR products were transfected into E. coli JM109. Both strands of the cloned PCR product were sequenced with pUC}M13 forward and reverse primers using an Amplicycle sequencing kit (Perkin Elmer) and an LKB ALF sequencer (Pharmacia). Analysis of the nucleotide sequences of segments S2, S1 and M6 showed them to be 675, 998 and 1884 bp long, respectively, and sequences were deposited in the GenBank database under accession numbers U53227 (S2 segment), U72694 (S1 segment) and AF000720 (M6 segment). Comparison of our sequences with those available from nucleic acid and protein databanks was done by using the BioSCAN (University of North Carolina, USA) and FASTA (Stanford University, USA) software programs. Sequence analysis of the three segments showed no significant homology with reported Reoviridae sequences at either nucleotide or amino acid levels. This is consistent with previous data which indicated that significant similarities (over 20 %) between sequences from members of the family Reoviridae exist only for genes encoding RNA polymerases (Koonin, 1992). Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Sun, 18 Jun 2017 07:19:49 Colorado tick fever virus sequences (a) (b) (c) Fig. 2. Terminal sequences of (a) CTF virus segment S2, (b) segment S1 and (c) segment M6. The possible secondary structures due to segmentspecific inverted terminal repeats were generated with the help of the MacStan software program. ‘ I ’ or ‘ ® ’ denote conventional base pairing while ‘ : : ’ denotes non-conventional, although possible, base pairing. In all three segments, conserved motifs were identified in the 5« NCR (a nonanucleotide, ACATTTTGT) and in the 3« NCR (a hexanucleotide, TGCAGT). Such conserved motifs can be detected in many viruses possessing multisegmented genomes. Virus- or segment-specific sequences would probably play an important role in transcription, replication and packaging of RNA as well as in virus maturation. It has been suggested that these motifs may act as sorting signals, bringing a single copy of each genomic segment into the viral capsid (Anzola et al., 1987 ; Xu et al., 1989). Moreover, the first three nucleotides in the 5« NCRs (GAC for segments S2 and M6 and CAC for segment S1) of each of the segments were found to be inverted complements of the last three nucleotides in the 3« NCRs (GTC for segments S2 and M6 and GTG for segment S1). A number of segment-specific inverted terminal repeats (ITRs), involving the conserved motifs, were detected. These ITRs could interact by homologous base pairing, thus forming secondary structures. Such possible secondary structures were generated with the help of the MacStan software program (Gast, 1994) and are displayed in Fig. 2. The complementarity of sequences in the 5« and 3« NCRs suggests that each RNA transcript could be held in a circular form either by itself or via protein components (Anzola et al., 1987 ; Theron & Nel, 1997). This finding has been reported previously for viruses such as infectious bursal disease virus (IBDV) (Mundt & Mu$ ller, 1995), wound tumour virus (Anzola et al., 1987) and influenza virus (Hsu et al., 1987), where a panhandle structure was described that might possibly function as a guiding site for the virus-specific RNA-dependent RNA polymerase. The motif CGTTCC was found in the 5« NCR of segment S1, and the motif CGTTCA was found twice with five intervening nucleotides in the 5« NCR of segment M6. These two motifs are partially complementary to the 3« end 18S human ribosomal RNA motif GGAAGG (GenBank accession number M10098, position 1916–1921). The significance of such partial complementarity is unclear, but was previously described for pestiviruses (Deng & Brock, 1993), picornaviruses (Le et al., 1993) and IBDV (Mundt & Mu$ ller, 1995). Analysis of the sequences of the three segments for open reading frames (ORFs) using the DNA Strider 1.1 software Table 1. Primers used in amplification of full-length segments Primer name Sequence Segment Map position Orientation M6COMPS M6COMPA S1COMPS S1COMPA S2COMPS S2COMPA GACATTTTGTGTCACGAACGTT GACTGCAAGGATTCCCTATATG CACATTTTGTCTCTGTGATCCC CACTGCATTTGTATCCAGGCC GACATTTTGTCTCAGAACGATGC GACTGCAATTACCCGTCCCGGGA M6 M6 S1 S1 S2 S2 1–22 1862–1884 1–22 978–998 1–23 653–675 Sense Antisense Sense Antisense Sense Antisense Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Sun, 18 Jun 2017 07:19:49 CIJH H. Attoui, P. De Micco and X. de Lamballerie program (Institut de Recherche Fondamentale, CEA, France) permitted identification of a 558 bp ORF in segment S2 and a 750 bp ORF in segment S1, encoding putative proteins of 185 and 249 amino acids, respectively. In segment M6 the analysis revealed two possible ORFs. The first extends from base 41 to base 1051, ending at the opal TGA stop codon, and encodes a putative protein of 337 amino acids. A second possible ORF (nt 41–1846) could exist if readthrough of the opal codon (nt 1052–1054), which is followed by a cytosine residue, occurs. The corresponding putative protein would consist of 602 amino acids. Similar situations have been reported in retroviruses (Feng et al., 1990) and alphaviruses (Strauss & Strauss, 1994). Such opal codons have been shown to be leaky, allowing a readthrough phenomenon resulting from the incorporation of arginine, cysteine or tryptophan in place of the opal codon (Feng et al., 1990). The suppression of this codon was found to be directly dependent on the presence of the flanking cytosine (Strauss & Strauss, 1994). The significance and the probability of readthrough of this opal codon remain unclear and require further investigation. Analysis of hydropathy profiles using the Kyte–Doolittle algorithm (Kyte & Doolittle, 1982) revealed that the putative proteins are hydrophilic. However, the absence of significant homologies with other viral proteins means that assigning specific functions to the predicted proteins requires further analysis. Three sets of specific primers for PCR amplification of the viral segments were selected from the determined full-length sequences with the help of the PrimerM software program (V. Proutski, University of Oxford, UK). These sets were used to amplify the complete sequence of the segments on which they are located (Table 1). Reverse transcription was performed as described above in the presence of 1 µM of each primer using genomic CTF virus RNA. PCR amplification was carried out using 35 cycles of denaturation at 94 °C for 50 s, annealing at 62 °C for 50 s and extension at 72 °C for 2 min, with a final extension step at 72 °C for 5 min. This generated full-length amplification products. The results are displayed in Fig. 1 (c). The molecular approach used in this study has contributed to our understanding of the CTF virus genomic segments S2, S1 and M6. Together with further sequencing data, these results should reveal the structural and antigenic properties of the virus and permit the elaboration of molecular diagnostic and epidemiological procedures. The authors wish to thank Drs C. Chastel and E. A. Gould who kindly provided viral strains and cell lines, respectively, for the coltivirus studies currently being carried out in our laboratory. This work was funded by a grant from the A. DE. RE. M. References Anzola, J. V., Xu, Z., Asamizu, T. & Nuss, D. L. (1987). Segment-specific inverted repeats found adjacent to conserved terminal sequences in wound tumour virus genome and defective interfering RNAs. Proceedings of the National Academy of Sciences, USA 84, 8301–8305. CIJI Brown, S. E., Gorman, M., Tesh, R. B. & Knudson, D. L. (1993). Coltiviruses isolated from mosquitoes collected in Indonesia. Virology 196, 363–367. Deig, E. F. & Watkins, H. M. S. (1964). Plaque assay procedure for Colorado tick fever virus. Journal of Bacteriology 88, 42–47. Deng, R. & Brock, K. V. (1993). 5« and 3« untranslated regions of pestivirus genome : primary and secondary structure analyses. Nucleic Acids Research 21, 1949–1957. Dyall-Smith, M. L., Azad, A. A. & Holmes, I. H. (1983). Gene mapping of rotavirus double-stranded RNA segments by Northern blot hybridization : application to segments 7, 8 and 9. Journal of Virology 46, 317–320. Feng, Y. X., Copeland, T. D., Oroszlan, S., Rein, A. & Levin, G. (1990). Identification of amino acids inserted during suppression of UAA and UGA termination codons at the gag–pol junction of Moloney murine leukemia virus. Proceedings of the National Academy of Sciences, USA 87, 8860–8863. Florio, L., Stewart, M. & Mugrage, E. R. (1946). The etiology of Colorado tick fever. Journal of Experimental Medicine 83, 1–10. Florio, L., Miller, M. S. & Mugrage, E. R. (1950). Colorado tick fever. Isolation of the virus from Dermacentor andersoni in nature and laboratory study of the transmission of the virus in the tick. Journal of Immunology 64, 257–263. Gast, F. U. (1994). A Macintosh program for the versatile generation of random nucleic acid sequences and their structural analysis. Computer Aplications in the Biosciences 10, 49–51. Hsu, M. T., Parvin, J. D., Gupta, S., Krystal, M. & Palese, P. (1987). Genomic RNA of influenza viruses are held in a circular conformation in virions and in infected cells by a terminal panhandle. Proceedings of the National Academy of Sciences, USA 84, 8140–8144. Joklik, W. K. (1983). The Reoviridae. New York : Plenum Press. Karabatsos, N., Poland, J. D., Emmons, R. W., Mathews, J. H., Calisher, C. H. & Wolff, K. L. (1987). Antigenic variants of Colorado tick fever virus. Journal of General Virology 68, 1463–1469. Koonin, E. V. (1992). Evolution of double-stranded RNA viruses : a case for polyphyletic origin from different groups of positive-stranded RNA viruses. Seminars in Virology 3, 327–339. Kyte, J. & Doolittle, R. F. (1982). A simple method for displaying the hydropathic character of a protein. Journal of Molecular Biology 157, 105–132. Laemmli, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680–685. Lambden, P., Cooke, S. J., Caul, E. O. & Clarke, I. N. (1992). Cloning of noncultivable human rotavirus by single primer amplification. Journal of Virology 66, 1817–1822. Le, S. Y., Chen, J. H., Sonenberg, N. & Mainzel, V. J., Jr (1993). Conserved tertiary structural elements in the 5« nontranslated region of cardiovirus, aphthovirus and hepatitis A virus RNAs. Nucleic Acids Research 21, 2445–2451. McCrae, M. A. (1985). Double-stranded RNA viruses. In Virology : A Practical Approach, pp. 151–168. Edited by B. W. J. Mahy. Oxford & Washington : IRL Press. Mertens, P. P. C., Burroughs, J. N. & Anderson, J. (1987). Purification and properties of virus particles, infectious subviral particles, and cores of bluetongue virus serotypes 1 and 4. Virology 157, 375–386. Mundt, E. & Mu$ ller, H. (1995). Complete nucleotide sequences of 5«and 3«-noncoding regions of both genome segments of different strains of infectious bursal disease virus. Virology 209, 10–18. Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Sun, 18 Jun 2017 07:19:49 Colorado tick fever virus sequences Murphy, F. A., Fauquet, C. M., Bishop, D. H. L., Ghabrial, S. A., Jarvis, A. W., Martelli, G. P., Mayo, M. A. & Summers, M. D. (editors) (1995). Virus Taxonomy. Sixth Report of the International Committee on Taxonomy of Viruses. Vienna & New York : Springer-Verlag. Ramig, R. F., Cross, R. K. & Fields, B. (1977). Genome RNAs and polypeptides of reovirus serotypes 1, 2 and 3. Journal of Virology 22, 726–733. Strauss, J. H. & Strauss, E. G. (1994). The alphaviruses : gene expression, replication, and evolution. Microbiological Reviews 58, 491–562. Theron, J. & Nel, L. H. (1997). Stable protein–RNA interaction involves the terminal domains of bluetongue virus mRNA but not the terminally conserved sequences. Virology 229, 134–142. Xu, Z., Anzola, J. V., Nalin, C. M. & Nuss, D. L. (1989). The 3«-terminal sequence of wound tumor virus transcripts can influence conformational and functional properties associated with the 5« terminus. Virology 170, 511–522. Received 13 May 1997 ; Accepted 1 July 1997 Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Sun, 18 Jun 2017 07:19:49 CIJJ