【病毒外文文獻(xiàn)】1996 The region between the M and S genes of porcine haemagglutinating encephalomyelitis virus is highly similar to huma

Ou fftOf TP Peptide UGUUGUGAUGAUUAUACUGGA ACCAGGAGUUUGUAAUCAAAACUUCACAUGACGAUUAAGUUCGUCUUUGUUUuACUGACUCUUGACCAUAUAGUAACU C A G g G HEV NT9 HEV VW572 HEV 67N 12 8 kDa UUAAGCAUUUGCCAAAGUUUUUAAGGCUACGCC CUAUUAAUGGACAUCUGGUG CCCUGAAAAGAAAUAUCUCCGUUAUACUAACGGAUUUAACGUCUCAG C U C U U HEV NT9 HEV VW572 HEV 67N AACUAGAAGAUGUCUGUUUUAAAUAUAACUAUCAAUUU CCAAAGUAGGAUAUUGUAGAGUUCCUAAUUAUGCUUGGUGCCGUAACCAAGGUAGUL u1 UG U U i00 I00 88 200 200 188 300 300 288 HEV NT9 HEV VW572 HEV 67N HEV NT9 HEV VW572 HEV 67N HEV NT9 HEV VW572 HEV 67N UGCUACAUUUACUuUGUACGGCAAAUCCAAACAUUAUGAUAAAUAUUUUGGAAUAAUAACUGGuuUCA AGCAUUCUCUAAUUCUUuAGAAGAAGCUGuU A U G 9 6 kDa AAUAAACUGGUUUuUUUAGCUGUUGACUU AuUACCUGGCGAAGCCAGAGUUUAAAUGUUUAUGGCUGAUGCuUAUCUUGCAGACACUGUGUGGUAUGUG U G U GGGCAAAU UL u UUAUAG GC UUUGuUuA GGWAU UAG GUAGUGGCAULrt JuGGC CuUL AAAWGUGUAWC CU y GCGGUAUGU C U 400 400 388 500 500 488 600 600 588 HEV NT9 HEV VW572 HEV 67N GUAAUA CUUAGUACUGUCCCCUUCUAUUUAUGUGUUUAAUAGAGGUAGGCAGUUUUAUGAGUUUUACAAUGAGGUAAAACCACCAGUCCUUGAUGuGGA U g M HEV NT9 UGACGUUUAGUUAAUC CAAACAUUAUGAGUAGUC CAACUACAC CAGUAC CA HEV VW572 HEY 67N Fi 8 1 Comparison of the nucleotide sequences between the M and S genes of HEV NT9 VW572 and 67N The sequence of HEV NT9 is written in full Positions having identical nucleotides are marked with dots positions of deleted nucleotides are marked with dashes Consensus intergenic sequences are underlined Asterisks indicate stop codons 700 700 688 751 751 739 of the structural and ns proteins of BCV and HCV OC43 revealed more than 91 identity Cox et al 1989 Kamahora et al 1989 K6nkel Labont6 et aI 1995 Lapps et al 1987 Mounir Zhang et a 1991a b 1992 In contrast no sequence data for HEV except for a small portion of the S gene have been published Vieler et al 1995 This short nucleotide sequence as well as hybridization studies using BCV S gene specific probes indicated a close genomic relationship to BCV and HCV OC43 Since the only major genomic difference between BCV and HCV OC43 identified so far was found in the region between the M and S genes it was of interest to study this genomic region in the closely related HEV We report here the sequence between the S and M genes of three HEV strains Only two ORFs with the potential to encode proteins of 12 8 and 9 6 kDa were identified in the HEV genome a situation similar to HCV OC43 and different from BCV The origin and cultivation of HEV strains 67N VW572 and NT9 BCV L9 and HCV OC43 as well as the HRT 18 cell line have been described previously Vieler eta 1995 Total RNA for RT PCR was isolated from virus infected cells by phenol chloroform extraction followed by ethanol precipitation Single stranded cDNA was synthesized at 47 5 C using SuperScript Plus RNase H reverse transcriptase with primer 6341 5 AACGTCATCCACATCAAGAAC 3 correspond ing to nucleotides 6321 6341 of the BCV 9 5 kDa sM protein EMBL accession number M30612 cDNA for preparation of the N gene probe used for Northern blot hybridization was synthesized basically under the same conditions but using primer 3 N 5 CTCCTGGTAAGC MATCCAGT 3 The digoxigenin labelled probe DIG dUTP Boehringer Mann heim corresponding to nucleotides 827 1376 of the BCV N gene EMBL M16620 was prepared by PCR from BCV L9 cDNA with primers 3 N and 5 N 5 TAGTAACCCT GAGGGAGTAC 3 For sequencing of the region between the M and S genes DNA was prepared by PCR using primers 6341 and 5327 5 ATGTGGTGGTTGTTGTGATGA 3 44 a HEV NT9 HEV VW572 HEV 67N HCV OC43 BCV Mebus MDIWCPEKKYLRYTNGFNVSELEDVCFKYNYQF VGYCRVPNYAWCRNQGSFCATFTLYGKSKHYDKYFGIITGFTAF N LEEAvNKLVFLAVDFITWRSQSLNVYGX 109 Q V V 109 109 R I A F SH R V N A TV D R E 109 R I A F K SH L V A TV R E 109 b HEV NT9 HEV VW572 HEV 67N HCV OC43 BCV Mebus MFMADAYLADTVWYvGQIIFIVAICLLVIIVVVAFLATFKLCIQLCGMCNTLVLSPSIYVFNRGRQFYEFYNE KPPVLDVDDVx 84 FG 84 D 84 T D 84 F G 84 c HEV NT9 HEV VW572 HEV 67N HCV OC43 BCV Mebus MT I KFVFVLLTLDH I VTLS I CQS FX 24 T D D X 24 N GF X 20 T DS HX ii T D AP D LHPFNHVKLIIRPIEVEHIIIATTMPAVX 43 Fig 2 Alignment of the predicted amino acid sequences of the 12 8 kDa ORF product o the 9 6 kDa ORF product b and the 24 and 20 aa peptide c of HEV strains NT9 VW572 and 67N compared to the corresponding sequences in HCV 0C43 Mounir Pall Prehybridi zation and hybridization were done at 50 C using the DIG Luminescent Detection kit for nucleic acids Boehringer Mannheim as recommended by the manufacturer After hybridization blots were washed twice for 5 min in 2 x SSC and 0 1 SDS at room temperature and then twice for 15 min at 75 C in 0 5 x SSC and 0 1 SDS The hybridization signals were immunologically detected according to the manu facturer s protocol As shown in Fig 1 the region between the S and M genes of HEV 67N NT9 and VW572 contained two ORFs potentially encoding proteins of 12 8 and 9 6 kDa The predicted amino acid aa sequences of these ORFs are shown in Fig 2 Additionally an ORF coding for a 24 aa HEV NT9 and VW572 and 20 aa 67N protein was observed upstream of the 12 8 kDa ORF beginning at nucleotide nt 50 The 12 8 kDa ORF nt 140 469 in NT9 and VW572 128 457 in 67N potentially encodes a protein of I09 aa with a molecular mass of 12 82 NT9 and VW572 or I2 77 kDa 67N Nudeotide and amino acid sequence alignments with the corresponding sequences in BCV Mebus Abraham et al I990 and HCV OC43 Mounir Fig 1 The 9 6 kDa ORF nt 450 710 in HEV NT9 and VW572 444 698 in 67N is predicted to encode an 84 aa protein with a molecular mass of 9 55 kDa in NT9 9 56 kDa in VW572 and 9 58 kDa in 67N Similarities with the corresponding regions and their products in BCV and HCV OC43 are more than 96 9 at the nucleotide and more than 95 2 at the amino acid level The UCCAAAC consensus intergenic sequence begins 129 nt upstream of the first potential start codon which is in the context UAAAUGU These features correspond to those reported for BCV and HCV OC43 Abraham et aI 1990 Mounir DNASTAR software package revealed a closer relationship between BCV and HCV OC43 than between either of them and the three HEV strains The only exception was the 9 6 kDa sM protein of HEV 67N which was more closely related to HCV OC43 than to all other coronaviruses tested The 24 and 20 aa encoding ORFs nt 50 121 and 50 109 respectively encode predicted peptides with molecular masses of 2 76 HEV NT9 2 75 VW572 and 2 30 kDa 67N Since only 645 nt of the HEV 2 gene were sequenced Vieler et aI 1995 the search for the consensus intergenic sequence of these peptides was restricted to this fragment However at the position where the consensus intergenic sequence UCAAAC nt 3760 3765 Mounir nt 1 6 Abraham et al 1990 the sequence CCAAUC nt 394 399 Vieler et al 1995 was found for HEV 67N No other potential consensus sequence was found within the HEV 67N 2 gene fragment Alignments of the first 10 aa of the predicted peptides revealed a high similarity with the 11 codon ORF in HCV OC43 and with the 3 end of the ORF encoding the 4 9 kDa protein in BCV Mebus Fig 2 Using Northern blot analysis Fig 3 HEV 67N and BCV L9 mRNAs 7 6 5 1 and 5 encoding the N M sM and 12 8 kDa ns proteins showed similar size mRNA 3 2 1 and 2 encoding the S HE and NS2 proteins migrated more slowly in BCV L9 than the corresponding mRNA species in HEV 67N The subgenomic RNA potentially encoding the 4 9 and 4 8 kDa ns proteins in BCV mRNA 4 was visible as a minor band but was absent in HEV 67N RNA Similar results were obtained for HEV NT9 and VW572 data not shown Mounir Hirano et aL 1993 Yagami et aL 1993 indicating that other mechanisms than the absence of these genes are involved in tissue tropism References Abraham S Kienzle T E Lapps W E Accepted 23 February 1996 44