【病毒外文文獻(xiàn)】2006 Human coronavirus 229E encodes a single ORF4 protein between the spike and the envelope genes

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BioMed Central Virology Journal Open Access Research Human coronavirus 229E encodes a single ORF4 protein between the spike and the envelope genes Ronald Dijkman 1 Maarten F Jebbink 1 Berry Wilbrink 2 Krzysztof Pyrc 1 Hans L Zaaijer 3 Philip D Minor 4 Sally Franklin 4 Ben Berkhout 1 Volker Thiel 5 and Lia van der Hoek 1 Address 1 Laboratory of Experimental Virology Department of Medical Microbiology Center for Infection and Immunity Amsterdam CINIMA Academic Medical Center University of Amsterdam The Netherlands 2 Laboratory for Infectious Diseases and Screening National Institute for Public Health and the Environment Bilthoven The Netherlands 3 Laboratory of Clinical Virology Department of Medical Microbiology Center for Infection and Immunity Amsterdam CINIMA Academic Medical Center University of Amsterdam The Netherlands 4 National Institute for Biological Standards and Controls NIBSC Hertfordshire UK and 5 Kantonal Hospital St Gallen Research Department St Gallen Switzerland Email Ronald Dijkman r dijkman amc uva nl Maarten F Jebbink m f jebbink amc uva nl Berry Wilbrink berry wilbrink rivm nl Krzysztof Pyrc k a pyrc Hans L Zaaijer h l zaaijer amc uva nl Philip D Minor pminor nibsc ac uk Sally Franklin pminor nibsc ac uk Ben Berkhout b berkhout amc uva nl Volker Thiel volker thiel kssg ch Lia van der Hoek c m vanderhoek amc uva nl Corresponding author Abstract Background The genome of coronaviruses contains structural and non structural genes including several so called accessory genes All group 1b coronaviruses encode a single accessory protein between the spike and envelope genes except for human coronavirus HCoV 229E The prototype virus has a split gene encoding the putative ORF4a and ORF4b proteins To determine whether primary HCoV 229E isolates exhibit this unusual genome organization we analyzed the ORF4a b region of five current clinical isolates from The Netherlands and three early isolates collected at the Common Cold Unit CCU in Salisbury UK Results All Dutch isolates were identical in the ORF4a b region at amino acid level All CCU isolates are only 98 identical to the Dutch isolates at the nucleotide level but more closely related to the prototype HCoV 229E 98 Remarkably our analyses revealed that the laboratory adapted prototype HCoV 229E has a 2 nucleotide deletion in the ORF4a b region whereas all clinical isolates carry a single ORF 660 nt in size encoding a single protein of 219 amino acids which is a homologue of the ORF3 proteins encoded by HCoV NL63 and PEDV Conclusion Thus the genome organization of the group 1b coronaviruses HCoV NL63 PEDV and HCoV 229E is identical It is possible that extensive culturing of the HCoV 229E laboratory strain resulted in truncation of ORF4 This may indicate that the protein is not essential in cell culture but the highly conserved amino acid sequence of the ORF4 protein among clinical isolates suggests that the protein plays an important role in vivo Published 28 December 2006 Virology Journal 2006 3 106 doi 10 1186 1743 422X 3 106 Received 08 December 2006 Accepted 28 December 2006 This article is available from 2006 Dijkman et al licensee BioMed Central Ltd This is an Open Access article distributed under the terms of the Creative Commons Attribution License http creativecommons org licenses by 2 0 which permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited Page 1 of 8 page number not for citation purposes Virology Journal 2006 3 106 Background Coronaviruses CoVs are enveloped plus strand RNA viruses belonging to the family Coronaviridae 1 The genomic RNA is 27 32 Kb in size capped and polyade nylated The virions are 80 150 nm in diameter and have a unique morphology with extended petal shaped spikes that give the virus a crown like projection Latin corona under the electron microscope 1 CoVs are classified into three groups based on phylogenetic and serological rela tionships Group 1 and 2 consist of different mammalian coronaviruses whereas bird viruses dominate group 3 All coronaviruses employ a common genome organization where the replicase gene encompasses the 5 two thirds of the genome and is comprised of two overlapping open reading frames ORFs ORF1a and ORF1b The structural gene region which covers the 3 third of the genome encodes the canonical set of structural protein genes in the order 5 spike S envelope E membrane M and nucleocapsid N 3 Expression of the replicase gene is mediated by translation of the genomic RNA that gives rise to the biosynthesis of two large polyproteins pp1a encoded by ORF1a and pp1ab encoded by ORF1a and ORF1b using a ribosomal frameshift at the ORF1a 1b junction Expression of the structural gene region is mediated via discontinuous transcription of subgenomic sg mRNAs a hallmark of coronavirus gene expression The number of sg mRNAs produced by a particular coro navirus usually exceeds the number of encoded structural proteins and consequently coronaviruses are able to express additional so called accessory genes formerly called group specific genes These genes are interspersed between the structural genes and their number and loca tion varies within coronavirus genomes The functions of coronavirus accessory proteins are largely unknown how ever reverse genetic analyses of Mouse Hepatits Virus MHV and Feline Infectious Peritonitis Virus FIPV sug gest that they are not required for virus replication 2 4 Moreover deletion of MHV and FIPV accessory genes results in attenuation in their respective hosts indicating that accessory genes represent pathogenicity factors 2 4 The group 1 coronaviruses can be divided into the two genetic subgroups 1a and 1b 5 Members of group 1a include canine coronavirus FIPV transmissible gastroen teritis virus TGEV and ferret enteric coronavirus Group 1b includes porcine epidemic diarrhea virus PEDV human coronavirus NL63 HCoV NL63 and human coronavirus 229E HCoV 229E All members of group 1b encode one or two accessory proteins between the S and E gene ORF3 protein for PEDV and HCoV NL63 and ORF4a and ORF4b proteins for HCoV 229E Figure 1 The numbering of the ORFs in HCoV 229E is based on Northern blot analysis of sg RNAs 6 The presence of an However the location of HCoV 229E ORFs 4a and 4b genes in the genome i e between S and E and sequence similarities to the group 1b ORF3 genes strongly support the notion that they are homologous Unfortunately very little information is currently available about the structure and function of the ORF3 proteins Several studies have linked the ORF3 protein of PEDV and TGEV to viral infec tivity and pathogenicity 7 8 PEDV and TGEV acquire truncated forms of their accessory proteins after exten sively passaging in cell culture and these laboratory adapted strains encoding truncated forms of ORF3 pro teins are less pathogenic than the corresponding wild type strains Figure 1 7 8 HCoV 229E contains two ORFs ORF4a and ORF4b between the S and E genes Figure 1 Since both genes share the same sg mRNA i e sg mRNA4 the expression of gene 4b would require alternative mechanisms of trans lation such as internal entry leaky scanning or transla tional reinitiation of ribosomes However comparison of the hydrophobic domains of both ORF4 parts with the single ORF3 homologs indicates that they encode a simi lar protein 9 suggesting a scenario in which HCoV 229E acquired an out of frame insertion or deletion It should be noted that the origin of full length genomic sequences of the group 1b coronaviruses PEDV and HCoV NL63 are derived from clinical isolates CV777 and Amsterdam 1 Amsterdam 057 and Amsterdam 496 respectively In contrast the HCoV 229E ORF4a b sequence 10 and the HCoV 229E full length genomic sequence 6 has been determined from a cell culture adapted virus more than 30 years after the initial isolation of HCoV 229E by Hamre and Procknow 11 We therefore hypothesized that HCoV 229E ORFs 4a b might actually had been a sin gle ORF that was truncated upon adaptation of HCoV 229E to cell culture Results Analysis of HCoV 229E cell culture adapted virus We first analyzed the ORF 4a b region of VR 740 a cell culture adapted virus that also originates from the initial HCoV 229E clinical isolate and which was deposited at the American Type Culture Collection ATCC in 1973 The ORF4a b sequence of VR 740 showed an overall 99 similarity at the nucleotide level with the published sequence of the cell culture adapted HCoV 229E and we detected only one single amino acid substitution in each protein ORF4a protein aa 94 Y D ORF4b protein aa 36 F L Still like the cell culture adapted HCoV 229E VR 740 encodes two ORFs between the S and E genes Fig ure 2 Analysis of current clinical HCoV 229E isolates Page 2 of 8 page number not for citation purposes additional sg mRNA in HCoV 229E infected cells i e sg mRNA3 shifts the numbering from ORF3 to ORF4a b The apparent identity of the ORF4a b region in both cell culture adapted viruses prompted us to elucidate this Virology Journal 2006 3 106 region in clinical HCoV 229E isolates of which no ORF4a b sequence data is available thus far We therefore studied nose throat swab and nasopharyngeal aspirate materials from 5 patients that were tested positive for HCoV 229E The clinical symptoms of the HCoV 229E infected patients were similar to those that are commonly observed for HCoV 229E infections with symptoms like rhinorrhoea fever and malaise Table 1 12 RT PCR sequencing analysis of the ORF4a b region revealed an overall nucleotide similarity of 97 of the Dutch clinical isolates to that of the cell culture adapted viruses Of most interest is the presence of a 2 nucleotide deletion within the ORF4a gene of the cell culture adapted viruses when aligned to the Dutch isolates Strikingly this Thymidine and Guanosine deletion at position 395 396 is absent in all clinical isolates Deduced protein sequences encoded by ORF4a and ORF4b from the published HCoV 229E sequence the sequence of VR 740 determined in this study and the sequences of the 5 clinical isolates were aligned This reveals that clinical isolates encode a single uninterrupted ORF between the S and E genes Figure 2 with a high similarity of the N and C terminal domains with ORF4a 93 and ORF4b 96 respectively All Dutch sequences have the same amino acid sequence Fig ure 2 but some silent mutations were observed at the nucleotide level excluding the possibility of PCR contam ination Analysis of early HCoV 229E isolates Although our data suggest that the deletion in the labora tory HCoV 229E strain and VR 740 is the result of cell culture adaptation we cannot rule out the possibility that the deletion might represent natural variation between different HCoV 229E isolates Since the VR 740 virus was deposited at the ATCC in 1973 HCoV 229E might have evolved over the years and current clinical isolates may thus differ from the prototype Alternatively the Dutch clinical isolates may represent a subgroup of HCoV 229E and VR 740 like clinical isolates may exist as a second HCoV 229E subgroup To investigate whether the 2 nucleotide deletion is also present in isolates that are more related to VR 740 we searched for other early Schematic overview of group 1b accessory protein genes between the S and E geneFigure 1 Schematic overview of group 1b accessory protein genes between the S and E gene PEDV NC 003436 HCoV NL63 NC 005831 and HCoV 229E NC 002645 the truncated forms of PEDV P40 and P80 are based on previous published data 7 Page 3 of 8 page number not for citation purposes This single accessory gene is 660 nucleotides in length and encodes an ORF4 protein that is 219 amino acids in size HCoV 229E isolates We were able to retrieve three early isolates that were originally collected at the common cold Virology Journal 2006 3 106 unit CCU Salisbury UK Two of these are laboratory adapted viruses of which it is unknown in which extend they were passaged HC LP was initially isolated in 1965 13 and HC Killick in the eighties 14 The third sample CCU T935 was obtained from an infected person that participated trial T935 in 1986 of which unfortunately all data were lost Phylogenetic analysis shows that the sequences of ORF4a b from the three early isolates were indeed more related to the cell culture adapted prototype viruses 98 99 than to the current Dutch isolates 98 Figure 2 and 3 Most importantly however the early isolate sequences of CCU T935 and HC Killick like those derived from the clinical isolates do not contain the 2 nucleotide deletion but carry the uninterrupted ORF between S and E Interestingly HC LP does not have this deletion was observed that is located halfway the ORF4a region Figure 2 Discussion Hypothetically an ORF4b protein could be translated via alternative translation mechanisms as described for some other coronavirus proteins 15 16 but those mecha nisms have not been described for this region nor has any evidence for the expression of HCoV 229E ORF4b protein ever been reported In addition our results show that a large fragment is deleted in the cell culture adapted HC LP virus which corresponds with the ORF4b region of the prototype virus As mentioned previously it has been shown for MHV and FIPV that accessory genes are dispen sable for virus growth in cell culture Moreover the dele Amino acid sequence alignment of ORF4 of laboratory adapted and clinical HCoV 229E strainsFigure 2 Amino acid sequence alignment of ORF4 of laboratory adapted and clinical HCoV 229E strains Denotes identical amino acid to the RIVM 04 037 sequence denotes stopcodon Page 4 of 8 page number not for citation purposes particlar 2 nucleotide deletion instead a larger 118 nt tion of accessory genes resulted in these cases in viruses that are attenuated in vivo Similarly attenuation of in Virology Journal 2006 3 106 vivo viral infectivity and pathogenicity has been linked to ORF3 truncation upon in vitro culturing of other group 1b coronaviruses For a virulent PEDV strain this occurred after 40 passages and more severe truncation and attenu ation was observed after 60 or more passages 7 Similar results have been reported for TGEV after at least 35 pas sages 8 Unfortunately no detailed information is avail able about the in vitro passaging of the cell culture adapted HCoV 229E strains It is tempting to speculate that ORF4 of HCoV 229E like ORF3 of PEDV is vital for efficient in vivo replication The fact that VR 740 con tains a truncated ORF4 may explain why this virus repli cates in vitro in murine cells expressing HCoV 229E receptor human CD13 but not in vivo in the human CD13 transgenic animals 17 18 It is of interest to inves tigate whether an HCoV 229E strain with a more severe truncated or a non truncated ORF4 gene can replicate in these mice Accompanied with the deletions we also observed several non silent nucleotide differences between the cell culture adapted viruses and the clinical isolates In our Dutch iso lates the ORF4 is highly conserved on the protein level The CCU T935 isolate that was collected in 1986 at the CCU Salisbury is a clinical isolate with high ORF4 simi larity to the cell culture adapted viruses Since we cannot reconstruct the experimental setting performed during the clinical trail T935 at the CCU in 1986 we cannot exclude the possibility that the CCU T935 sample was obtained from a volunteer inoculated with an HCoV 229E labora tory strain This strain might even have the same origin as the cell culture adapted viruses 11 In any case the CCU T935 sample is derived from an in vivo infection be it experimental or natural and this further supports a rele vant in vivo function of HCoV 229E full length ORF4 likely represents genetic drift over 20 30 years of evolu tion 19 Molecular clock analysis with the average muta tion rate of coronaviruses 20 21 supports this idea data not shown Given the long time of evolution the differ ences between the CCU T935 and Dutch isolates are remarkably small For HCoV NL63 we also observed a highly conserved ORF3 among different clinical isolates 22 and although for PEDV limited sequence data are available Song et al found only one nucleotide difference in ORF3 between two PEDV field isolates 7 Recently Tang et al reported on novel bat coronaviruses Bt CoVs of which several cluster with group 1b corona viruses They determined the full length genomic sequence of one of these wild type Bt CoVs Bt CoV 512 2005 23 The genome organization of this Bt CoV strain is similar to that of the other group 1b members with the exception of one putative gene at the 3 end of the genome However only one accessory protein encoded by ORF3 is identified between the structural genes S and E The ORF3 protein of Bt CoV 512 2005 is homologous to ORF3 proteins of PEDV HCoV NL63 and the ORF4 protein from our clinical HCoV 229E isolates These data show that all currently sequenced group 1b coronaviruses contain one homologous accessory gene between the S and E genes Conclusion We report the first sequences of the ORF4a b region of clinical HCoV 229E isolates The experimental data strongly support the hypothesis that a separation of a for merly single ORF4 had taken place upon adaptation of HCoV 229E to cell culture We observed two different types of deletions 2 or 118 nucleotides of the ORF4 gene only in cell culture adapted viruses whereas all clinical Table 1 Clinical symptoms of HCoV 229E infected patients Symptoms Patient Age Year of Sampling Acute start Cough Rhinorrhoea Sore Throat Fever Malaise Other Hospitalized Diagnosis RIVM 02 034 52 2002 Yes No Yes No No Yes Muscle joint pain No ARI Pharyngitis RIVM 02 041 54 2002 Yes No Yes No Yes No Hoarse No Common Cold Acute tonsillitis RIVM 03 224 68 2003 Yes No Yes Yes Yes No Respiratory allergy No Common Cold RIVM 04 037 54 2004 Yes Yes Yes Yes Yes Yes No ARI VS03 099 81 2003 Yes Yes U U No Yes Dyspnoea Yes Exacerbation of COPD CCU T935 U 1986 U U U U U U U U U The person was on treatment for an atypical Mycobacterium infection Unfortunately all administration on Trail No T935 is no longer available U Unknown ARI Acute Respiratory infection COPD Chronic Obstructive Pulmonary Disease Page 5 of 8 page number not for citation purposes protein We believe that the divergence between the cur rent Dutch isolates and the early CCU T935 strain most isolates including CCU T935 encoded a single ORF4 gene Both types of nucleotide deletion within the ORF4a Virology Journal 2006 3 106 b region of cell culture adapted HCoV 229E viruses cre ates a frame shift that introduces an early termination codon which either separates ORF4 to ORF4a and ORF4b or results in a truncated ORF4 a fragment HC LP Most likely the two types of deletion occurred independently and are not site specific Therefore the genome organiza tion for the group 1b coronaviruses HCoV NL63 PEDV Bt CoV and HCoV 229E is identical The amino acid sequence of the HCoV 229E ORF4 protein is highly con served among clinical isolates suggesting that the protein plays an important role during in vivo infection Methods Collection of patient material Patient materials were collected at the department of Med ical Microbiology Academic Medical Center AMC The Netherlands VS03 099 and from the Laboratory for Infectious Diseases and Screening National Institute of Public Health and the Environment RIVM Bilthoven The Netherlands RIVM02 034 RIVM02 041 RIVM03 224 and RIVM04 037 Table 1 One sample was col Viral RNA isolation Total viral RNA was isolated either from 200 l cell culture supernatant 100 200 l nose throat swab RIVM or nasopharyngeal aspirate AMC as previously described 24 RT PCR Reverse transcription and PCR reactions were performed as described 22 25 Amplification of the ORF4a b region was performed with the primer combination 5 229E ORF4ab 5 AAC TTC CTT ATT ACG ACG TT 3 and 3 229E ORF4ab 5 ATC CAC TAG CTT AAG GAA CA 3 If required a semi nested PCR was performed with the primers 5 229E ORF4abNested 5 CAT ACA GTA ATG GCT CTA GG 3 an