【病毒外文文獻】1983 Coronaviruses SD and SK share extensive nucleotide homology with murine coronavirus MHV-A59, more than that shared

《【病毒外文文獻】1983 Coronaviruses SD and SK share extensive nucleotide homology with murine coronavirus MHV-A59, more than that shared》由會員分享，可在線閱讀，更多相關(guān)《【病毒外文文獻】1983 Coronaviruses SD and SK share extensive nucleotide homology with murine coronavirus MHV-A59, more than that shared（9頁珍藏版）》請在裝配圖網(wǎng)上搜索。

VIROLOGY 126 669 677 1983 Coronaviruses SD and SK Share Extensive Nucleotide Homology with Murine Coronavirus MHV A59 more than That Shared between Human and Murine Coronaviruses SUSAN R WEISS 1 Department of Microbiology University of Pennsylvania Philadelphia Pennsylvania 19104 Received November 29 1982 accepted January 7 1983 A cDNA probe representing the genome of mouse hepatitis virus MHV strain A59 MHV A59 was used to measure nucleotide sequence homologies among murine and human coronaviruses and the SD and SK coronaviruses isolated by Burks et al Since SD and SK were isolated by inoculation of multiple sclerosis MS central nervous system CNS tissue into mice or cultured mouse cells it is important to determine their rela tionships to other murine and human coronavirus isolates Our results indicate that SD and SK share almost complete nucleotide homology approximately 90 with MHV A59 and generate subgenomic RNAs of the same sizes as MHV A59 The human coronavirus HCV strains tested show less homology with MHV A59 The immunologically unrelated HCV 229E shows no nucleotide homology with MHV A59 The immunologically cross reactive HCV OC43 shows nucleotide homology with MHV A59 by blot hybridization but not when hybridized in solution and assayed by S1 nuclease digestion INTRODUCTION Coronaviruses have been associated with acute and chronic neurological diseases in many species of animals McIntosh 1974 Infection of rodents with the murine co ronavirus mouse hepatitis virus MHV strain JHM has been used as a model sys tem to study virus induced demyelination Weiner 1973 Nagashima et al 1978 Stohlman and Weiner 1981 After initial panencephalitis caused by MHV JHM this virus produces a persistent infection with primary demyelination with some evi dence for remyelination Weiner 1973 Thus persistent MHV JHM infection of rodents has been cited as a model to study the human demyelinating disease multiple sclerosis MS Human coronaviruses HCV are ubi quitious in nature with a large portion of the human population possessing neutral izing antibodies McIntosh 1974 These viruses were isolated usually as respira tory and occasionally as enteric viruses To whom reprint requests should be addressed They are estimated to be responsible for 15 of common colds McIntosh 1974 There are no reports thus far of involve ment of human coronaviruses with per sistent neurological disease Some strains of HCV such as 0C43 are antigenically related to murine coronaviruses such as MHV strain JHM McIntosh 1974 Gerdes et al 1981a b and may be grown in the brains of suckling mice McIntosh et al 1967 Others such as HCV 229E are un related antigenically to MHV or HCV 0C43 McIntosh 1974 Pederson et al 1978 Because 1 murine coronaviruses are associated with chronic demyelinating disease in rodents Weiner 1973 Nagash ima et al 1978 2 antibody against HCV is very common in the human population McIntosh 1974 and 3 there is evidence suggesting that MS may be caused by a virus various workers have undertaken comparisons of human and murine coro naviruses and have started to search for coronaviruses in central nervous system CNS tissue from MS patients There is one report of particles with coronavirus 669 0042 6822 83 3 00 Copyright 9 1983 by Academic Press Inc All rights of reproduction in any form reserved 670 SUSAN R WEISS like morphology seen by electron micros copy in brain tissue from an MS patient Tanaka et al 1976 More recently Burks and co workers have isolated two corona viruses designated SD and SK by intra cerebral inoculation of unfrozen MS CNS autopsy tissue either into weanling mice or into 17CL 1 mouse cells in culture Burks et al 1980 Gerdes et al 1981a b and we this manuscript have examined the re lationship between these viruses and other known murine and human coronaviruses Gerdes et al 1981a b showed that SD and SK are antigenically related to MHV A59 and to HCV OC43 but not HCV 229E They were inconclusive about which strains their isolates were more related to We have further compared murine and human co ronaviruses and SD and SK by using mo lecular hybridization of virus specific RNA with cDNA probes Our results show ex tensive nucleotide homology between SD and SK and MHV A59 more than that be tween the human viruses and MHV A59 MATERIALS AND METHODS Viruses and cells MHV A59 was grown in 17CL 1 cells as previously described Weiss and Leibowitz 1983 SD and SK viruses Burks et al 1980 were obtained from Dr J Gerdes and were also grown in 17CL 1 cells HCV 229E was obtained from the American Type Culture Collec tion ATCC and grown in human embry onic lung L 132 cells also obtained from the ATCC These viruses were plaque pu rifled two times and grown in Dulbecco s medium with 10 fetal calf serum Robb and Bond 1979 HCV OC43 was obtained as a 20 suck ling mouse brain suspension from Dr J Hierholzer at the Center for Disease Con trol CDC Atlanta It was inoculated in tracerebrally into C57BL 6 suckling mice harvested 2 days later and a 10 brain suspension was made in phosphate buff ered saline PBS containing 0 75 bovine serum albumen The mothers of the suck ling mice were obtained from Jackson Labs as MHV free animals All were negative for antibodies against MHV A59 as de termined by an enzyme linked immuno sorbent assay and HCV OC43 as shown by a lack of 0C43 hemagglutination in hibiting activity in the sera of these ani mals Hierholzer et al 1969 and thus were considered uninfected by these co ronaviruses Virus in brain homogenates was assayed by hemagglutination of chicken red blood cells at room tempera ture Hierholzer et al 1969 Virus was further verified as HCV OC43 since hem agglutination was inhibited by an anti 0C43 reference antisera obtained from CDC HCV OC43 was also grown in human rectal tumor HRT cells Laporte et al 1980 obtained from Dr David Brian In fected mouse brain homogenates were ad sorbed onto monolayers of HRT cells for 1 hr at room temperature The cells were extensively washed medium added and the cells incubated at 33 Virus in the su pernatant was titered at various times postinfection by hemagglutination Hier holzer et al 1969 Mock infected cells were adsorbed with a brain homogenate from uninfected suckling mice cDNA probes cDNAs were synthesized using purified genome RNA as template oligomers of calf thymus DNA as primers and reverse transcriptase Taylor et al 1976 cDNAs were labeled with 32P dCTP to specific activity of approximately l0 s cpm g When used for liquid hybridiza tion cDNA was synthesized in the pres ence of actinomycin D and was 95 sin gle stranded Such cDNAs were validated to be highly virus specific and to represent the majority of the genome RNA as pre viously described in detail Weiss and Lei bowitz 1981 1983 RNA extraction MHV A59 SD SK and HCV 229E virus infections were carried out with a multiplicity of infection of be tween 0 1 and 1 plaque forming units per cell RNA was extracted 18 hr after infec tion with A59 SD and SK viruses when massive syncytia were present 229E in fected cells were labeled with 3H uridine in the presence of 10 ttg ml actinomycin D from 18 to 24 hr postinfection when RNA was extracted RNA was extracted from OC43 infected HRT cells at 3 days post HUMAN AND MURINE CORONAVIRUS RNA 671 infection RNA was extracted from the cy toplasm of infected cells by SDS protein ase K treatment followed by phenol ex traction as previously described Weiss and Leibowitz 1983 RNA was extracted from suckling mouse brain homogenates by SDS proteinase K treatment followed by phenol extraction Weiss Varmus and Bishop 1977 RNA analysis 1 Gel electrophoresis RNA was electrophoresed in 1 agarose gels in the presence of methyl mercury hydroxide Bailey and Davidson 1976 or formaldehyde Lehrach et al 1977 as de naturant Gels were either fluorographed with sodium salicylate Chamberlain 1979 or blotted onto nitrocellulose Thomas 1980 2 Blots Dot blots RNA was ad sorbed onto nitrocellulose filters in various amounts as designated in figure legends dried and the filters were baked and hy bridized with cDNA Thomas 1980 Northern blots RNA was electrophoresed in gels blotted onto nitrocellulose filters and hybridized with cDNA Alwine et al 1977 Thomas 1980 3 Hybridization in solution was carried out at 68 0 6 MNaC1 and assayed by 1 nuclease digestion as previously described Leong et aL 1972 In Crt curves increasing amounts of RNA were hybridized with a fixed amount of cDNA to achieve increasing Crt values where Crt concentration of RNA time of hybridization RESULTS 0C43 RNA HCV OC43 has been difficult to grow and assay in cell culture and this has impaired the study of viral nucleic acids This virus is usually grown in the brains of suckling mice and titered either by infection of suckling mice or by hemagglutination Hierholzer et al 1969 Schmidt et al 1979 have reported growing and plaquing HCV OC43 on human rhabdomyosarcoma RD cells Although we had difficulty with growing the virus in RD cells we have had some success with growth in human rectal tumor HRT cells Laporte et al 1980 We have used hemagglutination to detect and quantitate HCV OC43 in both infected suckling mouse brain homogenates and HRT cell supernatants As shown in Table 1 brain homogenates from infected mice contained 40 000 HAU ml of 0C43 and ho mogenates from control mock infected an imals had none This activity could be spe cifically inhibited by anti OC43 reference antisera but not by A59 antisera or preim mune sera data not shown Also shown in Table 1 after infection of HRT cells with OC43 infected mouse brain homogenates 800 hemagglutinating units 106 cells hemagglutinating activity could be measured in the medium As expected pretreatment of brain homogenates with antisera directed against OC43 prevented TABLE 1 TITERS OF HCV OV43 IN SUCKLING MOUSE BRAIN HOMOGENATES AND HRT CELL SUPERNATANTS Homogenate or supernatant HAU ml a OC43 infected suckling mouse brain homogenate 40 000 Mock infected suckling mouse brain homogenate b 0 OC43 infected HRT cells 1 day postinfection 0 2 days postinfection 0 3 days postinfection 80 4 days postinfection 1 280 5 days postinfection 5 120 Mock infected HRT cells b 5 days postinfection 0 HRT cells infected with antibody treated OC43 c 5 days postinfection 0 Hemagglutinating units ml of OC43 One HAU is defined as the amount of virus present in 0 05 ml of the highest dilution of brain homogenate or su pernatant capable of agglutinating 0 05 ml of 0 5 chicken erythrocytes in the standard assay described by Hierholzer et al 1969 b Mock infected mice or cells are mock infected with a 10 homogenate or uninfected suckling mouse brains c OC43 infected suckling mouse brain homogenate was incubated with anti OC43 reference antiserum for 1 hr at room temperature before infecting cells 672 SUSAN R WEISS growth of the virus We have used brain lysates of infected mice and infected HRT cells as sources of HCV OC43 RNA for the experiments described below a b Nucleotide Sequence Homologies among Coronavirus Strains Figure 1 illustrates the use of dot blot hybridizations to detect nucleotide se quence homologies between MHV A59 and various murine and human coronavirus strains including the SD and SK putative MS isolate strains of Burks et al 1980 A complementary DNA cDNA probe representing the majority of sequences of genome RNA of MHV A59 Weiss and Lei bowitz 1981 1983 hybridized to RNA ex tracted from cells infected with MHV JHM HCV OC43 SD SK and from brain homogenates of suckling mice infected with OC43 as well as to its homologous RNA There was no hybridization detected be tween A59 cDNA and RNA from cells in fected with HCV 229E Since there are re ports that the nucleocapsid protein of HCV 229E has some antigenic cross reac tivity with the other viruses Gerdes et al 1981b the reciprocal experiment using HCV 229E cDNA was also carried out As illustrated in Fig 2 cDNA representing the HCV 229E genome hybridized only to its homologous RNA and not to HCV OC43 RNA or to MHV A59 RNA Quantitation of Homology The blot experiments illustrated in the above sections show the homology be tween the nucleic acids of coronavirus strains MHV A59 and SD SK and HCV 0C43 and the lack of homology with HCV 229E These techniques however do not quantitate the percentage of the genome sequences that are homologous To be more quantitative solution hybridization ex periments were carried out and the extent of hybridization was measured by resis tance of hybridized cDNA to S1 nuclease digestion RNA extracted from cells in fected with MHV A59 or SK was hybrid ized to single stranded 32p cDNA repre senting MHV A59 Hybridization was car r d 0 f 9 h i J k I FIG 1 Dot blot hybridization of MHV A59 cDNA with coronavirus RNAs RNA from infected or mock infected cells or mouse brain homogenates was spot ted onto nitrocellulose filters In the case of intra cellular RNAs 5 0 0 5 and 0 05 ttg amounts were used In the case of purified genome RNA 0 1 0 01 and 0 001 g were used Filters were hybridized to 106 cpm 108 cpm g of A59 sUP cDNA and autora diographed Alwine et aL 1977 Thomas 1980 a SD infected 17CL 1 cellular RNA b SK infected 17CL 1 cellular RNA c A59 infected 17CL 1 cellu lar RNA d Uninfected 17CL 1 cellular RNA e A59 infected 17CL 1 cellular RNA f 229E infected L 132 cellular RNA g Uninfected L 132 cellular RNA h OC43 infected suckling mouse brain ho mogenate RNA i Mock infected suckling mouse brain homogenate RNA j Mock infected HRT cel lular RNA k OC43 infected HRT cellular RNA l A59 purified genome RNA Lanes a d e i and j 1 are from separate experiments ried out with varying amounts of RNAs to achieve the Crt values shown in Fig 3 Hy bridization of A59 cDNA to SK RNA was almost as extensive as to its homologous A59 RNA The shift in the SK curve to higher Crt values indicates less virus spe cific RNA in the SK infected cells relative HUMAN AND MURINE CORONAVIRUS RNA 673 to MHV A59 Uninfected cell RNA as ex pected showed no hybridization Similar hybridizations were carried out with RNAs from cells infected with the other viruses and the plateau values for the percentage cDNA hybridized are sum marized in Table 2 SD and SK are almost completely homologous to MHV A59 more so than another MHV strain JHM HCV 229E showed no homology with A59 cDNA as predicted from the blot experiments HCV OC43 showed no homology with the cDNA by this assay This is probably due to stringency of hybridization and S1 nu clease assay for hybridization see Dis cussion b c d Intracellular RNAs To further compare the RNA of the mu rine and human strains the intracellular subgenomic RNAs were examined by gel electrophoresis As illustrated in Fig 4 cells infected with MHV A59 contain six major subgenomic RNAs as well as ge nome sized RNA band 1 Cheley et al 1981a Lai et al 1981 Leibowitz et aL 1981 Spaan et al 1981 These range in molec ular weight from 0 63 106 daltons for RNA 7 to 6 1 106 daltons for RNA 1 Intraeellular RNAs extracted from eells infected with SD or SK were eleetropho resed in parallel with MHV A59 RNA blotted onto nitrocellulose and the virus specific species detected by hybridization with MHV A59 eDNA Seven RNA bands were observed all comigrating with the major MHV A59 RNAs RNAs 2 4 5 and in the ease of SK RNA 3 are less abun dant than with MHV A59 and the extra band between RNAs 2 and 3 is more prom inent The extra bands between RNAs 3 and 4 are also more prominent in the SD RNA The intraeellular RNAs of HCV 229E were also compared to those of MHV In this experiment MHV 3 RNA was used in stead of MHV A59 The genome of MHV 3 is 95 homologous to MHV A59 and MHV 3 generates the same size intraeel lular RNAs as MHV A59 Cheley et al 1981b Weiss and Leibowitz 1981 Since HCV 229E RNA does not cross hybridize FIG 2 Dot blot hybridizations of HCV 229E eDNA with coronavirus RNAs RNA from infected or mock infected cells or brain homogenates or from purified virions was spotted onto a nitrocellulose filter In the case of intracellular RNA 5 0 0 5 and 0 05 g were used and in the case of purified genome RNA 0 1 0 01 and 0 001 ug were used The filter was hybridized with 106 cpm 10 s cpm g of 229E 32P cDNA and auto radiographed Alwine et aL 1977 Thomas 1980 a 229E purified genome RNA b Uninfected L 132 cel lular RNA c 229E infected L 132 cellular RNA d OC43 infected suckling mouse brain homogenate RNA e Mock infected suckling mouse brain ho mogenate RNA f A59 infected 17CL 1 cellular RNA with A59 eDNA 229E intracellular RNA was labeled with 3H uridine in the presence of actinomycin D to inhibit host DNA dependent RNA synthesis As shown in Fig 5 229E also generates six subgenomic RNAs but they are of differ ent sizes from MHV 3 and hence from SD and SK Genome RNA was difficult to ob serve in this experiment probably due to some RNA degradation DISCUSSION Mammalian coronaviruses have been di vided into two antigenic groups Pederson et aL 1978 One includes MHV HCV OC43 674 SUSAN R WEISS I00 9 so r 6o Z 121 40 20 x 9 0 A59 X r s K Uninfected cells i0 I i0 0 i01 i0 10 3 io 4 Crt FIG 3 Kinetics of hybridization of A59 cDNA with cytoplasmic RNA from A59 and SK infected 17CL 1 cells Various amounts 80 g to 8 ng of intracellular RNA were hybridized with 2000 cpm 108 cpm g of A59 32P cDNA to the indicated Crt values Hybridization was assayed by digestion with S1 nuctease O A59 infected cells SK infected cells A Uninfected cells calf diarrhea coronavirus and hemagglu tinating encephomyelitis virus of swine The other includes HCV 229E feline in fectious peritonitis virus transmissible TABLE 2 HOMOLOGY AMONG CORONAVIRUS GENOMES a Source of RNA Percentage hybridization of A59 cDNA A59 purified virions JHM purified virions 229E infected L 132 cells OC43 infected suckling mouse brain homogenate SD infected 17CL 1 cells SK infected 17CL 1 cells 100 74 0 0 90 90 a RNA from virions infected cells or brain ho mogenates was hybridized with 2000 cpm 10 s cpm t g of A59 aZP cDNA to completion the plateau por tion of a Crt curve Hybridization was assayed by 1 nuclease digestion These values have been normal ized to 100 hybridization of A59 cDNA with its homologous A59 RNA The actual values of hybrid ization of A59 cDNA with A59 RNA ranged from 85 to 100 gastroenteritis virus of swine and canine coronavirus Gerdes et al 1981a b have shown that the SD and SK viruses fall into the first group They showed that all A59 intracellular proteins are immunoprecip itable with antisera directed against SD SK or HCV OC43 From these experi ments however it was impossible to de termine whether SD and SK were more related to human 0C43 or murine A59 viruses This is important in determining the origin of SD and SK and the possible link to MS We have used nucleic acid hybridization with cDNA to further explore the rela tionship among these viruses Our cDNAs are representative of most if not all of the genome RNA sequences Weiss and Lei bowitz 1981 1983 and thus are appropri ate reagents for quantitating sequence ho mologies The relationship between SD and SK and MHV A59 as determined by mo lecular hybridization experiments basi cally agrees with immunological studies The SD and SK viruses show extensive ho mology in nucleotide sequence approxi mately 90 to the A59 strain of MHV even when assayed by the stringent 1 nu HUMAN AND MURINE CORONAVIRUS RNA 675 clease assay This is more homology than that shared between MHV A59 and an other MHV strain JHM Lai and Stohl man 1981 Weiss and Leibowitz 1981 1983 Furthermore the pattern of intra cellular RNAs generated by SK and SD is very similar to that of MHV A59 This is not surprising considering that Gerdes et al 1981a b showed that cells infected by SD or SK have patterns of viral proteins similar to A59 infected cells Gerdes et al 1981a b An extra polypeptide of 42 000 daltons molecular weight was observed in A59 SD 1 2 3 U U 6 7 SK 28S 18S a b c FIG 4 Comparison of MHV A59 SD and SK in tracellular RNAs RNAs 10 g lane extracted from 17CL 1 cells infected with MHV A