【病毒外文文献】1982 Coronavirus 229E susceptibility in man-mouse hybrids is located on human chromosome 15

Somatic Cell Genetics Vol 8 No 1 1982 pp 83 94 Coronavirus 229E Susceptibility in Man Mouse Hybrids Is Located on Human Chromosome 15 Alan Y Sakaguchi and Thomas B Shows Department of Human Genetics Roswell Park Memorial Institute New York State Department of Health Buffalo New York 14263 Received 15 July 1981 Final 14 September 1981 Abstract Human coronavirus 229E an enveloped RNA containing virus causes respiratory illness in man and is serologically related to murine coronavirus JHM which causes acute and chronic demyelination in rodents 229E displays a species specific host range restriction whose genetic basis was studied in human mouse hybrids 229E replicated in human WI 38 cells but not in three mouse cell lines tested RAG LM TK and A9 Human coronavirus sensitivity HCVS was expressed as a dominant phenotype in hybrids indicating that mouse cells do not actively suppress 229E replica tion HCVS segregated concordantly with the human chromosome 15 enzyme markers mannose phosphate isomerase MPI and the muscle form of pyruvate kinase PKM2 and analysis of hybrids containing an 15 translocation t X 15 p11 q11 localized HCVS to the qll qter region of chromosome 15 HCVS might code for a specific surface receptor allowing 229E to be absorbed to and received within the host cell INTRODUCTION Several human genes exerting control over the replication of a number of RNA and DNA viruses have been identified through parasexual means using cultured somatic cell hybrids 1 5 The somatic cell genetic approach has been a successful and currently necessary alternative to the use of animal hosts in studying the role of host genes in virus replication One group of human viruses that are amenable to genetic analysis using somatic cell hybrids is the coronaviruses Coronaviruses are enveloped RNA containing viruses causing a diverse group of diseases in man and other animals 6 7 In 83 0098 0366 82 0100 0083503 00 0 9 1982 Plenum Publishing Corporation 84 Sakaguchi and Shows man they are causative agents of a high proportion of respiratory illness and may be involved in diseases of other organs 6 8 9 Relatively little is known about the genetic factors regulating infection caused by coronaviruses in their natural hosts The prototype human coronavirus strain 229E provides a unique opportunity for investigating the genetics of susceptibility to coronavi rus in vitro 229E as do most other coronaviruses displays a narrow species specific host range restriction since it is capable of growing in a limited number of human cell types including cultured cells but not in rodent cells 6 8 This property allows a genetic dissection of host range restriction and cellular susceptibility to 229E using man mouse cell hybrids since the virus permissive state is usually dominant in cell hybrids Because cell hybrids preferentially lose human chromosomes each hybrid in a set of independent cell hybrids possesses a reduced complement of human chromosomes while collectively the whole genome is represented 10 Correlation of susceptibility to infection with the presence of a specific chromosome in hybrids 1 3 5 chromosomally assigns the gene Using this strategy the ability of 229E to form plaques on cell hybrids with different numbers and combinations of human chromosomes derived from several different human and mouse parental cells was determined Our analysis indicates that the presence of the ql 1 qter region of human chromosome 15 is required for susceptibility to this virus in vitro The widespread occurrence of coronaviruses in the animal kingdom suggests that these viruses have evolved and diversified in parallel with the speciation of their respective animal hosts more specifically in parallel with a common host cell product necessary for virus infection Based upon the known properties of 229E we suggest that the product of HCVS might comprise part of a virus receptor mediating entry of the virus into the cell The importance of identifying host genes involved in coronavirus infec tion derives from the observation that in certain animal species coronaviruses display a tropism for tissues of the nervous system For example the murine coronavirus JHM can experimentally cause acute and chronic demyelination in rodents 11 14 and persistent infection of neural derived cells in vitro 15 16 Moreover 229E is serologically related to the neurotropic murine coronavirus JHM 6 8 These virus host cell systems are being studied as possible models for human demyelinating diseases such as multiple sclerosis which might have a viral etiology 17 18 MATERIALS AND METHODS Parental and Hybrid Cells WI 38 lung fibroblasts ATCC CCL 75 were used as representative parental human cells Mouse parental cells with selectable markers were RAG HPRT LM TK LTP HPRT TK a derivative of LM TK and A9 HPRT Cells were grown in Dulbecco s Human Coronavirus Sensitivity on Chromosome 15 85 modified Eagle s medium with 10 fetal calf serum and antibiotics 19 Human fibroblast mouse hybrids used in this study included DUA DUV x A9 DUM DUV x RAG WlL WI 38 x LTP RAS SH 421 x RAG XTR GM 194 x RAG ALR AnLy x RAG and TSL GM 2808 LM TK DUV 28 SH 421 42 GM 194 Mutant Cell Repository Camden New Jersey GM 2808 Mutant Cell Repository and AnLy 19 are skin fibroblasts while WI 38 is derived from fetal lung tissue These six cell strains were originally isolated from unrelated individuals The isolation and propagation of these cell hybrid series in hypoxanthine aminopterin thymidine HAT selection medium has been described 19 21 Virus Assay Human coronavirus 229E NIAID research reference reagent Cat No V 361 001 021 was grown in WI 38 fibroblasts and was assayed by plaque titration 22 Virus pools had titers ranging from 105 to 106 plaque forming units PFU ml Parental human and mouse cells and their derived hybrids were tested for susceptibility by either a cytopathic effect CPE assay or by plaque assay For CPE assay 2 4 x 104 cells in 0 1 ml of medium were seeded in 96 well Microtest plates Falcon The following day triplicate wells were exposed to 0 1 ml of virus dilutions and CPE was scored over the next 7 days of incubation at 33 For plaque assay 1 1 5 x 10 6 cells were seeded into 35 mm plates and used the following day Overlay medium consisted of fortified Eagle s medium 22 containing 2 bovine serum 0 6 agarose Seakem hypoxanthine 0 272 mg ml and thymidine 0 078 mg ml Plaques were visualized after 7 days of incubation of plates at 33 using overlay medium containing 0 033 mg ml neutral red Each dilution of virus was tested on triplicate plates Karyotype Analysis of Hybrids Human metaphase chromosomes were identified in hybrid cells using the trypsin Giemsa banding techniques 19 Enzyme Marker Electrophoresis The chromosome 15 markers mannose phosphate isomerase MPI and pyruvate kinase PKM2 were determined by vertical starch gel electrophoresis as described 23 Gel electrophoresis procedures for enzyme markers assigned to each of the other 21 autosomes and the X chromosome have been described 24 25 RESULTS VirusHost Range Human rodent cell hybrids often express a variety of phenotypes characteristic of the parental cells from which they are derived including susceptibility to viruses exhibiting a narrow host range 1 5 Human coronavirus 229E replicates in a limited number of human cell types but not in mouse cells 9 11 Human WI 38 cells and several mouse cell lines and their derivative human mouse hybrid lines were screened for susceptibility to 229E to determine if human mouse hybrids were susceptible to 229E and whether they could be utilized to study the basis for host range 86 Sakaguchi and Shows PLAQUE TITRATION OF HUMAN CORONAVIRUS 2 29E IN PARENT AND HYBRID CELLS Fig 1 229E was titrated by plaque assay in parental WI 38 and RAG cells and in RAS 14 and RAS 1 two human mouse hybrids WI 38 and RAS 14 supported plaque production whereas RAS 1 and RAG did not Two other mouse parental lines A9 and LM TK also did not support plaque production C control cells not exposed to virus UD cells exposed to the undilute virus pool restriction for this virus If the nonpermissive state in mouse cells was dominant 229E would not be expected to replicate in man mouse hybrid cells regardless of their human chromosome composition A plaque titration of 229E in human WI 38 mouse RAG and two hybrid lines designated RAS Human Coronavirus Sensitivity on Chromosome 15 87 are shown in Fig 1 The upper row of plates are control cells not exposed to virus whereas the next four rows are cells exposed to 10 fold dilutions of 229E beginning with the undiluted virus sample The phenotype of RAS 14 was similar to that of WI 38 whereas RAS 1 resembled RAG cells which yielded no plaques after exposure to the undiluted virus sample approxi mately 105 PFU plate The plaque titers of the virus sample in WI 38 cells and RAS 14 were similar and the number of plaques obtained was propor tional to the virus dose The results indicate that some hybrids can be infected with 229E and they suggest that a human gene retained in some hybrid lines determined susceptibility to 229E 229E Plaque Production in Human Mouse Hybrids Thirty of the 32 hybrids used in this study were tested for susceptibility to 229E by plaque assay The remaining two hybrids did not survive under agar for the 7 days required for the plaque assay and were therefore examined by CPE assay These hybrids were constructed from four different mouse cell lines RAG LM TK LTP and A9 and human parental cells from six unrelated individuals Eighteen of the hybrids supported 229E plaque production and yielded dilution titers similar to those obtained when the same virus sample was titrated in parallel in WI 38 cells Table 1 demonstrates examples of Table 1 229E Plaque Production in Human Mouse Hybrid Lines a No of different Presence of Hybrid human Sensitivity chromosome 15 line chromosomes to 229E and or X 15 b Plaque titer of virus pool PFU ml Hybrid WI 38 DUA 1 3 5 x 104 1 x 105 DUA 5 11 5x 106 2 9x 106 DUA 5 BSAG A 7 0 2 9 10 6 ALR 1 18 0 1 9 10 6 ALR 2 20 4 4 X 10 6 5 5 10 6 RAS M4 18 0 1 9 106 RAS 8 20 3 9 x 106 5 5 x 106 XTR 1 16 0 1 9 x 106 XTR 8 23 4 5 105 4 105 WIL 8 20 3 5 x 105 1 8 x 105 WIL 12 12 0 4 7 x 104 a229E was titrated by plaque assay on hybrid lines and WI 38 cells using 10 fold dilutions of virus The figures represent the titer of a given virus pool when assayed simultaneously in WI 38 and the indicated hybrid line A zero indicates that no plaques were observed in the hybrid line 229E grows to relatively low titers in human cells and it was therefore necessary to use several different virus pools for screening all the hybrids This accounts for the variation in titers observed after plaque assays The human chromosome content of hybrids was determined by enzyme assay and or karyotyping and indicates the presence and absence respectively of virus susceptibility and chromosome 15 or X 15 in the indicated hybrids bDUA hybrids are derived from a human fibroblast containing an X 15 translocation 46 X t X 15 pl 1 ql 1 28 88 Sakaguc andShows 229E sensitive and insensitive hybrids derived from the different sets of parental cells No plaques were observed in RAG LM TK or A9 cells exposed to comparable virus doses These results indicate that sensitivity to 229E was transferred to these different mouse cell lines by fusion to several independently derived human parental cells obtained from the skin or lung Analysis of the replication of viruses with narrow host range has often revealed a diminution in virus output from hybrids formed from permissive and nonpermissive cells For example polyoma virus replicates in mouse cells but not in hamster cells In mouse hamster hybrids late events in the polyoma virus replication cycle are suppressed by increased numbers of hamster chromosomes even when these hybrids contain a diploid number of mouse chromosomes 26 No suppressive effects of the mouse genome on 229E plaque production could be detected in hybrid cells using this qualita tive assay For example the hybrid XTR 8 that contained all of the human chromosomes except the Y was as capable of supporting 229E plaque production as were parental WI 38 cells Table 1 It appears here that sensitivity to 229E is a dominant phenotype in human mouse hybrids One might surmise from the chromosome content of certain of the hybrids listed in Table 1 that sensitivity to 229E varies with small changes in the number of different human chromosomes that each line possesses For example ALR 2 which contained two more human chromosomes than ALR 1 20 vs 18 respectively was sensitive to 229E while the latter hybrid was not A similar result was obtained when RAS 9 sensitive was compared to RAS M4 not sensitive Because 229E grows to relatively low titers in cultured cells it has not been practicable to determine virus outputs from infected hybrids We have observed that plaque size varied in different hybrid clones but could not be correlated in a consistent fashion with the number of human chromosomes retained However 229E infection of a hybrid clone containing one human chromosome DUA 1A Table 3 yielded variegated lysis during plaque assays rather than distinct plaques The results suggest that a single human chromosome determines suscep tibility to 229E in hybrids although they do not rule out the involvement of other human chromosomes in virus replication Thus it is possible that other host cell functions that might be required for virus replication can be provided by the mouse genome in hybrids Segregation of Virus Sensitivity with Human Enzyme Markers For each hybrid line tested a corresponding homogenate was prepared from the same passage of cells used for plaque or CPE assay Each homogenate was analyzed by starch gel electrophoresis for the presence of enzyme markers previously assigned to each of the 22 human autosomes and the X chromo some By comparing the results of such enzyme analyses with the virus assay data it was possible to identify and assign a gene determining sensitivity to Human Coronavirus Sensitivity on Chromosome 15 89 Table 2 Segregation of Human Coronavirus Sensitivity HCVS and Human Enzyme Markers in Human Mouse Hybrid Clones a HCVS expression Chromosome Marker enzymes Concordant Discordant 1 AK2 PEPC 23 9 2 ACP1 IDH1 20 12 3 ACY1 19 11 4 PEPS 18 13 5 HEXB 21 10 6 ME1 20 12 7 GUSB 19 13 8 GSR 16 15 9 AK1 ACO1 16 16 10 GOT1 22 10 11 ACP2 LDHA 18 14 12 LDHB PEPB 21 11 13 ESD 19 13 14 NP 19 13 15 MPI PKM2 31 1 16 APRT 20 11 17 GALK 21 11 18 PEPA 24 10 19 GPI 18 14 20 ADA 23 9 21 SOD1 19 13 22 ACO2 18 7 X G6PD 21 11 Symbols of enzymes their chromosome assignments and gel electrophoresis procedures have been previously described 24 25 ACY1 aminoacylase 1 was determined by bioautography 25 Enzyme marker analysis and virus assays were performed on independent cell hybrid clones of the same passage The figures under the concordant column represent hybrid clones in which HCVS and the enzyme markers were present or absent together Figures under the discordant column represent hybrids in which the expression of HCVS did not correlate with the given enzyme markers The involvement of the Y chromosome in determining HCVS could be eliminated since hybrids were derived from female human parental cells 229E Table 2 compares the segregation of human coronavirus sensitivity to 229E The HCVS phenotype best correlated with the expression in hybrids of the human chromosome 15 markers mannose phosphate isomerase MPI and the muscle form of pyruvate kinase PKM2 HCVS segregated indepen dently of all other human chromosomes The single discordancy in which MPI and PKM2 were present while HCVS was absent is most likely due to chromosome breakage or could arise if only a very small proportion of hybrid cells in the population contained human chromosome 15 Such small discor dancy rates 3 have been observed for other linked genes using these hybrid cells 19 21 Chromosome Analysis of HCVS and HCVS Hybrids Hybrid cells containing translocations involving the human X chromosome have proven to be powerful tools for gene mapping and regional gene assignments 27 The Table 3 Chromosome Distribution of Human Mouse Hybrids Segregating HCVS and MPI and PKM2 a Chromosome Hybrid HCVS PKM2 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 b 16 17 18 19 20 21 22 X X 15 b 15 X b DUA 1 d DUA 1A c DUA 1 BSAG B DUA 3 BSAG A DUA 5 BSAG A DUM 6 DUM 13 RAS 8 RAS 9 DT WIL 6 ALR 2 TSL 2 aVirus assays enzyme analyses and karyotyping of clones were performed on the same cell passage HCVS MPI and PKM2 and human chromosomes were scored for their presence or absence in hybrid cells Human metaphase chromosomes were identified in hybrid cells using the trypsin43iemsa banding techniques bDUA and DUM hybrids are derived from a human fibroblast containing an X 15 chromosome translocation t X 15 pll qll 28 which is retained in HAT medium The normal chromosome 15 and the reciprocal 15 X translocation are retained or lost independently of the X 15 translocation CPlaque assays of DUA 1A yielded variegated lysis of the monolayers dEight percent of the cells contained the 15 X translocation 00 gl O Human Coronavirus Sensitivity on Chromosome 1 91 karyotypes of 12 hybrid lines were analyzed to confirm the results of marker enzyme analyses Table 3 and these data indicated that HCVS segregated with the qll qter portion of chromosome 15 This conclusion was based upon the following observations Two series of hybrids designated DUM and DUA were constructed by fusing a human fibroblast strain containing an X 15 translocation 28 with RAG cells and A9 cells respectively HAT supplemented medium and 8 azaguanine could therefore be employed to select for and against 27 respectively the retention of the X 15 transloca tion Of the 12 hybrids seven were susceptible to 229E and contained an intact chromosome 15 and or X 15 translocation The five hybrid lines in this group that were not susceptible to 229E did not contain an intact chromosome 15 DUA 1 contained human chromosome 7 and the X 15 translocation but not an intact chromosome 15 and was sensitive to 229E Table 3 DUM 6 contained the X 15 translocation but not the intact 15 and was sensitive to 229E Both DUM 6 and DUA 1 also contained the reciprocal 15 X translocation However it can be deduced that susceptibility to 229E segregates with the X 15 translocation DUA 1A and not with the 15 X reciprocal translocation DUA 1 CSAZB The independent secondary clones DUA 3 BSAG A and DUA 5 BSAG A both counterselected in 8 azaguanine supplemented medium contained neither an intact chromo some 15 nor the X 15 translocation and both were not sensitive to 229E The possible involvement of chromosomes X and 7 in determining the HCVS phenotype can be eliminated by the combined enzyme and karyotype data Tables 2 and 3 Thus the results of karyotyping are consonant with the enzyme marker data and support the idea that a gene or genes on the ql 1 qter region of chromosome 15 regulate susceptibility to 229E DISCUSSION Human mouse hybrids exposed to human coronavirus 229E confirmed the prediction that sensitivity to this virus is determined by the presence of a specific human chromosome in cell hybrids Our results demonstrate that a gene or genes in the ql 1 qter region of human chromosome 15 determines susceptibility to 229E in human mouse hybrids The results of plaque assays indicate that HCVS is expressed as a dominant phenotype in hybrid cells and that the virus replicated in hybrids containing human chromosome 15 or X 15 in three different mouse genomic backgrounds RAG LTP and A9 Thus these parental mouse cell lines and likely rodent cell lines in general do not actively restrict 229E replication We conclude accordingly that the human genome contributes products that render human mouse hybrids susceptible to 229E Interspecies hybrids have been used for identifying genes controlling virus host cell interactions at different steps in the virus replication cycle 92