Inactivation of Expression of Gene 4 of Mouse Hepatitis Virus Strain JHM Does Not Affect Virulence in the Murine CNS
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Virology 289, 230–238 (2001) doi:10.1006/viro.2001.1167, available online at http://www.idealibrary.com on View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Elsevier - Publisher Connector Inactivation of Expression of Gene 4 of Mouse Hepatitis Virus Strain JHM Does Not Affect Virulence in the Murine CNS Evelena Ontiveros,* Lili Kuo,† Paul S. Masters,† and Stanley Perlman*,‡,1 *Interdisciplinary Program in Immunology, University of Iowa, Iowa City, Iowa 52242; †Wadsworth Center for Laboratories and Research, New York State Department of Health, Albany, New York 12201; and ‡Departments of Pediatrics and Microbiology, University of Iowa, Iowa City, Iowa 52242 Received July 27, 2001; returned to author for revision August 27, 2001; accepted August 31, 2001 The protein encoded by ORF 4 of mouse hepatitis virus (MHV) is not required for growth of some strains in tissue culture cells, but its role in pathogenesis in the murine host has not been defined previously in a controlled manner. MHV strain JHM causes acute and chronic neurological diseases in susceptible strains of rodents. To genetically manipulate the structural proteins of this and other strains of MHV, we have generalized an interspecies-targeted RNA recombination selection that was originally developed for the A59 strain of MHV. Using this approach, a recombinant MHV–JHM was constructed in which gene 4 was genetically inactivated. Virus lacking gene 4 expression replicated in tissue culture cells with similar kinetics to recombinant virus in which gene 4 expression was not disrupted. Both types of viruses exhibited similar virulence when analyzed in a murine model of encephalitis. These results establish a targeted recombination system for inserting mutations into MHV–JHM. Furthermore, the protein encoded by ORF 4 is not essential for growth in tissue culture cells or in the CNS of the infected host. © 2001 Academic Press INTRODUCTION of the genome and encodes the RNA replicase. All down- stream ORFs are expressed by synthesis of a set of The genome of mouse hepatitis virus (MHV), a coro- nested subgenomic mRNAs, each containing a common navirus, is a 31-kb positive-strand RNA molecule. The leader sequence and 3Ј terminus (Lai and Cavanagh, large size of the genome has until recently made its 1997). Homology between the leader sequence and a genetic manipulation difficult. The development of target region upstream of each ORF [the intergenic sequence recombination has facilitated site-directed mutagenesis (IGS)] is involved in mRNA formation, although the mech- of the structural genes of MHV, which reside in the 3Ј-most 9 kb of the genome (Koetzner et al., 1992; Kuo et anism of this process is not well established (Mizutani et al., 2000; van Marle et al., 1999). In general, only the al., 2000; Masters, 1999; Phillips et al., 1999). More re- Ј cently, infectious cDNA clones have been developed for 5 -most ORF in each mRNA is translated. At least three of other coronaviruses (Almazan et al., 2000; Thiel et al., the downstream ORFs of the MHV genome, ORF 2a, ORF 2001; Yount et al., 2000) and it is likely that one will 4, and ORF 5a, are nonessential for growth in tissue become available for MHV as well. In targeted recombi- culture, as demonstrated by analysis of natural variants nation, a donor RNA transcript containing the 3Ј end of of various strains (Schwarz et al., 1990; Yokomori and Lai, the genome is introduced into cells infected with a re- 1991). cipient virus. Recombination between the donor RNA A previous study showed that transcription of gene 4 and the recipient virus results in recombinants that can was not required for growth in the mouse (Yokomori and be selected based on either the loss of temperature Lai, 1991). This conclusion was based on an analysis of sensitivity or the host cell specificity. This method has MHV strain S, a strain of MHV that causes hepatitis in been used most widely to create recombinant viruses of mice. MHV-S was shown not to express mRNA 4, most MHV strain A59 (Fischer et al., 1997; Kuo et al., 2000; likely due to a point mutation in the IGS (AAUUUAAAC, Masters, 1999; Phillips et al., 1999) and transmissible instead of the canonical AAUCUAAAC). In addition, ORF gastroenteritis virus (Sanchez et al., 1999). 4 of another strain, MHV-A59, contains a single nucleo- The first gene of MHV occupies the 5Ј-most two-thirds tide deletion that results in premature termination after amino acid 19. A second potential ORF is present down- stream of this frameshift, but only the upstream 19 amino acid peptide was detected after translation in a cell-free 1 To whom correspondence and reprint requests should be ad- dressed at Department of Pediatrics, University of Iowa, Medical Lab- system (Weiss et al., 1993). oratories 2042, Iowa City, IA 52242. Fax: 319-335-8991. E-mail: Stanley- MHV–JHM is a neurotropic strain that causes both [email protected]. acute viral encephalitis and chronic demyelination. In 0042-6822/01 $35.00 Copyright © 2001 by Academic Press 230 All rights of reproduction in any form reserved. MOUSE HEPATITIS VIRUS STRAIN JHM DOES NOT AFFECT CNS 231 contrast to MHV-A59 and MHV-S, ORF 4 of MHV–JHM JHM recipient was not tested, since this portion of the encodes a protein of 139 amino acids, rich in threonines genome was to be exchanged back to MHV–JHM in and containing a highly hydrophobic amino-terminal re- subsequent mutant construction. One recombinant, gion (Skinner and Siddell, 1985). This protein has been fMHV-JHM B3b, was chosen for further work since its HE detected in infected cells, although its function is un- gene contained the largest 5Ј extent of the MHV–JHM HE known (Ebner et al., 1988). The presence of a full-length gene, crossing over into MHV-A59 sequence in a region ORF 4 protein in MHV–JHM-infected cells raises the of identity between the two strains between 159 and 149 possibility that this protein contributes to the neurotro- nt upstream of the HE gene stop codon. pism exhibited by the JHM strain. Since an infectious cDNA clone for MHV–JHM is not yet available, we de- Construction of MHV–JHM gene 4 KO and control veloped a system to introduce mutations into this virus recombinants by targeted recombination. This method was used to mutate gene 4, and the resulting virus was analyzed for To generate mutants of MHV-JHM, a vector for the growth in tissue culture cells and in the murine CNS. synthesis of donor RNA for targeted recombination was constructed. This vector was created by the stepwise RESULTS replacement in plasmid pMH54 of MHV-A59 genes 3–7 Construction of a general recipient virus for site- (the S gene through the N gene) with their MHV–JHM directed mutagenesis of MHV–JHM counterparts (Fig. 2). In the resulting construct, pJHM, the fragment of the HE gene of MHV-A59 was retained be- The original development of targeted RNA recombina- cause it is minimally divergent from that of MHV–JHM tion as a means to manipulate the coronavirus genome and because it is not expressed in either MHV-A59 or in was enabled by the discovery of a thermolabile N gene our laboratory strain of MHV–JHM. Likewise, the 3Ј-UTR mutant of MHV-A59, Alb4, which could be counter- of MHV-A59 was retained, since it differs from that of selected to obtain recombinants (Koetzner et al., 1992; MHV–JHM at only a single nucleotide. To inactivate ex- Masters, 1999). Since similar counterselectable markers pression of gene 4, three sets of mutations were intro- have not been identified in other coronaviruses, the ap- duced into pJHM to produce pJHM-gene4KO (Fig. 2). plication of targeted recombination has largely been lim- First, the IGS upstream of gene 4 was made identical to ited to MHV-A59. More recently, however, it was shown the one present in MHV-S, the natural variant that does that the stringent species specificity of MHV-A59 could not transcribe gene 4 (Yokomori and Lai, 1991). Second, be switched from murine to feline cells by replacement of a premature stop codon was engineered in place of the ectodomain of the MHV S protein with that of the amino acid 18. Finally, a downstream AUG at codon 23 feline coronavirus FIPV (Kuo et al., 2000). This created an was mutated to CGA. A termination codon is present in interspecies chimera, designated fMHV, from which, in MHV-A59 at approximately the same location in ORF 4 turn, new MHV mutants could be generated by restora- and may result in usage of the downstream AUG. Al- tion of the MHV S protein ectodomain and selection for though usage of this AUG has never been demonstrated, the reacquisition of the ability to grow in murine cells. In this mutation removed it from our construct. This last principal, a similar procedure should allow the construc- change also introduced a SalI restriction site, useful for tion of mutants of any strain of MHV. To apply this method to the JHM strain of MHV, we screening purposes. used the same donor RNA, from vector pFM1, that had Targeted recombination was then used to generate been used to create fMHV (strain A59) (Kuo et al., 2000). gene 4 KO recombinants (RJHM.gene4KO) and wild-type MHV–JHM-infected mouse cells were transfected with MHV–JHM controls (RJHM.wt) (Fig. 1B). Donor RNA was pFM1 RNA and plated onto feline cell monolayers. Re- transcribed from pJHM or pJHM-gene4KO and was trans- sulting recombinant viruses were plaque-purified in fe- fected into FCWF cells that had been infected with line cells, and six recombinants, representing three in- fMHV–JHM B3b.