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The Reovirus Ml Gene, Encoding a Viral Core Protein, Is Associated with the Myocarditic Phenotype of a Reovirus Variant BARBARA Sherrylt* and BERNARD N

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The Reovirus Ml Gene, Encoding a Viral Core Protein, Is Associated with the Myocarditic Phenotype of a Reovirus Variant BARBARA Sherrylt* and BERNARD N JOURNAL OF VIROLOGY, Nov. 1989, p. 4850-4856 Vol. 63, No. 11 0022-538X/89/114850-07$02.00/0 Copyright C) 1989, American Society for Microbiology The Reovirus Ml Gene, Encoding a Viral Core Protein, Is Associated with the Myocarditic Phenotype of a Reovirus Variant BARBARA SHERRYlt* AND BERNARD N. FIELDS12'3 Department of Microbiology and Molecular Genetics, Harvard Medical School,' Department of Medicine (Infectious Disease), Brigham & Women's Hospital,2 and the Shipley Institute of Medicine,3 Boston, Massachusetts 02115 Received 16 May 1989/Accepted 31 July 1989 Reoviruses contain a genome composed of 10 double-stranded RNA gene segments. A reovirus reassortant, 8B, derived from type 1 Lang (TlL) and type 3 Dearing (T3D), displayed a phenotype unlike that of either of its parents in that it efficiently induced numerous macroscopic external cardiac lesions in neonatal mice (B. Sherry, F. J. Schoen, E. Wenske, and B. N. Fields, J. Virol. 63:4840-4849, 1989). A panel of TlL/T3D reassortants and a panel of reassortants derived from 8B were used to determine whether novel TlL/T3D gene associations in 8B were responsible for its myocarditic phenotype. The results eliminated the possibility that any TlL/T3D gene combination found in 8B, from 2 genes to all 10 genes, was the explanation for its induction of cardiac lesions. This suggested that a mutation(s) in an 8B gene(s) might be responsible for induction of the myocarditis. Statistical analysis of experiments with 31 reassortants derived from 8B revealed a highly significant association (P = 0.002) of the 8B Ml gene with induction of cardiac lesions. The reovirus Ml gene encodes a viral core protein of unknown function, although evidence suggests a potential role in core structure and/or viral RNA synthesis. This represents the first report of the association of a viral gene with induction of myocarditis. Reoviruses, members of the family Reoviridae, have a responsible (44). Coxsackieviruses, members of the family genome composed of 10 double-stranded RNA gene seg- Picornaviridae, have been implicated most frequently (3). ments (for a review, see reference 39). The segmented nature Coxsackieviruses which are myocarditic or nonmyocarditic of the genome has been used to great advantage in the past in the mouse have been studied for a number of years (for a in the production of reassortant viruses which contain dif- review, see reference 15), and although there has been an ferent combinations of gene segments derived from different extensive investigation into the role of the host immune parental viruses (for a review, see reference 26). When two system in the generation of the disease, there has been little parent viruses display distinguishable phenotypes, the prog- insight into what viral factors are responsible for the myo- eny reassortant viruses can be used to determine which gene carditic phenotype. segment(s) cosegregates with the phenotype of interest. The myocarditic reovirus variant 8B offers the opportunity Reovirus reassortants have been previously used to iden- to use genetics to identify the viral components important in tify the genes associated with phenotypes in vitro, for the generation of the disease. We present here the first report example, inhibition of host DNA, RNA, and protein synthe- of the association of a viral gene with induction of myocardi- sis (34, 35). Reassortants have also been used to identify tis. genes associated with aspects of viral pathogenesis in the neonatal mouse, such as growth at a primary site of entry in MATERIALS AND METHODS the animal (1), mode of spread through the animal (40), tissue Virus stocks and cells. Mouse L929 (L) cells were propa- tropism in the central nervous system (42), and viral shed- gated as Spinner cultures the ding from the animal (14). Our overall goal is to combine in Joklik modification of Eagle the minimal essential medium (MEM) supplemented with 2 mM in vitro results which reveal gene function with the in vivo results to better understand the molecular basis for viral glutamine, 1 U of penicillin per ml, 1 ,ug of streptomycin pathogenesis. (Irvine Scientific, Santa Ana, Calif.) per ml, and either 5% fetal calf serum (Hyclone Laboratories, Logan, Reoviruses cause a variety of diseases in the neonatal Utah) or 2.5% fetal calf serum and 2.5% viable serum mouse (for a review, see reference 39). The work protein described (agamma VSP; Biocell, Carson, Calif.) (completed here stems from the fortuitous isolation of a reovirus variant MEM). All viruses, including reassortants, were triply plaque which was derived as follows. Type 1 Lang (T1L) and type puri- fied and then passaged twice (to increase virus stock on 3 Dearing (T3D) were coinjected perorally into neonatal titer) mouse L cells. The second-passage (P2) virus stocks were mice, and a reassortant virus, called 8B, was isolated from clarified by low-speed centrifugation before titration and the brain on day 13 postinjection (43). This reassortant was efficiently myocarditic when reintroduced into neonatal use. Virus stocks were stored at 4°C. All gene segment mice, in contrast to the parental strains TlL and T3D, which derivations were confirmed by polyacrylamide gel electro- were nonmyocarditic (36). phoresis of viral double-stranded RNA (see below). TlL and T3D were from standard laboratory stocks (24). Myocarditis is an important human disease, and viruses Generation of virus reassortants. T1L/T3D have been suspected for many years to be the primary agent reassortants were generated previously (4, 8), using a protocol similar to the one described below. For the EW and DB series of * Corresponding author. reassortants, viruses were mixed in a 1:1 or 4:1 ratio and t Present address: College of Veterinary Medicine, North Caro- used to inoculate L-cell monolayers (24-well tissue culture lina State University, Raleigh, NC 27606. dishes; Costar, Cambridge, Mass.) at a total multiplicity of 4850 VOL. 63, 1989 GENETIC BASIS FOR REOVIRUS MYOCARDITIS 4851 infection of 10 PFU per cell. After 1 h of infection at 37°C, % MICE WITH the monolayers were overlaid with 1 ml of completed MEM GENE SEGMENT (8B OR WILD TYPE) EXTERNAL l Ka LI La CARDIAC and incubated for 22 to 24 h at 37°C. Virus was released from VIRUS fl£1 2 1i3. L3 LESIONS cells by disruption with a microtip probe at 50% maximal 8B * * * * * * * * * 0 100% setting (W225R Cell Disruptor; Heat Systems-Ultrasonics WT O 0 0 0 0 0 0 0 0 0 0% Inc., Plainview, N.Y.). The virus suspensions were then EW2 9 * O * * 0 0 0 0 0 0% 0 0 0 0 0% diluted in gel saline (137 mM NaCl, 0.2 mM CaCl2, 0.8 mM EW4 3 * * 0 0 0 EW27 O 0 0 0 0 * 0 0 0 0% MgCl2, 19 mM H3BO3, 0.1 mM Na2B407, 0.3% [wt/vol] DB93A O 0 0 0 0 * 0 0 0 0 0% gelatin [Fisher, Medford, Mass.]) and plated in plaque as- DB93B O 0 0 *0 *0 * 0 0 0% 0 0 0 0 0 0 0 0% Virus was recovered from DB188 O * * says as described previously (41). EW50 O 0 0 0 0 0 0 0 0 0% plaques in neutral red-stained monolayers. A Pasteur pipette DB95 * 0 0 0 * 0 0 0 0% 0% was used to transfer the cell debris and overlying agar to a EW116 O * O * 0 0 0 * 0 2-dram (-7-ml) vial containing either 1 ml of completed EW4 6 O 0 0 * 0 0 0 * 0 0% EW10 O 0 0 * S 0 * 0 0 0% MEM (DB series of reassortants) or an L-cell monolayer in EW26 O O 0 0 0 0 0 0 0 0% 1 ml of MEM series of Sam- EW102 * 00 0 0 * 0 0 0% completed (EW reassortants). EW38 * * 0 0 0 0 0 0% ples of the plaque suspensions of the DB series of reassor- DB7 6 O * 0 0 0 0 0 0 0 0 0% tants were used to infect L-cell monolayers in 2-dram vials, EW4 0 O 0 0 0 * 0 0 0 0 8% and these vials as well as those containing the EW series of EW47 * 0 0 0 0 0 0 0 0 12% reassortants were incubated at 37°C until there was exten- DB62 O 0 0 0 0 0 0 * 0 0 18% sive cytopathic effect, resulting in amplified plaque suspen- DB68 O 0 0 0 0 0 0 0 0 0 18% EW9o * 0 0 0 0 * 0 0 0 19% sions. EW67 * 6 * 0 0 0 0 0 0 33% Samples of plaque were used to EW9 3 * * * 0 * 0 0 0 * 43% amplified suspensions 0 0 0 0 0 0 44% infect L-cell monolayers in six-well tissue culture dishes, EW100 * * * EW2 5 * 0 * * 0 0 0 * 0 53% overlaid with 3 ml of completed MEM, and incubated at DB69A O 0 0 0 * 0 0 0 * 0 56% 37°C. These infected monolayers were used to isolate viral EW96 O * 0 0 0 0 0 0 0 80% EW112 * 0 0 0 0 0 0 0 0 82% double-stranded RNA and to determine the gene segment EW89 * 0 0 0 * 0 0 0 0 82% derivations as described below. Suspensions containing re- DB181 O 0 0 0 0 0 0 * 0 * 89% EW60 O 0 0 * 0 0 * 0 0 94% assortant viruses were identified, and the original plaque 0 0 0 0 0 0 0 100% suspension (DB series of reassortants) or the amplified DB88 O * * plaque suspension (EW series of reassortants) was used as FIG. 1. Frequency of macroscopic external cardiac lesions.
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