An encephalomyocarditis virus epizootic in a baboon colony.

Approximately 80 baboon deaths were caused by encephalomyocarditis virus (EMCV) infection in a 3060 member research and production colony. The epizootic extended over a 9-month period and occurred in baboons ranging from 1 day to 22 years of age. Acute death was the most common history. When clinical disease was detected, it was characterized by labored respiration associated with acute congestive heart failure. The salient necropsy findings were pulmonary congestion and edema, hydropericardium, hydrothorax, ascites, lymph node and splenic hypertrophy, and pale white-to-tan mottled hearts. The most significant histologic lesion was nonsuppurative necrotizing myocarditis. Placental infection with fetal loss occurred. Diagnosis was confirmed by light microscopy, transmission electron microscopy, virus culture, and serology. Rarely, EMCV-induced antibody persisted in surviving baboons for more than 24 months. EMCV-infected feral rats were the probable source of the virus and their control stopped the epizootic. No EMCV neutralizing antibody was detected in colony support personnel or chimpanzees.     

Cell-free synthesis of encephalomyocarditis virus

We developed a system for complete replication of encephalomyocarditis virus (EMCV) in a test tube by using an in vitro translation extract from Krebs-2 cells. Efficient virus synthesis occurred in a narrow range of Mg(2+) and EMCV RNA concentrations. Excess input RNA impaired RNA replication and virus production but not translation. This suggests the existence of a negative-feedback mechanism for regulation of RNA replication by the viral plus-strand RNA or proteins.     

Chloroquine enhances replication of Semliki Forest virus and encephalomyocarditis virus in mice.

Chloroquine (CHL) has been suggested to play an important role in the development of Burkitt's lymphoma by enhancing Epstein-Barr virus expression. Herpes zoster virus incidence is markedly increased following malaria infection in children being treated with CHL. Recently, CHL has also been shown to dramatically increase the transactivation of Tat protein purified from human immunodeficiency virus. These previous studies indirectly suggest that CHL may be involved in the enhancement of virus replication. This study demonstrates for the first time that CHL indeed enhances Semliki Forest virus and encephalomyocarditis virus replication in mice. These results raise the possible connection between the increased spread of AIDS in endemic malaria areas and the wide use of CHL in those areas for the chemotherapy of malaria.     

Mutational analysis of the encephalomyocarditis virus primary cleavage.

Sixteen substitution mutations of the conserved DvExNPGP sequence, implicated in cardiovirus and aphthovirus primary polyprotein cleavage, were created in encephalomyocarditis virus cDNA, expressed, and characterized for processing activity. Nearly all the mutations severely decreased the efficiency of the primary cleavage reaction during cell-free synthesis of viral precursors, indicating a stringent requirement for the natural sequence in this processing event. When representative mutations were tested in full-length genomic contexts, they were lethal and no revertants were observed. Not only were the primary cleavage reactions deficient in these polyproteins, but subsequent cleavage of P1 by endogenous or exogenous 3C pro was also impaired. This indicates that primary cleavage has a role in the proper processing of the viral capsid precursor.     

Functional characterization of cleavage-defective mutants of encephalomyocarditis virus.

We characterized seven temperature-sensitive capsid cleavage (cleavage-defective) mutants of encephalomyocarditis virus. Our experimental approach was to monitor in vitro proteolysis reactions of either wild-type or cleavage-defective mutant capsid precursors mixed with cell-free translation products (containing the viral protease) of either wild-type or mutant viral RNA. The cell-free translation reactions and in vitro proteolysis reactions were done at 38 degrees C, because at this temperature cleavage of the capsid precursors was restricted in reactions containing cleavage-defective mutant viral RNA as the message, relative to those reactions containing wild-type viral RNA as the message. Wild-type or cleavage-defective mutant capsid precursors were prepared by adding cycloheximide to cell-free translation reactions primed with wild-type or mutant viral RNA, respectively, 12 min after the initiation of translation. In vitro proteolysis of wild-type capsid precursors with cell-free translation products of either wild-type or cleavage-defective mutant viral RNA led to similar products at 38 degrees C, indicating that the cleavage-defective mutant viral protease was not temperature sensitive. As a corollary to this, at 38 degrees C cleavage-defective mutant capsid precursors were not cleaved as completely as were wild-type capsid precursors by products of cell-free translation of wild-type viral RNA. The results from these in vitro proteolysis experiments indicate that all seven of the cleavage-defective mutants have capsid precursors with a temperature-sensitive configuration.     

Absence of 5'' terminal capping in encephalomyocarditis virus RNA.

The nature of the 5' terminus of encephalomyocarditis (EMC) virion RNA has been investigated. We have failed to detect any capped products or nucleoside polyphosphates arising upon complete digestion of the RNA with T1, T2, and pancreatic ribonucleases, and it would therefore appear that the 5' terminus of EMC virus RNA is not phosphorylated and not capped with m7G. EMC virions do contain, however, large amounts of all four 5'-monosubstituted nucleoside triphosphates (4.2M pppG; 16.4M pppA; 3.OM pppU and 2.5M pppC), of which at least a proportion (about 15-20%) appear to remain bound to fully denatured RNA in the presence of divalent cations.     

Site of attachment of encephalomyocarditis virus on human erythrocytes.

Previous studies of the attachment of encephalomyocarditis (EMC) virus to human erythrocytes concluded that the glycophorins, a family of human erythrocyte sialoglycoproteins, act as EMC virus receptors. Evidence is presented that the major glycophorin species, glycophorin A, is the receptor for EMC virus attachment to human erythrocytes. Comparison of the structures of glycophorins A and B and sialoglycopeptides released by chymotrypsin and trypsin treatment of erythrocytes confirmed our previous suggestion (A. T. H. Burness and I. U. Pardoe, J. Gen. Virol. 64:1137-1148, 1983) that attachment of EMC virus to glycophorin A involves the region containing amino acids 35 to approximately 70 (numbered from the NH2 terminus), four of which (amino acids 37, 44, 47, and 50) are glycosylated. In addition, we provide evidence that the segment containing amino acids 35 to 39 with an oligosaccharide side chain on threonine-37 is particularly important for EMC virus attachment.     

Persistent infection of K562 cells by encephalomyocarditis virus.

Infection of human erythroleukemic K562 cells by encephalomyocarditis virus readily resulted in establishment of persistently infected cultures. In contrast to the usual typical lytic infection by encephalomyocarditis virus, in which trypan blue staining of cells reaches close to 100% by about 15 h postinfection, K562 cell cultures required 3 to 4 days postinfection to reach a maximum of about 80 to 90% cell staining. The proportion of K562 cells taking up stain gradually decreased to about 10% of those present by about 13 days postinfection; during this time, virus yield per day measured by either plaque or hemagglutination titration fell about 10-fold. The decrease in percent staining was followed by waves of increased staining accompanied by increased virus production. Virus-producing cultures were maintained for over 3 months. Evolution of both virus and cells accompanied establishment of persistence in that plaque size changed from about 7 mm in diameter for the original virus to less than 1.5 mm by day 20 postinfection and most of the cells cloned from persistently infected cultures were resistant to superinfection with the original virus. Resistance was due, at least in part, to reduced virus attachment in that binding of 3H-labeled virus to cloned resistant cells was about 2% of that to uninfected cells.     

Effects of cDNA hybridization on translation of encephalomyocarditis virus RNA.

Cell-free translation of the RNA of encephalomyocarditis virus was examined after hybridization of chemically synthesized cDNA fragments to different sites of the 5' noncoding region of the viral RNA. The following results were obtained. The binding of cDNA fragments to the first 41 nucleotides, to the poly(C) tract (between nucleotides 149 and 263), and to the sequence between nucleotides 309 and 338 did not affect translation of the viral RNA; the binding of cDNA fragments to the sequence between nucleotides 420 and 449 caused a slight inhibition; and the binding of fragments to eight different sites between nucleotides 450 and the initiator AUG codon (nucleotide 834) caused high degrees of inhibition. The results suggest that the first part of the 5' untranslated region, at least to nucleotide 338, may not be required for encephalomyocarditis viral RNA translation; however, the region near nucleotide 450 is important for translation of the viral RNA. The possibility that initiation occurs at an internal site is discussed.     

Dissociation of interferon effects on murine leukemia virus and encephalomyocarditis virus replication in mouse cells.

Two subclones of Swiss mouse cells infected with Moloney murine leukemia virus (M-MuLV) were tested for their response to interferon (IFN). Whereas M-MuLV production in the two subclones was inhibited to the same extent, one of the subclones was significantly more sensitive to IFN when the antiviral effect was measured by replication of encephalomyocarditis (EMC) virus. The same subclone was also more sensitive to the anticellular activities of IFN. Additionally, NIH 3T3 cells infected with M-MuLV were completely resistant to IFN actions when EMC virus replication or the anticellular activities were tested. However, under the same conditions, M-MuLV production was completely inhibited by IFN. These results indicate that IFN may affect cell growth functions and EMC replication through mechanisms different from those by which MuLV production is inhibited.     

Evidence for the existence of protomers in the assembly of encephalomyocarditis virus.

Two capsid precursor subunits, which sediment on glycerol gradients at 13S and 14S, respectively, have been identified in cytoplasmic extracts of encephalomyocarditis virus-infected HeLa cells. The 13S subunit, which was detected after a 10-min pulse label with -3H-labeled amino acids, contained only capsid precursor chain A (mol wt 100,000). When the 10-min pulse label in such cells was chased for 20 min, the A-containing 13S subunit in the cytoplasmic extracts was replaced by a 14S subunit containing equimolar proportions of three chains: epsilon, gamma, and alpha. This (epsilon, gamma, alpha)-containing 14S subunit could be dissociated into 6S subunits with the same polypeptide composition. The sedimentation properties and the polypeptide stoichiometry of these three precursor subunits, when compared with those of the 13S, (beta, gamma, alpha)(5), and 5S, (beta, gamma, alpha), subunits derived by acid dissociation of purified virions, suggest the following structural assignments: 13S, (A)(5); 14S, (epsilon, gamma, alpha)(5), 6S, (epsilon, gamma, alpha). The molecular weights of the individually isolated capsid chains were determined by gel filtration in 6 M guanidine hydrochloride to be: epsilon, 36,000; alpha, 32,000; beta, 29,500; gamma, 26,500; and delta, 7,800. With the exception of the delta-chain, these values are in reasonable agreement with the values previously determined by electrophoresis on sodium dodecyl sulfatepolyacrylamide gels. These data support the hypothesis that picornavirus capsids are assembled from identical protomers according to the following scheme: (A) leads to (A)(5) leads to (epsilon, gamma, alpha)(5) leads to (delta, beta, gamma, alpha)60-n(epsilon, gamma, alpha)n where n is the number of immature protomers per virion.     

Size and location of poly (A) in encephalomyocarditis virus RNA.

Encephalomyocarditis (EMC) virus RNA contains a covalently bound sequence of polyriboadenylic acid (poly(A). This was determined by two-dimensional gel electrophoresis of complete T1 and pancreatic RNase digests of formamidesucrose gradient-purified RNA and subsequent analysis of the product by alkaline hydrolysis. The size of the EMC virus genomic poly(A) sequence was estimated by formamide-polyacrylamide gel electrophoresis of the RNase-resistant product, or by [3H-]poly(U) hybridization to freshly purified virion RNA, to be, on average, 40 nucleotides in length. The evidence obtained from [3H-]isoniazid labelling and other experiments would indicate that the poly(A) sequence is located at the 3'-terminus of EMC virus RNA.     

Specificity of protein synthesis inhibitors in the inhibition of encephalomyocarditis virus replication.

Effect of protein synthesis inhibitors on encephalomyocarditis virus production in L-cells was studied. Inhibition of initiation by hypertonicity, harringtonine, or pactamycin decreased viral protein synthesis to a lesser extent than that of host. Virus yield was unaffected or actually enhanced by low concentrations of these inhibitors. On the contrary, the elongation inhibitors cycloheximide, anisomycin, and emetine, shown previously to inhibit viral protein synthesis preferentially, had a greater effect on virus yield than on overall protein synthesis. These results support our earlier proposal that the antiviral activity of cycloheximide derives from its specific effect on the rate of elongation of protein synthesis, and that elongation inhibitors in general may show varying degrees of specific antiviral activity.     

Protein-RNA interaction in encephalomyocarditis virus as revealed by UV light-induced covalent linkages.

UV irradiation of encephalomyocarditis virus led to an increase in the buoyant density of the virus in CsCl gradients from 1.34 to 1.46 g/cm3. Heat treatment of the irradiated virus (20 min at 54 degrees C) reduced the density to 1.40 g/cm3 and led to the loss of approximately 55% of the labeled RNA from the virions. The non-irradiated virions were converted by such heating into empty capsids. Irradiation also resulted in an increase in the accessibility of RNA inside the virions to the action of pancreatic RNase. An increase in the UV dose did not enlarge the fraction of RNA molecules covalently linked to protein; this was revealed by the lack of any secondary increase in the apparent RNase resistance of the labeled RNA in the irradiated virions. Destruction of the irradiated virus with sodium dodecyl sulfate and 2-mercaptoethanol allowed the isolation of a 40S structure containing viral RNA and RNA-linked proteins. The latter comprised no more than 2.5% of the whole protein content of the virion. Polyacrylamide gel electrophoretic analysis of the RNase-treated 40S structure revealed at least three viral structural proteins in the same ratio as was present in the intact virions.