Coxsackievirus B4-induced development of antibodies to 64,000-Mr islet autoantigen and hyperglycemia in mice.

Diabetogenic Coxsackievirus B4 infection produces a diabetes syndrome in susceptible mice resembling insulin-dependent diabetes mellitus. We reported a two- to threefold increased expression of a 64,000-Mr (64 K) islet autoantigen in the infected mice preceding the development of hyperglycemia, suggesting a possible role of the virus in autoimmunity. To assess if the virus could be a trigger of autoimmunity, 64 K autoantibody development was correlated with hyperglycemia and virus replication in islets during early and late infection. Additionally, the effects of blood removal from these mice on the incidence of hyperglycemia and antibody production were evaluated. Noninfected control mice were essentially 64 K antibody negative, the infected consistently positive. Approximately 30% of the animals developed antibodies by 72 h postinfection (p.i.) and 90% by 4-6 wk p.i. Virus-induced hyperglycemia appeared bimodal: hyperglycemia in 50% of the mice by 1 wk p.i., which decreased to 30% by 3 wk and then increased to 80-100% by 6 wk p.i. Infectious virus was abundant in the islets at 72 h but undetectable later. Hyperglycemia seen at 6 wk decreased dramatically (67-73%) if the mice were bled once between 72 h and 2 wk p.i. Only 50-60% of the mice bled once were 64 K positive compared to 90% positive nonbled mice. Coxsackievirus may initiate or enhance the autoimmune response.

Pancreatic D-cell disorder in coxsackievirus B4-induced diabetic mice.

Pancreatic D-cell disorder was analyzed in Coxsackievirus B4-induced diabetic mice employing molecular hybridization with a radiolabelled probe to quantitate somatostatin mRNA, and specific immunoprecipitation to measure somatostatin synthesis and its release. Many infected mice showed blood glucose lower than noninfected control animals at 72 h postinfection and 85% became hyperglycemic at 6-8 weeks postinfection. Pancreatic somatostatin decreased by 24% and 43% at 72 h and 6 weeks postinfection, respectively, while somatostatin release in islets from the infected mice increased by 2-fold or more. Residual islet somatostatin content after release was initially higher than control at 72 h and then declined at 6 weeks. Islet cellular RNA content decreased by 35% at 6 weeks, somatostatin mRNA content decreased by approximately 45% at 72 h and 6 weeks postinfection. D-cell disorder - somatostatin mRNA supply, synthesis, and release - is clearly evident in this model, which could be of significance in type I diabetes.

Effect of coxsackievirus B4 infection in mice on expression of 64,000-Mr autoantigen and glucose sensitivity of islets before development of hyperglycemia.

Diabetogenic strains of Coxsackievirus B4 (CB4) produce a diabetes syndrome in susceptible mice that resembles insulin-dependent diabetes mellitus. To assess the possible role of autoimmunity, the expression of a 64,000-Mr islet antigen in SJL/J and CD1 mice infected with a diabetogenic strain of CB4 was monitored in early and late infection. Additionally, virus-induced abnormalities in glucose metabolism were correlated with several changes in purified islets to assess beta-cell physiology. Over 80% of the mice exhibited subnormal blood glucose at 72 h postinfection (p.i.) and were hyperglycemic at 6 and 8 wk p.i. Islet yield in infected mice decreased by 29-47% at 72 h and 6 wk p.i. compared to noninfected mice. Insulin release stimulated by 16.7 mM glucose increased greater than twofold at 72 h p.i. but declined at 6 wk well below the level of noninfected mice. Likewise, residual islet insulin content after release also increased at 72 h and then declined. Total protein synthesis in the islets decreased by 30% at 72 h and by 60% at 6 wk p.i. Although the synthesis of five proteins of heterogeneous molecular weights, including tubulin, was severely depressed in the infected islets at 72 h p.i. compared with control islets or islets at 6 wk p.i., synthesis of the 64,000-Mr component and another protein of 36,000 Mr increased by two- to threefold. It is possible that CB4 infection may initiate or enhance an autoimmune reaction by increased expression of the 64,000-Mr antigen.

Purification and characterization of a strain of coxsackievirus B4 of human origin that induces diabetes in mice.

A diabetogenic strain of coxsackievirus B4 of human origin has been purified to study its biochemistry and diabetogenicity. Tissue culture cells infected with the virus contain two distinct types of particles--virions and membrane-bound virions (MBV). MBVs are lighter (p = 1.29) than virions (p = 1.34), and they contain relatively more protein than RNA. Virons contain four capsid proteins, VPI-4, of various molecular weights: VP1, 37,500; VP2, 36,000; VP3, 26,000; and VP4, 5,500. MBVs contain three of these proteins and several additional proteins of molecular weights 45,000 to greater than 92,500, possibly of host or viral origin. The RNA in each type of particle is a 35S molecule; T1 oligonucleotide fingerprint profiles suggest minor differences in the two RNAs. Hybridization experiments show a great deal of sequence homology between the RNA of the diabetogenic strain and the RNA of prototype CB4, which does not induce overt diabetes. MBVs are 10-70 times less infective than virions, yet they are more pathogenic in mice and induce significantly higher glucose intolerance (hyperglycemia). The hyperglycemic response appears to be lower in mice infected with both types of particles than in mice infected with MBVs alone. Thus, the two subpopulations of virions present in the diabetogenic strain differ biochemically and in their ability to induce diabetes.

Insulin mRNA content in pancreatic beta cells of coxsackievirus B4-induced diabetic mice.

Molecular hybridization was used to measure poly(A)-containing mRNA and insulin mRNA, and to evaluate viral persistence, in pancreatic beta cells of coxsackievirus B4-induced diabetic mice. Cellular RNA was hybridized with [3H]poly(U) to measure poly(A)-containing total mRNA, 32P-labeled preproinsulin I and II probes to measure insulin mRNA, and a 32P-labeled virus-specific probe to evaluate persistence. The infected mice (80-90%) showed subnormal blood glucose at 72 h postinfection and were hyperglycemic at 6 and 8 weeks. Poly(A)-containing total mRNA decreased by about 26% at 72 h and 6 weeks and by 49% at 8 weeks, while preproinsulin I mRNA by 30% and preproinsulin II by 46% at 8 weeks postinfection compared to control. Viral sequences were abundant at 72 h and in fair amounts later. It appears that persistent viral infection produces a pathological state, which impairs beta cell function to reduce insulin mRNA and consequently insulin synthesis apparently leading to hyperglycemia.

Protein kinase in nondiabetogenic coxsackievirus B4.

Alkali-dissociated, purified preparations of prototype coxsackievirus B4 release a protein kinase that catalyzes the incorporation of gamma-phosphate from 32P-labeled ATP into three virus capsid proteins (VP1, VP3, VP4), several additional proteins of the particle, and exogenous acceptor proteins. Using protamine sulfate as an acceptor protein, we detected nearly 20-fold more enzyme activity in membrane-bound virions (MBV) than in virions of the virus. The activity in the MBV is cyclic nucleotide-independent, divalent cation-dependent, and has a pH optimum of 8.0. Phosphoserine is labeled with 32P. The enzyme activity sediments at about 5S and is separated into at least two peaks of heterogeneous proteins by ion-exchange chromatography. The patterns of phosphorylation by these enzyme peaks are somewhat similar. Coxsackievirus-associated protein kinase appears to be located internally in the virus and may be host-cell-coded. The enzyme appears to be lacking in a variant of the virus that produced diabetes in mice.

Functional alterations in pancreatic beta cells as a factor in virus-induced hyperglycemia in mice.

Alterations in the functional capacity of pancreatic beta cells appear to contribute to coxsackievirus B4-induced, long-term hyperglycemia in mice. Mice infected with prototype B4 or its diabetogenic E2 variant were monitored for abnormalities in sugar metabolism (by the glucose tolerance test), for total protein and insulin synthesis in intact beta cells, for alterations in beta cell proteins, and for virus replication. The infected mice were hypoglycemic at 72 h postinfection and hyperglycemic at 6 weeks. At 8 weeks postinfection, few of the prototype- but most of the E2-infected mice remained hyperglycemic. Total protein and synthesis of immunoprecipitable insulin decreased during early infection. At 8 weeks postinfection, insulin synthesis in the prototype-infected mice increased almost to the level of control mice. Although insulin synthesis increased likewise in the E2-infected mice, it remained well below the control level. Two-dimensional gel electrophoresis revealed the disappearance of many cellular proteins in beta cells from E2-infected mice but of very few in cells from prototype-infected mice at 72 h postinfection. Many of the disappearing proteins reappeared gradually in the E2-infected group. Infectious virus was recovered from the infected beta cells only at 72 h postinfection. Functional impairment in these cells appears to be a factor in virus-induced hyperglycemia.

Membrane-bound virions of coxsackievirus B4: cellular localization, analysis of the genomic RNA, genome-linked protein, and effect on host macromolecular synthesis.

Hela cells infected with several group B coxsackieviruses contain, in addition to standard virions, a population of virus-specific ribonucleoprotein particles which we (5) designated membrane-bound virions (MBV). MBVs differ from standard virions in buoyant density, yield, appearance, protein composition and infectivity. Here we present several new features of MBVs of coxsackievirus B4. The MBVs are lighter (rho about 1.30) and are localized in rough membranes, intermixed with virions. They contain 35S virion RNA covalently linked with a small protein, VPg. The VPg contain two proteins of different charge. MBV VPg is considerably smaller than the 5300-dalton virion VPg. MBV RNA is homologous to the base sequence present in B4 virus double-stranded RNA. The T1 oligonucleotide fingerprint of MBV RNA is distinguishable from that of virion RNA by one oligonucleotide. Several oligonucleotides of virion RNA appear to occur in submolar quantities in MBV RNA. MBVs are 75 to greater than 200 times less infective; they inhibit host cell macromolecular synthesis less efficiently than virions. In coinfected cells, the extent of inhibition of host synthesis is less severe than in cells infected with virions alone, which suggest interference by MBV particles.

RNA and protein synthesis and kinetics of processing and release of insulin in cultures of mouse pancreatic beta cells.

Monolayer cultures of mouse pancreatic beta cells were used to study protein composition, the extent and nature of RNA and protein synthesis, and the kinetics of insulin precursors synthesis, precursor processing, and the release of nascent insulin. The cells synthesized both RNA and proteins actively, but the amounts synthesized declined with the age of the culture. The size and spectrum of proteins synthesized in cells soon after isolation and after maintenance in culture were very similar. At least 15 proteins (mol. wt. 66,000 to less than 14,000) were synthesized, including insulin precursors and insulin. Radioactive labeling and chase experiments revealed the synthesis of insulin precursors and insulin continuously. Radioactivity present in the precursors decreased rapidly during the early times of chase. This reduction in radioactivity was partly due to the processing of insulin precursors into insulin and partly due to the release of proinsulin into the medium along with newly synthesized insulin. The residual labeled precursors turned over at a slower rate. It appears that the processing of insulin precursors is two-phase-initially rapid and then slow and prolonged, which could have resulted from two populations of precursors with different half-lives.