Virus-induced Diabetes Mellitus. XXV. Difference in the RNA Fingerprints of Diabetogenic and Non-diabetogenic Variants of Encephalomyocarditis Virus

Abstract

SUMMARYThe genomes of diabetogenic and non-diabetogenic variants of encephalomyocarditis virus were analysed by nucleic acid hybridization and RNA fingerprinting. Hybridization and thermal elution profiles failed to show any difference between the RNAs of the two variants, whereas fingerprinting of the T 1-digested RNAs revealed at least one oligonucleotide, 20 to 25 nucleotides long, missing in the non-diabetogenic variant.Encephalomyocarditis (EMC) virus produces diabetes in mice by infecting and destroying pancreatic beta cells (Notkins, 1977). Recently, we showed that our virus pool contained two antigenically similar variants which could not be distinguished by hyperimmune sera (Yoon et al., 1980). One of the variants, designated D, produced diabetes, whereas the other variant, designated B, failed to produce diabetes. The present study was initiated to analyse the genomes of these two variants by nucleic acid hybridization and RNA fingerprinting. We found that both viral cDNA probes hybridized equally well to homologous and heterologous RNAs, but that the RNAs differed by at least one spot in the unique sequence region of their oligonucleotide fingerprints.Viruses were purified from infected L929 cells by sucrose and CsCI gradient centrifugation . RNA was obtained from purified virions after phenol-cresolchloroform extraction and ethanol precipitation (Kacian & Myers, 1976). The viral RNA was treated with methylmercury hydroxide to enhance transcription according to the method of Payvar & Schimke (1979). Then, labelled cDNA was synthesized using [~-32p]dCTP and avian myeloblastosis virus reverse transcriptase. Oligo(dT) and oligo(dG) were used as primers and the cDNA was purified by Sephadex G-75 and DEAE column chromatography. Foldback sequences were removed by hydroxylapatite column chromatography as previously outlined by Aulakh & GaUo (1977). The single-stranded cDNA probes of EMC-D and EMC-B were shown to be representative of the complete viral genomes by their ability to protect the labelled viral RNAs from digestion with ribonucleases A and T1. The respective homologous cDNAs protected greater than 94~o of EMC-D-and 95~ of EMC-B-labelled RNAs.Hybridizations were performed using 20 ~tg/ml viral RNA and approximately 0-05 ~tg/ml cDNA probe (50000 ct/min) in 0.48 M-sodium phosphate buffer pH 6.8, 3 mM-EDTA, and 0.4~ SDS. After denaturing at 105 °C for 5 min, the hybrids were allowed to anneal at 65 °C for 10 h. Hybrids were separated from unhybridized strands by hydroxylapatite chromatography.When EMC-D cDNA was hybridized with EMC-D and EMC-B RNA, 90~o and 91~, respectively, of the cDNA formed hybrids. Similarly, when EMC-B cDNA was hybridized with homologous and heterologous viral RNA, 90~ and 89~ of the cDNA formed hybrids. The hybrids were also analysed for thermal stability to evaluate mismatching in base pairing.

Picornavirus Infection Leading to Immunosuppression

“…Further examination of the M variant by plaque purifcation led to the identifcation of two stable variants, EMC-D and EMC-B. EMC-D was found to induce IDDM/T1D in approximately 90% of infected mice compared with mice inoculated with the EMC-B variant, which did not acquire IDDM/T1D [33,34]. Furthermore, Jun et al [35] demonstrated that the change of a single amino acid (Thr to Ala) at position 776 of the EMC polyprotein, located in the capsid protein VP1, caused diabetes in mice.…”

Molecular Identification of Diabetogenic Viral Gene

“…All of the 35 mice infected with EMC-D (3 x 10 5 pfu/mouse) developed diabetes (mean glucose index 437 ± 34 mg/dl), whereas none of the 30 mice infected with EMC-B (3 x 10 5 pfu/mouse) became diabetic (mean glucose index 146 ± 19 mg/dl). Viral RNAs were prepared from the purified virus by phenol/chloroform extraction (12). Molecular cloning.…”

“…In contrast, none of the mice inoculated with EMC-B became diabetic. Despite these distinct differences in diabetogenic activity, available data indicate that the two variants are extremely similar in their capsid proteins and nucleic acids (12,13). Therefore, complete nucleotide sequences of both viral genomes are required to find the fine differences between these two variants.…”

Determination of encephalomyocarditis viral diabetogenicity by a putative binding site of the viral capsid protein.

“…Viruses and viral RNA. Diabetogenic EMC-D and nondiabetogenic EMC-B viruses were grown in L-929 cells (13), purified by CsCl and sucrose gradient ultra-centrifugation, and used in the preparation of viral RNA by phenol-chloroform extraction (14,18). The integrity and purity of the viral RNA was confirmed by denaturing gel electrophoresis and measurement of the A 260 /A 280 ratio (19).…”