Accumulation of 2',5'-oligoadenylates in encephalomyocarditis virus-infected mice

Abstract

Levels of 2',5'-oligoadenylates (2-5A) in various tissues of murine encephalomyocarditis virus (EMCV)infected mice were determined and compared with those found in pathogen-free mice and in mice treated with the interferon inducer poly(I) * poly(C). In control, pathogen-free mice, liver, spleen, brain, and kidney tissues possessed levels of 2-5A below 1 pmol/g of tissue, demonstrating that 2-5A was not a major component of uninfected mouse tissue. All control tissues had low basal levels (0.3 to 2.0 pmol/h per g) of 2-5A synthetase, the enzyme responsible for 2-5A production. After mice were injected intravenously with the interferon inducer poly(I) * poly(C), circulating interferon, 2-5A synthetase, and 2-5A were elevated with increasing doses of double-stranded RNA. The greatest response to poly(I) poly(C) occurred in the kidney, in which enyme levels increased 5-fold and 2-5A levels increased 24-fold to 15 pmol/g. Mice that were infected with EMCV also * Corresponding author. MATERIALS AND METHODS Reagents. Poly(I) poly(C), pppA2'pA2'pA, and A2'pA2'pA were purchased from Pharmacia, Inc., Piscat-1586 Vol. 61, No. 5 on September 17, 2020 by guest http://jvi.asm.org/ Downloaded from 2',5'-OLIGOADENYLATES IN EMCV-INFECTED MICE 1587 away, N.J. C18 and silica Sep-paks and C18 ,uBondapak columns were obtained from Waters Associates, Inc., Milford, Mass. Microtiter plates (Immunolon I) were purchased from Dynatech Laboratories, Inc., Alexandria, Va. Biotinylated goat anti-rabbit immunoglobulin G, biotinylated rabbit anti-mouse immunoglobulin G, and streptavidin horseradish peroxidase were obtained from Bethesda Research Laboratories, Inc., Gaithersburg, Md. Radioactive pppA2'pA2'pA2'pA3'[32P]pCp was generously provided by

Recurrent loss-of-function mutations reveal costs to OAS1 antiviral activity in primates

“…In the absence of infection, however, RNase L deficient mice display significantly increased longevity, potentially due to the lack of chronic low level activation of OAS proteins by cellular dsRNAs 37 . Indeed, RNA extracted from various cell types weakly activates OAS1 in vitro 38 , consistent with detectible 2-5A in uninfected tissues 39 . In addition, recent work demonstrated that cellular RNAs activate OAS proteins in the absence of RNA editing by ADAR1 12 .…”

Enzyme assays for synthesis and degradation of 2-5As and other 2′-5′ oligonucleotides

“…Enzyme measurements of 2-5A nucleases have mainly relied on short substrates unable to activate RNase L, which requires oligoadenylates of trimer length and above [18,[40][41][42][43][44]. We used oligoadenylates of tetramer length and above in our assays, thereby increasing the significance with regard to the 2-5A system.…”

“…Also, the reaction products have mainly been resolved by HPLC using reverse-phased C 18 columns or thin-layer chromatography with PEI-Cellulose, in which the former has been used for production purposes. Likewise, many 2-5A nuclease assays have been reported [18,[40][41][42][43]. They rely on very short 2′-5′ oligoadenylate substrates, especially the 2-5A core dimer ApA, which despite being commercially available, is not biologically relevant in the sense that oligoadenylates of a minimum of three AMP moieties (i.e.…”

Alternative splicing coupled with transcript degradation modulates OAS1g antiviral activity

“…EMCV is a (+)ssRNA member of the Picornaviridae family 121 that replicate through partially dsRNA intermediates (Carocci and Bakkali-Kassimi 2012). Infection 122 has been shown to cause accumulation of 2-5A, and viral replication is sensitive to the OAS/RNase 123 L pathway (Hearl and Johnston 1987;Zhou et al 1997). As oligoadenylate synthetases bind viral 124 dsRNA, the RNA activators in EMCV-infected cells are believed to be the viral replicative 125…”