Claude La Bonnardière

Interferon-delta: The first member of a novel type I interferon family

We have recently described a novel type I interferon (IFN) co-expressed with IFN-gamma by the trophectoderm of the pig conceptus between day 12 and day 18 of gestation, a development stage that corresponds to implantation in the uterus. This IFN, now officially named IFN-delta, is recognized as the first member of a novel type I IFN family. This paper reviews the main published data on IFN-delta, together with some new data, showing that IFN-delta, while being a true type I IFN, has some very specific structural and biological properties. Sequences related to IFN-delta coding sequence were found in the genome of man and other ungulates but the only other potentially functional gene was found, so far, in the horse. The pig IFN-delta mature protein, with 149 amino acids, is the smallest of all known type I IFNs. It is unusually rich in cysteines (seven residues), and has a very basic isoelectric point. Recombinant IFN-delta expressed in insect cells is glycosylated and has a high antiviral activity on porcine cells, but not on human cells. It has high antiproliferative activity, which is significantly enhanced in the presence of IFN-gamma. This new IFN was shown to bind on pig cells to the same type I receptor as IFN-alpha. IFN-delta and IFN-gamma genes are co-regulated in the pig trophectoderm, whose cells on day 14-16 of development simultaneously secrete both IFN proteins. The biological role of porcine IFN-delta in early pregnancy has been found unrelated to the known antiluteolytic effect of trophoblastic IFN-tau in ruminants.

Cloning of the porcine interferon-γ receptor and its foeto-endometrial expression in early pregnancy

In early gestation, trophoblastic cells of porcine preimplanting conceptuses transiently and massively secrete two distinct interferons (IFNs), one of which is IFN‐γ. In order to localize possible cellular target(s) for this IFN‐γ, the expression of the porcine IFN‐γ receptor and its developmental regulation have been investigated on the maternal endometrium and on the embryonic tissues. A cDNA encoding the porcine IFN‐γ binding‐chain (pIFNGR1) was isolated. When expressed in COS‐7 cells, it displayed a specific binding to radiolabelled pIFN‐γ and was shown to be a glycosylated membrane protein with an apparent molecular mass of 92 kDa. Porcine IFNGR1 mRNA was detected by RT‐PCR not only in uterine epithelial cells but also in embryonic tissues from at least as early as day 10 of gestation. Moreover, membrane expression of the pIFN‐γ receptor quantified by binding and crosslinking of 32P‐pIFN‐γ was demonstrated in uterine epithelium and in the trophoblast. In the trophoblast, expression of the receptor was found to be developmentally regulated: although expression was weak on days 12 and 15 of gestation, it reached a level similar to that found on some IFN‐γ–sensitive cells on day 16. This study shows that maternal endometrium is not the only possible target for trophoblastic IFN‐γ: the induction of pIFN‐γ receptor expression in the trophoblast around day 16 of gestation could suggest the appearance of responsiveness to pIFN‐γ in this implanted tissue and therefore a possible delayed autocrine effect of trophoblastic pIFN‐γ. Mol. Reprod. Dev. 51:225–234, 1998.© 1998 Wiley‐Liss, Inc.

Vesicular stomatitis virus mRNA methylation in Vivo: Effect of cycloleucine, an inhibitor of S-adenosylmethionine biosynthesis, on viral transcription and translation

Abstract Cycloleucine is a potent and specific inihibitor of S -adenosylmethionine biosynthesis and subsequent RNA methylations in Chinese hamster ovary (CHO) cells. When added in the course of an infection of CHO cells with vesicular stomatitis virus (VSV), cycloleucine does not significantly modify the rate of viral RNA and protein synthesis or the production of mature particles. Under the same conditions cycloleucine will reduce by 83% the methyolation of newly synthesized polyadenylated 11–16 S viral RNA. The mode of action of cycloleucine and cycloheximide are clearly distinguishable under these conditions. In contrast when cells are treated with cycloleucine, immediately after virus adsorption, the effect on viral RNA synthesis is very similar to that of cycloheximide. That is, a 90% inhibition in the production of viral messenger RNAs is observed along with a complete disappearance of newly synthesized 38 S RNA species, suggesting that secondary transcription is inhibited. Viral protein synthesis and virus production are similarly inhibited during this period. The undermethylated mRNAs of VSV synthesized in the presence of cycloleucine are 80% undertranslated as compared to mRNAs synthesized in untreated cells. Our results suggest that mRNA methylation in VSV is required for translation in vivo . The mode of action of cycloleucine during the infectious process is discussed.

Contribution of molecular biology to the study of the porcine interferon system

Abstract We have performed molecular studies on the pig interferon (IFN) system (i) to analyse the role played by endogenous IFN in neonatal viral enteritis such as transmissible gastroenteritis and possibly to obtain, via recombinant DNA technology, a new anti-infectious and immunomodulatory agent in this species, (ii) to characterize the structure and biological functions of the IFN-like antiviral activity produced by the porcine embryo at the time implantation in the uterus. By probing porcine genomic libraries with human and porcine IFN-α probes to isolate related genes, we have shown that the porcine IFN-α multigene family included, like several other mammalian species, two subfamilies of related but distinct genes. Class I subfamily contains at least 11 loci, located on chromosome no. 1, among which nine have been cloned and two (potentially functional) sequenced. Class II subfamily, which is specifically expressed by the embryo of ruminants before implantation, contains at least seven loci among which six have been cloned. One of the sequenced class I loci: PoIFN-α1 encodes a 189 amino acids (AA) preprotein. After removal of the sequence encoding the putative signal peptide (23 N-terminal AA) this gene was inserted into an Escherichia coli bicistronic expression vector allowing intracellular synthesis of mature porcine IFN-α1 (methionyl IFN-α1). Expression of the recombinant protein was optimized by insertion of a seven base pairs long random synthetic sequence in the intercistronic region, followed by cloning in E. coli and immunodetection of clones expressing high amounts of recombinant protein. The E. coli strain obtained produced high levels of a 18 000 Da protein exhibiting the same in vitro overall biological properties as leucocyte derived porcine IFN (LeuIFN). However, it had a stronger antiviral effect on porcine cells than LeuIFN. After immunoaffinity purification to a specific activity of 5−10 × 107 International Units (IU)/mg of protein, pharmacokinetic and pharmacological studies were realized to determine the in vivo half life of this rIFN-α in the pig. These experiments revealed no major toxic effects in newborn (given 5 × 106IU/kg) or adult (1 × 106IU/kg) pigs. A significant pyrogenic effect (+ 1.5°C) was noted only in the adults.