Role of Methylation in the Induced and Spontaneous Expression of the Avian Endogenous Virusev-1: DNA Structure and Gene Products

Abstract

The endogenous avian provirus ev-1 is widespread in white leghorn chickens. Although it has no major structural defects, ev-1 has not been associated with any phenotype and is ordinarily expressed at a very low level. In this report, we describe a chicken embryo (Number 1836) cell culture containing both ev-1 and ev-6 which spontaneously expressed the ev-1 provirus. This culture released a high level of noninfectious virions containing a full complement of virion structural (gag) proteins but devoid of reverse transcriptase activity or antigen. These virions contained 70S RNA closely related to the genome of Rousassociated virus type 0, but identifiable as the ev-1 genome by oligonucleotide mapping. A fraction of the RNA molecules in the 70S complex were unusual in that they were polyadenylated 100 to 200 nucleotides downstream of the usual polyadenylation site. Eight sibling embryo cultures did not share this unusual phenotype with 1836, indicating that it was not inherited. However, an identical phenotype was inducible in the sibling cultures by treatment with 5-azacytidine, an inhibitor of DNA methylation, and the induced expression was stable for more than 10 generations. Analysis of chromatin structure and DNA methylation of the ev-1 provirus in 1836 cells revealed the presence (in a fraction of the proviruses) of both DNase I hypersensitive sites in the long terminal repeats and in gag and a pattern of cleavage sites for methyl-sensitive restriction endonuclease not found in a nonexpressing sibling. These results lend strong support to the role of DNA methylation in the control of gene expression. Additionally, they explain the lack of phenotype associated with ev-1 as due to a combination of its low expression and defectiveness in pol and env.The endogenous viruses of chickens, which are inherited in chromosomal DNA, are closely related to the exogenous avian leukosis proviruses (for review, see 26). At least 13 distinct endogenous proviruses have been identified by DNA restriction endonuclease mapping (3, 18). Although closely related to one another, proviruses at different endogenous virus (ev) loci differ in DNA content and transcriptional activity. Thus, they may provide a set of genes for the study of elements, both viral and host, that contribute to the control of gene expression.The nondefective endogenous proviruses have a structure similar to integrated proviral DNA found after exogenous infection. That is, they retain the gene order 5'-gag-pol-env-3' of genome RNA and are flanked by two long terminal repeat (LTR) sequences, consisting of sequences derived from the 5' (U5) and 3' (U3) ends of viral RNA, as well as a sequence (R) which is itself repeated at the termini of genome RNA. Thus the overall structure is U3-R-U5-gag-pol-env-U3-R-U5 (17, 28; for review, see 6). The U3 region contains sequences important for regulation of growth rate (39) and appears to include the promoter for viral RNA synthesis (10,21,50).The ev-1 provirus is found in more than 99% of white leghorn chickens (38) and does...

Proliferation and Differentiation Requirements for the Induction of Two Retroviral Loci during B-Cell Activation

“…It has been suggested that alterations in DNA methylation might provide a means of controlling gene expression (Razin & Riggs, 1980). Consistent with this idea, an inverse relationship has been observed between the level of proviral DNA methylation and virus expression (Niwa & Sugahara, 1981 ;Groudine et al, 1981 ;Conklin et al, 1982). It should be noted, however, that studies of the oestrogen responsive chicken gene vitellogenin have suggested that oestrogen treatment results in a specific demethylation even in a tissue which does not express the vitellogenin gene (Wilks et al, 1982).…”

Azacytidine-induced reactivation of a DNA repair gene in Chinese hamster ovary cells.

“…AC is also capable of reactivating silent genes on the inactive X chromosome (13,29), which is known to be hypermethylated at the glucose 6-phosphate dehydrogenase and hypoxanthine guanine phosphoribosyl transferase loci (45,48,50). It also activates latent retroviruses, and these events have also been shown to be associated with the loss of 5-methylcytosine (5mC) (5,14,31). All these results support the hypothesis that hypermethylation of DNA sequences can shut off transcription and that AC reactivates genes by demethylating these sequences.…”

Varied interactions between proviruses and adjacent host chromatin.

“…For example, we previously reported that ev-1 is highly methylated, while ev-3 is not (8,18,19). In addition, both ev-1 and ev-2 are inducible with the methylation inhibitor 5-azacytidine (19; unpublished data), indicating that methylation plays a role in at least maintaining provirus repression.…”