Circles with two tandem long terminal repeats are specifically cleaved by pol gene-associated endonuclease from avian sarcoma and leukosis viruses: nucleotide sequences required for site-specific cleavage

We have analyzed the genome of the domestic chicken for the presence of genetic sequences related to the envelope protein-encoding genes of avian sarcoma/leukosis retroviruses to determine the organization, structure, potential functionality, and distribution of such sequences. We have previously identified in the genus Gaus an extensive group of endogenous avian retroviruses termed EAV-0. Southern blot and sequence analysis presented here of EAV-0 elements revealed that the majority of the EAV-0 elements in the domestic chicken genome have large deletions in their env genes. Screening of a line 0 chicken genomic DNA library for potential full-length env gene-containing endogenous elements yielded three provirus clones of a previously unrecognized group of endogenous retroviruses. These three clones, E13, E33, and E51, are more closely related to each other (80%o or more sequence identity) than to other avian retroviruses (70%o or less sequence identity). The E13 element has a large deletion in env, but the E51 element has full-length and highly divergent SU-and TM-coding domains. Complete sequence analysis of the E51 env gene region revealed a defective SU-coding domain and an intact TM-coding domain. Sequence analysis of the E51, E33, and E13 3' termini revealed highly distinctive long terminal repeats of approximately 360 bp which appear to be the products, in part, of long terminal repeat domain shuffling. Hybridization analysis with E51 and E33 env gene probes

Section: Discussionmentioning

confidence: 99%

Multiple complex families of endogenous retroviruses are highly conserved in the genus Gallus

Boyce-Jacino

O'Donoghue

Faras

1992

“…Other methods. Conditions for measuring endonuclease activity and mapping specific cleavage sites by a primer extension assay have been described previously (3,7).…”

Section: Methodsmentioning

confidence: 99%

Properties of avian sarcoma-leukosis virus pp32-related pol-endonucleases produced in Escherichia coli

Terry

Soltis

Katzman

et al. 1988

Self Cite

The gag-pol precursor protein of the avian sarcoma-leukosis virus is processed into three known pol-encoded mature polypeptides; the 95-and 63-kilodalton (kDa) and a subunits, respectively, of reverse transcriptase and the 32-kDa pp32 protein. The pp32 protein possesses DNA endonuclease activity and is produced from the precursor by two proteolytic cleavage events, one of which removes 4.1 kDa of protein from the C terminus. A 36-kDa protein (p36P°') which retains this C-terminal segment is detectable in small quantities in virions. We have constructed Escherichia coli plasmid clones that express the C-terminal domains of pol corresponding to pp32 and p36. These proteins have been purified by column chromatographic methods to near homogeneity. No significant differences could be detected in the enzymatic properties of the bacterially produced p32P"' and p36&"" proteins. Both possess DNA endonuclease activity and, like the pp32 protein isolated from virions, can cleave near the junction of two tandem avian sarcoma-leukosis virus long terminal repeats in double-stranded supercoiled DNA substrates. In the presence of Mg2+, both p32P"' and viral pp32 cleave either strand of DNA 2 nucleotides 5' to the junction.

“…Both RT, which exists as an up heterodimer, and IN (pp32) have been purified to homogeneity (14,22,36), and each has in vitro DNA endonuclease activity (10,11,14,21). Using a primer extension runoff assay to map cleavage sites, RT and IN have been shown to nick supercoiled DNA substrates that contain covalently linked tandem LTR sequences near the LTR-LTR junction (3,7,36). In the presence of the divalent cation Mn2+, RT nicks each strand 3 nucleotides 5' to the junction, whereas IN nicks each strand at sites 2 or 3 nucleotides 5' to the junction.…”

mentioning

confidence: 99%

The avian retroviral integration protein cleaves the terminal sequences of linear viral DNA at the in vivo sites of integration

Katzman

Katz

Skalka

et al. 1989

Self Cite

306

207

The purified integration protein (IN) of avian myeloblastosis virus is shown to nick double-stranded oligodeoxynucleotide substrates that mimic the ends of the linear form of viral DNA. In the presence of Mg2+, nicks are created 2 nucleotides from the 3' OH ends of both the U5 plus strand and the U3 minus strand. Similar cleavage is observed in the presence of Mn2' but only when the extent of the reaction is limited. Neither