Nucleotide sequence of cloned unintegrated avian sarcoma virus DNA: viral DNA contains direct and inverted repeats similar to those in transposable elements.

Abstract

We have determined the nucleotide sequence of portions oftwo circular avian sarcoma virus (ASV) DNA molecules cloned in a prokaryotic host-vector system. The region whose sequence was determined represents the circle junction site-i.e., the site at which the ends ofthe unintegrated linear DNA are fused to form circular DNA. The sequence from one cloned molecule, SRA-2, shows that the circle junction site is the center of a 330-base-pair (bp) tandem direct repeat, presumably representing the fusion ofthe long terminal repeat (LTR) units known to be present at the ends of the linear DNA. The circle junction site is also the center ofa 15-bp imperfect inverted repeat, which thus appears at the boundaries of the LTR. The structure of ASV DNA-unique coding region flanked by a direct repeat that is, in turn, terminated with a short inverted repeat-is very similar to the structure ofcertain transposable elements. Several features ofthe sequence imply that circularization to form the SRA-2 molecule occurred without loss of information from the linear DNA precursor. Circularization of another cloned viral DNA molecule, SRA-1, probably occurred by a different mechanism. The circle junction site of the SRA-1 molecule has a 63-bp deletion, which may have arisen by a mechanism that is analogous to the integration of viral DNA into the host genome. Flanking one side of the tandem direct repeat is the binding site for tRNAT"P, the previously described primer for synthesis of the first strand of viral DNA. The other side of the direct repeat is flanked by a polypurine tract, A-G-G-G-A-G-G-G-G-G-A, which may represent the position of the primer for synthesis of the second strand of viral DNA. An A+T-rich region, upstream from the RNA capping site, and the sequence A-A-T-A-A-A are present within the direct repeat sequence. These sequences may serve as a promoter site and poly(A) addition signal, respectively, as proposed for other eukaryotic transcription units.Three major types of virus-specific DNA have been identified in cells after infection with retroviruses: linear duplex (form III) DNA, the initial product of synthesis by RNA-directed DNA polymerase; covalently closed circular (form I) DNA, derived from linear precursors; and DNA covalently integrated into the host genome (proviral DNA) (1, 2).Physical maps of the unintegrated and integrated forms of avian sarcoma virus (ASV) DNA have helped to clarify the structural relationships among these forms (cf. Fig. 1). Form III and proviral DNA are coextensive with a subunit of the RNA genome (10-13). These forms also contain a long terminal repeat (LTR) ofabout 300 base pairs (bp) that is not present in the RNA genome (10-13). The repeated domain is composed of sequences unique to each end of the RNA (U5 and U3) joined by a short sequence, R, that is present as a terminal repeat in the RNA (7). Two principal classes ofcircular DNA have been characterized (10, 11). One class bears a single copy of the LTR sequence and presumably arises by recombination between the LTRs at...

The encephalomyocarditis virus internal ribosome entry site allows efficient coexpression of two genes from a recombinant provirus in cultured cells and in embryos.

“…In cells infected with the wild-type RSV, about half of the primary transcripts are spliced to generate env and v-src subgenomic mRNAs, although the precise ratio of spliced to unspliced RNA varies among strains (49). For an internal promoter, we used a fragment of the U3 region of the RSV LTR (50). This fragment includes promoter and enhancer sequences but excludes both the inverted repeat sequence at the 5' end, which is required for integration, and the polyadenylation signal at the 3' end.…”

“…The upstream gene was always fused in frame with the gag polyprotein and expressed from the LTR of the SRA-2 clone of RSV (50). The downstream gene was expressed in one of three ways: from a spliced subgenomic message, from an internal promoter, or from the EMCV IRES.…”

Cellular DNA Surrounding Integration Sites of an Avian Retrovirus

“…Structural and sequence analysis of the LTRs of several different retroviruses (Dhar et al, 1980;Ju & Skalka, 1980;Reddy et al, 1980;Sutcliffe et al, 1980;Van Beveren et al, 1980;Yamamoto et al, 1980;Donehower et al, 1981 ;Swanstrom et al, 1981) and the observation that proviruses generate short direct repeats of host DNA upon insertion Van Beveren et al, 1982;Hishinuma et al, 1981 ;Hughes et al, 1981 ;Majors & Varmus, 1981 ;Shoemaker et al, 1981) have revealed a remarkable resemblance between retroviruses and transposable elements (Calos & Miller, 1980). It has been proposed that the mechanism of integration of retroviruses may be similar to transposition and that retroviruses evolved from cellular movable genetic elements .…”

Frequent Dual Initiation in Human Immunodeficiency Virus-Based Vectors Containing Two Primer-Binding Sites: a Quantitative In Vivo Assay for Function of Initiation Complexes

“…However, it is possible that both nonrecombining and recombining virions are capable of initiating and completing synthesis of two full genomic DNAs. Inefficient minus-strand DNA transfer, intermolecular minus-strand DNA template switching (3,34,39), degradation of viral DNA, formation of one and two long terminal repeat (LTR) circles (41,61,65), autointegration (46,62), and inefficient integration (19,20,29,63) could reduce the efficiency of provirus formation.…”