Characterization of Rous sarcoma virus sequences essential for viral gene expression

Abstract

Using the Escherichia coli lacZ gene product ,-galactosidase as an indicator of gene expression, we analyzed sequences that are required for expression of the Rous sarcoma virus (RSV) genome in avian cells. The RSV long terminal repeat (LTR) and leader region were sufficient to direct the synthesis of high levels of enzymatically active gag-lacZ fusion proteins. A portion of U3 greater than 140 nucleotides upstream from the cap site was essential for gene expression. This element functioned in either orientation, but its activity was attenuated when it was relocated further away from the cap site. The insertion of exogenous LTRs 3' of lacZ augmented the expression of that gene by increasing the level of stable gag-lacZ transcripts. Furthermore, 3' LTRs could partially compensate for certain defects within the 5' LTR. Insertion of various fragmentary LTRs allowed the identification of at least three synergistically acting domains within the 3' LTR that influence gene expression. Interestingly, the gag-lacZ expression was only stimulated by a 3' LTR when the exogenous 3'-untranslated region was adjacent. Our results imply that the two LTRs of a provirus interact in a complex manner to promote high levels of stable transcripts. It was also found that gag-lacZ expression was independent of viral gene products, suggesting that trans-activation is not a key mechanism regulating RSV expression in avian cells.

VBP and RelA regulate avian leukosis virus long terminal repeat-enhanced transcription in B cells

“…These observations were surprising, as similar mutations of the corresponding Pr-RSV-C LTR CCAAT/enhancer elements all decreased LTR-driven transcription in CEF (59). Moreover, previous studies have shown that the RSV LTR enhancer supplies approximately 90% of the LTR transcription activity in avian or mammalian fibroblasts (18,47). This could reflect differences in the cell types analyzed or sequence differences between the closely related ALV and RSV LTRs (9).…”

“…The avian leukosis virus (ALV) long terminal repeat (LTR) contains compact enhancer and promoter sequences which form a powerful transcription unit in many cell types (42,53). The enhancer region is important for high rates of LTR-driven transcription in fibroblasts (19,47) and is essential for hyperexpression of the c-myc oncogene during ALV induction of bursal lymphomas in chickens (52). ALV infects and produces a high level of viral expression in many tissues (56), but the tumors that develop are bursal in origin, suggesting that cellspecific host factors are important in tumor induction.…”

Proposed gag-encoded transcriptional activator is not necessary for Rous sarcoma virus replication or transformation

“…trans Activation of the RSV LTR by RSV proteins has not been confirmed by other workers. Norton and Coffin (22) looked for trans activation of a P-galactosidase gene under control of the RSV LTR. Expression of ,-galactosidase was found to be substantially lower in RSV-infected turkey cells than in uninfected cells.…”

The putative trans-activator in the MAgag region of Rous sarcoma virus is not required for cell transformation

“…NOTES 4769 (5), in RSV-infected turkey embryo fibroblasts (12), and in tdlO7-infected CEF (unpublished results) on the preproinsulin promoter (5) and the RSV LTR (12; unpublished results). Therefore, the totally normal replication and transforming activity of NY MXD2 suggest that the putative transactivator in the MA9'9 region of RSV does not play a role in the replication of RSV or the transformation of primary CEF by RSV.…”