The Mason-Pfizer monkey virus (MPMV) constitutive transport element (CTE) is a cis-acting RNA element located in the 3' untranslated region (UTR) of the viral genome. The HIV-1 and SIV Rev/RRE regulatory system can be replaced with MPMV CTE (Bray et al., 1994; Zolotukhin et al., 1994; Rizvi et al., 1996a); similarly, CTE function can also be replaced by the HIV or SIV Rev/RRE regulatory system (Rizvi et al., 1996b; Ernst et al., 1997). In addition, we have shown that in the context of the SIV genome, position is important for CTE function (Rizvi et al., 1996a). To determine the importance of position for CTE function in the context of the MPMV genome, MPMV molecular clones were generated by deleting CTE or removing it from the 3' UTR and placing it in the approximately 40 bp of intervening sequences between the pol termination codon and env initiation codon. A test of these molecular clones in a single round of replication assay revealed that deletion or displacement of CTE in the intervening sequences between pol and env completely abrogated virus replication. Western blot analysis of cell lysates and pelleted culture supernatants revealed negligible amounts of Pr78 Gag/Pol precursor and the processed p27(gag) when CTE was deleted or displaced. Slot blot analysis of fractionated RNAs revealed entrapment of the viral Gag/Pol mRNA in the nucleus with CTE deletion or displacement. Upon reinsertion of CTE in the original genomic position of clones with the deleted or displaced CTE, virus replication, Gag/Pol protein production, and nucleocytoplasmic transport of viral mRNA were restored to normal levels. Displacement of CTE to the 5' UTR immediately upstream of the Gag initiation codon also resulted in aberrant Gag/Pol protein production and nucleocytoplasmic transport of viral RNA. Reinsertion of CTE at the original genomic position of the clone with CTE displacement at the 5' UTR restored normal Gag/Pol protein production and RNA transport, demonstrating that the 3' terminal position of CTE is important for its function. To explore why the 3' terminal location of CTE is important, heterologous DNA sequences of increasing lengths were inserted between CTE and the polyadenylation (poly(A)) signal of the virus to augment the distance between the two cis-acting elements. Test of these constructs revealed that CTE function was progressively lost with incremental increase in distance between CTE and poly(A). To explore this relationship further, CTE was displaced to the env region ~2000 bp upstream of the poly(A) signal which abrogated CTE function. However, cloning of poly(A) signal to ~200 bp downstream of CTE in the env region (the natural distance between CTE and poly(A)) restored CTE function. Together, these results demonstrate that the close proximity of CTE to the poly(A) signal is important for CTE function, suggesting a functional interaction between CTE and the polyadenylation machinery.
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