Efficient transcription and replication of simian immunodeficiency virus in the absence of NF-kappa B and Sp1 binding elements

Ten mutants of the simian immunodeficiency virus (SIV) SIVmac239 bearing deletions (Delta) or substitutions (subst) in the NF-KB and/or Spl binding elements were created, and the replicative capacities of the mutants were analyzed. All mutants, including one extensively mutagenized strain entirely missing the NF-KB and four Spl binding elements, replicated with wild-type kinetics and to a wild-type level in peripheral blood mononuclear cell cultures in 50 to 100% of the experiments. One group of mutants replicated very similarly to SIVmac239 in kinetics and yield in CEMx174 cells (2xNF kappa B greater than or equal to SIV mac239 approximate to Delta NF kappa B approximate to Delta Sp1234 approximate to substNF kappa B approximate to substSp12 approximate to substSp23), while a second group replicated with delayed or slightly delayed kinetics in CEMx174 cells (SIVmac239>substSp34>Delta NF kappa B Delta Sp1234 approximate to Delta NF kappa B Delta Sp1234). Reversions or additional mutations were not detected in the U3 and R regions of proviral DNA from CEMx174 cells infected with the SIVmac239 mutants. Similar results were obtained when mutants of SIVmacMER (a macrophage-competent derivative of SIVmac239) were tested in peripheral blood mononuclear cell and CEMx174 cultures. However, the growth of most mutated viruses was suppressed in primary rhesus monkey alveolar macrophages (SIVmacMER approximate to 2xNF kappa B approximate to substNF kappa B>Delta NF kappa B>Delta NF kappa B Delta Sp1234 approximate to Delta NF kappa B Delta Sp1>Delta Sp1234 approximate to substSp12>substSp23 approximate to substSp34 approximate to substSp1234 greater than or equal to SIVmac239). Thus, changes in the Sp1 binding sites had the most dramatic effects on SIVmac replication in primary macrophage cultures, Analysis of long terminal repeat driven secreted alkaline phosphatase activity in transient assays showed that, unlike human immunodeficiency virus type 1, the SIV long terminal repeat possesses an enhancer region just upstream of the NF-KB element which maintains significant levels of basal transcription in the absence of NF-kappa B and Spl sites. This region is responsive to transactivation by Tat, In addition, the SIV TATA box was shown to be stronger than that of human immunodeficiency virus type 1, Therefore, the surprisingly high replicative capacity of NF-kappa B and Spl binding site mutants of SIVmac is due to unique features of the enhancer/promoter region.