Efficient transcription and replication of simian immunodeficiency virus in the absence of NF-kappaB and Sp1 binding elements.
Ten mutants of the simian immunodeficiency virus (SIV) SIVmac239 bearing deletions (delta) or substitutions (subst) in the NF-kappaB and/or Sp1 binding elements were created, and the replicative capacities of the mutants were analyzed. All mutants, including one extensively mutagenized strain entirely missing the NF-kappaB 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 CEMxl74 cells (2xNFKappaB > or = SlVmac239 approximately deltaNFkappaB approximately deltaSpl234 approximately substNFkappaB approximately substSpl2 approximately substSp23), while a second group replicated with delayed or slightly delayed kinetics in CEMxl74 cells (SIVmac239 > substSp34 > deltaNFkappaBdeltaSpl234 approximately deltaNFkappaBdeltaSp1 > substSpl234). Reversions or additional mutations were not detected in the U3 and R regions of proviral DNA from CEMxl74 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 approximately 2xNFkappaB approximately substNFkappaB > deltaNFkappaB > deltaNFkappaBdeltaSpl234 approximately deltaNFkappaBdeltaSpl > deltaSpl234 approximately substSpl2 > substSp23 approximately substSp34 approximately substSpl234 > or = 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-kappaB element which maintains significant levels of basal transcription in the absence of NF-kappaB and Sp1 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-kappaB and Sp1 binding site mutants of SIVmac is due to unique features or the enhancer/promoter region.
Ilyinskii
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New England Regional Primate Research Center, Harvard Medical School, Southborough, Massachusetts 01772-9102, USA.
Desrosiers
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D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D002478:Cells, Cultured; D002522:Chlorocebus aethiops; D017931:DNA Primers; D015754:Genes, nef; D015497:HIV-1 / Q000502:physiology; D006801:Humans; D007700:Kinetics; D008214:Lymphocytes; D008253:Macaca mulatta; D008264:Macrophages; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D016328:NF-kappa B / Q000235:genetics / Q000378:metabolism*; D011033:Podophyllin / Q000031:analogs & derivatives* / Q000235:genetics / Q000378:metabolism; D011034:Podophyllotoxin / Q000031:analogs & derivatives; D016133:Polymerase Chain Reaction; D017384:Sequence Deletion; D015302:Simian Immunodeficiency Virus / Q000378:metabolism / Q000502:physiology*; D013997:Time Factors; D014158:Transcription, Genetic; D014779:Virus Replication
D017931:DNA Primers; D016328:NF-kappa B; C003170:mitopodozide; D011033:Podophyllin; D011034:Podophyllotoxin