Transcriptional activation of minimal HIV-1 promoter by ORF-1 protein expressed from the SalI-L fragment of human herpesvirus 6.
The SalI-L fragment of human herpesvirus 6 (HHV-6) strain U1102 transformed rodent cells and transactivated the HIV-1 LTR 10- to 15-fold in both monkey fibroblasts and human T-lymphocytes. In this report, the SalI-L transactivator of the HIV-1 LTR was localized to ORF-1 which codes for a protein of 357 amino acids. To determine if ORF-1 required functional Sp1 binding sites or the TATA box element of HIV-1 LTR for transactivation, 5'-deletion mutants of the HIV-1 LTR were employed. Plasmids pBS/SalI-L, pBS/SalI-L-SH, and pC6/ORF-1(S), a mammalian expression vector containing ORF-1, all transactivated a deletion mutant of HIV-1 LTR lacking functional Sp1 binding sites (CD-54). These studies demonstrate that transactivation occurred in the absence of Sp1 binding sites and required only a minimal HIV-1 promoter which contains the TATA box element. The specificity of the SalI-L transactivator for HIV-1 LTR was demonstrated by its inability to transactivate the human papillomavirus type 16 or 18 early promoters. The ORF-1 gene was cloned into and expressed from the pET17b bacterial expression vector. Purified ORF-1 protein was obtained by ammonium sulfate precipitation, Mono-S chromatography, and anti-T7. Tag immunoaffinity chromatography. Transactivation of the HIV-1 LTR by ORF-1 protein was demonstrated by electroporation studies in vivo and by transcription studies in vitro. To substantiate the putative biological role of ORF-1, pBS/SalI-L, pBS/SalI-L-SH, and pC6/ORF-1 all reactivated tat-defective HIV-1 provirus from latently infected cells expressing CD4. Thus, the data presented suggest that HHV-6 infection could have a cofactor role in the progression of AIDS.
Kashanchi
F
F
Department of Microbiology and Immunology, Georgetown University Medical Center, Washington, DC 20007.
Thompson
J
J
Sadaie
M R
MR
Doniger
J
J
Duvall
J
J
Brady
J N
JN
Rosenthal
L J
LJ
D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D017353:Gene Deletion; D005814:Genes, Viral / Q000235:genetics; D016655:HIV Core Protein p24 / Q000032:analysis; D016325:HIV Long Terminal Repeat / Q000235:genetics*; D015497:HIV-1 / Q000235:genetics* / Q000502:physiology; D015654:Herpesvirus 6, Human / Q000235:genetics*; D006801:Humans; D008969:Molecular Sequence Data; D009857:Oncogenes; D016366:Open Reading Frames / Q000235:genetics; D011401:Promoter Regions, Genetic / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000032:analysis / Q000235:genetics; D016415:Sequence Alignment; D016329:Sp1 Transcription Factor / Q000235:genetics; D015534:Trans-Activators / Q000032:analysis / Q000737:chemistry / Q000235:genetics*; D015533:Transcriptional Activation / Q000235:genetics*; D014162:Transfection; D014407:Tumor Cells, Cultured; D014764:Viral Proteins / Q000032:analysis / Q000737:chemistry / Q000235:genetics*; D014775:Virus Activation; D017735:Virus Latency
D016655:HIV Core Protein p24; C087150:ORF-1 protein, Human herpesvirus 6; D011993:Recombinant Fusion Proteins; D016329:Sp1 Transcription Factor; D015534:Trans-Activators; D014764:Viral Proteins