Abstract
The human immunodeficiency virus type 1 (HIV-1) Tat protein (hTat) activates transcription initiated at the viral long terminal repeat (LTR) promoter by a unique mechanism requiring recruitment of the human cyclin T1 (hCycT1) cofactor to the viral TAR RNA target element. While activation of equine infectious anemia virus (EIAV) gene expression by the EIAV Tat (eTat) protein appears similar in that the target element is a promoter proximal RNA, eTat shows little sequence homology to hTat, does not activate the HIV-1 LTR, and is not active in human cells that effectively support hTat function. To address whether eTat and hTat utilize similar or distinct mechanisms of action, we have cloned the equine homolog of hCycT1 (eCycT1) and examined whether it is required to mediate eTat function. Here, we report that expression of eCycT1 in human cells fully rescues eTat function and that eCycT1 and eTat form a protein complex that specifically binds to the EIAV, but not the HIV-1, TAR element. While hCycT1 is also shown to interact with eTat, the lack of eTat function in human cells is explained by the failure of the resultant protein complex to bind to EIAV TAR. Critical sequences in eCycT1 required to support eTat function are located very close to the amino terminus, i.e., distal to the HIV-1 Tat-TAR interaction motif previously identified in the hCycT1 protein. Together, these data provide a molecular explanation for the species tropism displayed by eTat and demonstrate that highly divergent lentiviral Tat proteins activate transcription from their cognate LTR promoters by essentially identical mechanisms.
Highlights
The human immunodeficiency virus type 1 (HIV-1) Tat protein activates transcription initiated at the viral long terminal repeat (LTR) promoter by a unique mechanism requiring recruitment of the human cyclin T1 cofactor to the viral TAR RNA target element
To investigate the basis for this human cell-specific defect, we used reporter plasmids based on the wild-type equine infectious anemia virus (EIAV) LTR or on an HIV-1 LTR in which the TAR element remained intact, was replaced with eTAR sequences, or was replaced by the SLIIB minimal HIV-1 Rev RNA binding site
The intact EIAV Tat (eTat) protein potently transactivated chloramphenicol acetyltransferase (CAT) expression from both the pEIAV/ eTAR/CAT and pHIV/eTAR/CAT indicator plasmids in canine cells but not from either pHIV/HIV-1 TAR (hTAR)/CAT or pHIV/ SLIIB/CAT (Fig. 2A). These data demonstrate that replacement of the hTAR element with the eTAR equivalent is, as previously reported [4], entirely sufficient to confer eTat responsiveness in permissive cells and confirm that no DNA sequence specific to the EIAV LTR is required for eTat function
Summary
The human immunodeficiency virus type 1 (HIV-1) Tat protein (hTat) activates transcription initiated at the viral long terminal repeat (LTR) promoter by a unique mechanism requiring recruitment of the human cyclin T1 (hCycT1) cofactor to the viral TAR RNA target element. Critical sequences in eCycT1 required to support eTat function are located very close to the amino terminus, i.e., distal to the HIV-1 Tat-TAR interaction motif previously identified in the hCycT1 protein Together, these data provide a molecular explanation for the species tropism displayed by eTat and demonstrate that highly divergent lentiviral Tat proteins activate transcription from their cognate LTR promoters by essentially identical mechanisms. While both eTat and hTat have the ability to activate transcription via an RNA target, it has remained unclear whether the activities of these two proteins involve similar or different mechanisms, given the sequence and tropism differences outlined above In this context, it is noteworthy that in addition to the hTat cofactor P-TEFb, at least two other proteins, cdk and the herpes simplex virus transactivator VP16, have been shown to activate transcription when artificially recruited to a promoter-proximal RNA element [13, 16, 17, 33]. Recruitment of CycT1–P-TEFb to a promoterproximal RNA element represents a general mechanism by which highly divergent lentiviral Tat proteins activate transcription directed by either primate or ungulate lentivirus LTR promoter elements
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