Cyclin T1 expression is mediated by a complex and constitutively active promoter and does not limit human immunodeficiency virus type 1 Tat function in unstimulated primary lymphocytes

Cyclin T1 (CycT1), a component of positive-transcription-elongation factor- b (P-TEFb), is an essential cofactor for transcriptional activation by lent ivirus Tat proteins. It is thought that low CycT1 expression levels restric t human immunodeficiency virus type I (HIV-1) expression levels and replica tion in resting CD4(+) lymphocytes. In this study, we undertook a functiona l analysis of the cycT1 promoter to determine which, if any, promoter eleme nts might be responsible for cellular activation state-dependent CycT1 expr ession. The cycT1 gene contains a complex promoter that exhibits an extreme degree of functional redundancy: five nonoverlapping fragments were found to exhibit significant promoter activity in immortalized cell lines, and th ese elements could interact in a synergistic or redundant manner to mediate cycT1 transcription. Reporter gene expression, mediated by the cycT1 promo ter, was detectable in unstimulated transfected primary lymphocytes and mul tiple sites within the promoter could serve to initiate transcription. Whil e utilization of these start sites was significantly altered by the applica tion of exogenous stimuli to primary lymphocytes and two distinct promoter elements exhibited enhanced activity in the presence of phorbol ester, over all cycT1 transcription was only modestly enhanced in response to cell acti vation. These observations prompted a reexamination of CycT1 protein expres sion in primary lymphocytes. In fact, steady-state CycT1 expression is only slightly lower in unstimulated lymphocytes compared to phorbol ester-treat ed cells or a panel of immortalized cell lines. Importantly, CycT1 is expre ssed at sufficient levels in unstimulated primary cells to support robust T at activity. These results strongly suggest that CycT1 expression levels in unstimulated primary lymphocytes do not profoundly limit HIV-1 gene expres sion or provide an adequate mechanistic explanation for proviral latency in vivo.