ON THE ROLE OF THE 2ND CODING EXON OF THE HIV-1 TAT PROTEIN IN VIRUS-REPLICATION AND MHC CLASS-I DOWN-REGULATION

Tat is an essential protein of human immunodeficiency virus type 1 (HI V-1) and activates transcription from the viral long terminal repeat ( LTR) promoter. The tat gene is composed of two coding exons of which t he first, corresponding to the N-terminal 72 amino acid residues, has been reported to be sufficient for its transcription function. We intr oduced a stop codon at the end of the first Tat-coding exon in an expr ession vector that produces a truncated 71-amino acid Tat protein. Thi s Q72stop mutant displays reduced transcriptional activity of approxim ately 54% in transient LTR-CAT transfection assays. To test the contri bution of the second Tat-coding exon to virus replication, the Q72stop mutation was also introduced in the infectious pLAI molecular clone. The effect on virus replication was analyzed in primary cells and in a transformed T cell line, The fitness of the mutant virus was calculat ed to be approximately 75% compared with the wild-type control. Thus, a small contribution of the C-terminal Tat domain to viral fitness was measured. It has been proposed that the second Tat-coding exon is inv olved in transcriptional downregulation of the MHC class I gene of the infected host cell. Cell surface expression of the MHC protein was an alyzed in T cells infected with the wild-type LAI virus and the replic ation-competent Q72stop mutant. MHC expression was transiently reduced on infection with either virus, indicating that the second Tat-coding exon is not involved in this downregulation.