On the role of the second coding exon of the HIV-1 Tat protein in virus replication and MHC class I downregulation.
Tat is an essential protein of human immunodeficiency virus type 1 (HIV-1) and activates transcription from the viral long terminal repeat (LTR) promoter. The tat gene is composed of two coding exons of which the first, corresponding to the N-terminal 72 amino acid residues, has been reported to be sufficient for its transcription function. We introduced a stop codon at the end of the first Tat-coding exon in an expression vector that produces a truncated 71-amino acid Tat protein. This Q72stop mutant displays reduced transcriptional activity of approximately 54% in transient LTR-CAT transfection assays. To test the contribution 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 calculated 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 involved in transcriptional downregulation of the MHC class I gene of the infected host cell. Cell surface expression of the MHC protein was analyzed in T cells infected with the wild-type LAI virus and the replication-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.
Verhoef
K
K
Department of Human Retrovirology, Academic Medical Center, University of Amsterdam, The Netherlands.
Bauer
M
M
Meyerhans
A
A
Berkhout
B
B
D000818:Animals; D019556:COS Cells; D002478:Cells, Cultured; D015536:Down-Regulation; D005091:Exons; D015696:Gene Products, tat / Q000235:genetics*; D016325:HIV Long Terminal Repeat; D015497:HIV-1 / Q000235:genetics* / Q000502:physiology; D006367:HeLa Cells; D015395:Histocompatibility Antigens Class I / Q000235:genetics*; D006801:Humans; D014158:Transcription, Genetic; D014779:Virus Replication; D054322:tat Gene Products, Human Immunodeficiency Virus
D015696:Gene Products, tat; D015395:Histocompatibility Antigens Class I; D054322:tat Gene Products, Human Immunodeficiency Virus