Gene, 116 (1992) 151-158 © 1992 Elsevier Science Publishers B.V. All rights reserved. 0378-1119/92/$05.00 GENE 06502 151 Functional analysis of two long terminal repeats from the HTLV-I retrovirus (Promoter comparison; reporter gene; virus tropism; transient expression; leukemia virus; sequence variation; Tax trans activation) Daniel Gonzalez-Dunia, Sylvie Chirinian-Syan, Michel Brahic and Simona Ozden Unitd des Virus Lents, Ddpartement des Rdtrovirus. UA CNRS 1157, lnstitut Pasteur. 75724 Paris Cedex 15 (France) Received by D.T. Denhardt: 28 December 1991; Revised/Accepted: 16 February 1992; Received at publishers: 16 March 1992 SUMMARY Human T-cell leukemia virus type I (HTLV-I) is associated with a large spectrum of clinical manifestations in man. Viral and host factors are probably involved in determining the consequences of infection. Although most of the genome of HTLV-I appears remarkably stable, considerable variation is observed in the long terminal repeat (LTR) which harbors the promoter region. So far, no correlation between specific mutations and pathogenesis has been found, and the current opinion is that sequence variations reflect the geographical origin ofthe isolate more than the associated pathology. To assess whether the mutations observed between two HTLV-I LTRs were functionally significant, two LTRs, which differ by ten mutations, were coupled to the highly sensitive eukaryotic luciferase-encoding reporter gene, luc, and tested by transfection in a variety of cell lines. Marked differences in promoter activity were observed in some of the cells tested, whereas in others both LTRs were equally active. This result demonstrates that the minor differences observed between two HTLV-I LTRs can affect the activity level of the promoter in some cellular environments, a result which could point to the LTR as one determinant of HTLV-I cell tropism in vivo. INTRODUCTION Human T-cell leukemia virus type I (HTLV-I) causes a variety of diseases including ATL, a chronic neurological Correspondence to: Dr. D. Gonzalez-Dunia, Unit6 des Virus Lents, ln- stitut Paste, ur, 28 rue du Dr. Roux, 75724 Paris Cedex 15 (France). Tel. (33-1)45.68.87.71; Fax (33-1)45.68.88.85. Abbreviations: A, absorbance; ATL, adult T-cell leukemia; bp, base pair(s); BSA, bovine serum albumin; CMV, cytomegalovirus; DMEM, Dulbecco's modified Eagle's medium; FCS, fetal calf serum; HAM, HTLV-l-associated myelopathy; HTLV-I, human T-cell leukemia virus; IU, international unit(s); kb, kilobase(s) or 1000 bp; Lb, lysis buffer; Luc, fireflyluciferase; luc, gene encoding Luc; LTR, long terminal repeat; LU, luciferase unit(s); MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetra- zolium bromide; nt, nucleotide(s); Poilk, Klenow (large) t~agment of E. coil DNA polymerase I; RPML Roswell Park Memorial Institute; SCP, sheep choroid plexus; Tax, trans activating protein of HTLV-I; tax, gene encoding Tax; TRE, Tax-responsive elements; TSP, tropical spastic para- paresis; U3, promoter region of the HTLV-I LTR. disease called either TSP or HAM, and various other dis- orders (Gessain et al., 1985; Hall et al., 1991; Hinuma et al., 1981; Ijichi et al., 1990; Lagrenade et al., 1990; Morgan et al., 1989; Nishioka et al., 1989). The vast majority of infected individuals, however, do not develop any disease. Understanding the complex interactions between viral and host factors that determine the different outcome of this infection is a central question of pathogenesis. The genome of HTLV-I is remarkably stable. All the proviruses which have been totally sequenced (Evangelista et al., 1990; Malik et al., 1988; Seiki et al., 1983; Tsujimoto et al., 1988) are more than 97% homologous; direct se- quencing after amplification of several regions of proviruses obtained from HTLV-I-infected individuals confirmed this observation (Daenke et al., 1990; Komurian et al., 1991). We do not know whether the small number of mutations that distinguish different isolates have biological signifi- cance. The majority of these mutations are found in the U3 region of the LTR, a region that contains the viral pro-