Upregulation of c-Fos in activated T lymphoid and monocytic cells by human
immunodeficiency virus-1 Tat protein
D Gibellini, A Caputo, S Capitani, M La Placa and G Zauli
Institute of Microbiology, University of Bologna, Italy.
The regulatory Tat protein of the human immunodeficiency virus type-1
(HIV-1) is essential for viral replication and also shows pleiotropic
activities on various cell functions. To get further insights into the
molecular mechanisms underlying the biological activity of Tat, we
investigated the effect of endogenous and exogenous Tat protein on c- fos
gene expression in T lymphoblastoid (Jurkat) and monocytic (U937) cell
lines, as well as in primary peripheral blood mononuclear cells (PBMC).
Transient cotransfection of tat cDNA in sense orientation (tat/S), together
with a plasmid containing the c-fos promoter (FC3, from -711 to +42) in
front of the bacterial chloramphenicol acetyltransferase (CAT) gene
significantly enhanced CAT activity in Jurkat cells activated by the
addition of 15% fetal calf serum (FCS) or 5 micrograms/mL
phytohemagglutinin plus 10(-7) mol/L phorbol myristate acetate (PMA) and
U937 cells activated by 15% FCS or 10(-7) mol/L PMA. This effect was
specifically due to Tat, since Jurkat and U937 cells cotransfected either
with tat cDNA in antisense orientation (tat/AS), tat carrying a mutation in
the aminoacid cys22-gly22 (tat 22/S) or with the backbone vector alone
(pRPneo-SL3) did not show any significant difference in c-fos promoter
activity as compared to cells transfected with FC3 plasmid alone. By using
deletion mutants of the c-fos promoter, we found that the minimal DNA
sequence required for Tat activity was located between nucleotides
-404/-220 and that the serum responsive element (SRE, -317/-288), present
within this region, was still responsive to Tat. A single point mutation in
the SRE completely abrogated the responsiveness to tat/S. Exogenous
recombinant Tat protein was also able to upregulate c-fos promoter activity
in serum- activated Jurkat and U937 cells, as well as endogenous c-fos mRNA
expression and c-Fos protein synthesis in both serum-activated cell lines
and primary PBMC. c-Fos protein was shown essential for an optimal
transactivation of the HIV-1 long terminal repeat (LTR) by Tat: incubation
of Jurkat cells with antisense, but not sense, c-fos oligonucleotides
significantly reduced either the Tat-enhanced expression of an LTR-CAT
reporter construct or the levels of gag p24 in the culture supernatants of
Jurkat cells and PBMC acutely infected with HIV-1. Our data suggest that
the c-fos upregulation mediated by Tat might play a significant role in the
control of viral gene transactivation.
Volume 89,
Issue 5,
pp. 1654-1664,
03/01/1997
Copyright © 1997 by The American Society of Hematology
