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Journal of Virology, February 2001, p. 1736-1743, Vol. 75, No. 4
Basic Research Laboratory, National Cancer
Institute, National Institutes of Health, Bethesda, Maryland
20892,1 and UMDNJ-New Jersey Medical
School, Newark, New Jersey 071032
Received 1 August 2000/Accepted 20 November 2000
The human immunodeficiency virus type 1 (HIV-1) Tat protein has
been reported to transactivate several cellular genes, including the
potent chemotactic factor interleukin-8 (IL-8). Consistent with these
in vitro assays, elevated levels of IL-8 protein are found in the serum
of HIV-infected individuals. We now extend these observations by
demonstrating that Tat induction of IL-8 is linked to the cell cycle.
Cells that constitutively express the Tat(1-86) protein (eTat) and
control cells (pCEP) were reversibly blocked at the G1/S
border with hydroxyurea or thymidine. The cells were subsequently
released, and IL-8 expression was monitored by RNase protection assays
and enzyme-linked immunosorbent assay (ELISA). RNase protection assays
demonstrated that IL-8 mRNA expression is transiently induced,
approximately fourfold, as the Tat-expressing cells enter S phase.
Consistent with the RNase protection assay, an increase in IL-8 protein
was observed in the cell supernatant using an IL-8 ELISA. Similar
experiments were performed following a reversible block at the
G2/M border with nocodazole and release into
G1. Using the RNase protection assay and ELISA, little or no increase in IL-8 expression was observed during G1.
Using gel shift as well as an immobilized DNA binding assay, we
demonstrate that the increase in IL-8 gene expression correlates with a
specific increase in p65 NF-
0022-538X/01/$04.00+0 DOI: 10.1128/JVI.75.4.1736-1743.2001
Cell Cycle Regulation of Human Interleukin-8 Gene
Expression by the Human Immunodeficiency Virus Type 1 Tat
Protein
B binding activity only in the nucleus
of the Tat-expressing cells. Moreover, the CREB-binding protein
coactivator is present in the complex in the Tat cell line. Finally, we
demonstrate that the presence of the proteasome inhibitor MG-132
inhibits the induction of NF-
B binding, as well as IL-8 expression,
supporting the role of NF-
B.
*
Corresponding author. Mailing address: Virus Tumor
Biology Section, Basic Research Laboratory, DBS, NCI, NIH, Building 41, Room B201, Bethesda, MD 20892. Phone: (301) 496-0986. Fax: (301) 496-4951. E-mail: bradyj{at}exchange.nih.gov.
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