HUMAN RETROVIRUSES AND COORDINATE GENE EXPRESSION IN T CELLS 
Gary J. Nabel, M.D., Ph.D., Assistant Investigator 
A major interest of Dr. Nabel's laboratory is to 
understand tiow viruses interact with host cell fac- 
tors to replicate successfully in eukaryotic cells. 
Viral replication is often dependent on transcrip- 
tion factors relevant to cell proliferation and differ- 
entiation. These studies therefore not only address 
mechanisms of viral gene regulation but also pro- 
vide insights into eukaryotic gene expression. 
Dr. Nabel previously showed that activation of 
T cells, which increased human immunodeficiency 
virus (HIV) expression up to 50-fold, correlated with 
the induction of a DNA-binding protein, NF-kB. The 
laboratory has begun the identification of cellular 
genes regulated by NF-kB, to understand the mech- 
anism of transcriptional activation by NF-kB, to de- 
termine its role in HIV activation in other cell types 
(e.g., monocytes), and to identify genes encoding 
kB binding proteins. The cellular genes regulated 
by NF-kB in non-B cells were previously unknown, 
although related sites are present in the enhancers 
of several genes, including class I and II major his- 
tocompatibility genes and p^'^^i'^i'oglobulin. A se- 
quence related to the kB upstream region of the 
interleukin-2 receptor a (IL-2Ra) gene, designated 
IL-2R kB, has been identified. It is identical to kB at 
9 of 11 base pairs and competes for binding to 
NF-kB. Unlike kB in Ig light chain or Hiy mutation 
of this site does not affect induction by phorbol my- 
ristate acetate (PMA); however, this site is required 
for stimulation of the IL-2Ra enhancer by tax^ of 
human T cell leukemia virus I (HTLV-I). These data 
suggested that a KB-like site may play an important 
role in the regulation of a T cell-specific gene and 
that there is heterogeneity in function that may be 
mediated by related kB sites. 
To understand potential mechanisms of HIV acti- 
vation. Dr. Nabel has examined viral transactivators 
that stimulate the HIV enhancer. Viral genes may 
provide insight into how HIV expression could be 
activated and may also be clinically relevant to co- 
factors that accelerate HIV disease. Cotransfection 
of the adenovirus ElA gene had previously been 
shown to increase HIV-CAT (chloramphenicol ace- 
tyltransferase) expression in Jurkat cells, and activa- 
tion by ElA was dependent on the TATA box. Be- 
cause ElA stimulated the HIV enhancer through 
the TATA box, the possibility was considered that 
the TATA box mediated activation by NF-kB in this 
enhancer. When HIV-CAT plasmids with mutant 
TATA boxes were transfected into Jurkat cells, they 
remained responsive to PMA activation through 
NF-kB and used the same transcriptional initiation 
site, even though basal levels of expression were re- 
duced 10- to 50-fold. Inversion of the TATA box or 
substitution with the SV40 TATTT sequence re- 
vealed a similar response, suggesting that the TATA 
sequence in the HIV enhancer functions as a posi- 
tion- and orientation-dependent, positive regula- 
tory element. 
Although cells of the monocyte lineage provide a 
reservoir for HIV production in vivo, the regulation 
of HIV transcription in these cells had not been de- 
fined. When regulatory elements required for HIV 
gene expression in the monocyte lineage were ex- 
amined. Dr. Nabel and his colleagues found that it 
was regulated by NF-kB; however, the regulation 
of NF-kB in the monocyte lineage differed from 
T cells. NF-kB binding became active at a discrete 
stage of monocyte differentiation and was thereaf- 
ter present constitutively in the mature monocyte 
and macrophage. Its binding activity correlated 
with the transition from promonocyte to monocyte. 
In a chronically infected promonocytic cell, Ul, 
HIV-1 replication was also activated by cellular dif- 
ferentiation, and this activation correlated with 
NF-kB binding activity. These findings suggested 
that NF-kB binding and subsequent HIV expression 
are associated with differentiation in the monocyte 
lineage. Other factors, such as granulocyte macro- 
phage colony-stimulating factor (GM-CSF), appar- 
ently exert their control through a mechanism 
independent of NF-kB, since GM-CSF does not in- 
duce NF-kB binding, and activate HIV expression in 
mature macrophages, which already contain NF-kB. 
These findings suggest that at least two signals 
modulate synthesis and secretion of HIV in mono- 
nuclear cells, with NF-kB induction in the mono- 
cytic lineage providing one signal that may initiate 
viral transcription. 
NF-kB binds to the IL-2R kB site, but the IL-2R kB 
site differs from kB because it does not respond to 
induction by PMA in Jurkat T leukemic cells, sug- 
gesting possible heterogeneity among proteins that 
bind to related KB-like sites. To define the biochem- 
ical basis of kB specificity, the laboratory has devel- 
oped binding conditions for the electrophoretic 
mobility shift assay to detect other kB binding pro- 
teins. This approach was used to define an IL-2R kB 
binding protein distinct from NF-kB. Binding of this 
factor to the IL-2R kB site under these conditions is 
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