HUMAN RETROVIRUSES AND CELLULAR GENE EXPRESSION 
Gary J. Nabel, M.D., Ph.D., Associate Investigator 
The replication of viruses in a susceptible host is 
critically dependent on host cell factors essential to 
the function of eukaryotic cells. These factors often 
function to regulate cell proliferation and differen- 
tiation. This laboratory previously showed that 
a cellular transcription factor is responsible for 
transcriptional activation of the human immu- 
nodeficiency virus (HIV) in T lymphocytes and 
macrophages. 
Activation of T cells, w^hich increases HIV expres- 
sion up to 50-fold, correlates with the induction of a 
DNA-binding protein, NF-/cB. Many cellular genes 
that are regulated by NF-kB have been identified, 
and the mechanism of transcriptional activation by 
NF-/cB defined. Furthermore, the role of NF-zcB in 
HIV activation in T cells and monocytes has now 
been established, and the identification of genes en- 
coding /cB-binding proteins achieved. Knowledge 
derived from the study of retroviral and cellular 
gene expression has also been adapted to deliver 
recombinant genes to specific sites in vivo. These 
studies therefore address mechanisms of viral gene 
regulation, provide insight into the regulation of eu- 
karyotic gene expression in vivo, and contribute to 
the treatment of human disease. 
T Cell Activation and HIV Replication 
The transcription factor NF-kB is a protein com- 
plex comprising a DNA-binding subunit of ~ 50 kDa 
and an associated transactivation protein of ~65 
kDa. Both the 50- and 65-kDa subunits have similar- 
ity with the rel oncogene and the Drosophila mater- 
nal-effect gene dorsal. The 50-kDa DNA-binding 
subunit was previously thought to be a unique pro- 
tein derived from the 105-kDa gene product, pi 05. 
This laboratory has isolated a complementary DNA 
that encodes an alternative DNA-binding subunit of 
NF-/CB. 
The new subunit is more similar to pi 05 NF-kB 
than are other family members and defines a new 
subset of re/-related genes. Synthesized as an ~ 1 00- 
kDa protein, pi 00 is expressed in different cell 
types, contains cell cycle or ankyrin motifs, and 
(like pl05) must be processed to generate a 50-kDa 
form. 
A 49-kDa product (p49) can be generated inde- 
pendently from an alternatively spliced transcript. It 
has specific kB DNA-binding activity and can form 
heterodimers with other rel proteins. In contrast to 
the ~ 50-kDa protein derived from pi 05, p49 acts 
in synergy with p65 to stimulate the HIV enhancer 
in transiently transfected Jurkat cells. Thus p49/ 
pi 00 NF-kB could be important in the regulation of 
HIV and other KB-containing genes. 
Because it is possible that specific interactions of 
different subunits can allow selective gene activa- 
tion, the specificity of transcriptional activation by 
different combinations of these subunits has now 
been characterized. The laboratory finds that an 
~ 50-kDa form of pi 00 (p49) binds weakly to kB 
alone but associates with p65 to bind efficiently to 
this site. Furthermore, p49 acts in combination with 
either p65 or a Rel-VPl6 fusion protein to activate 
/cB-dependent transcription in Jurkat T leukemia 
cells. 
The p49-p65 or p49-Rel combination stimulated 
transcription mediated by the canonical kB site but 
did not stimulate interIeukin-2 receptor-a (IL-2Ra) 
or histocompatibility complex kB reporters, despite 
its ability to bind to these sites. Transactivation me- 
diated by the p49/plOO and p65 NF-kB products is 
therefore sensitive to minor changes in the se- 
quence of the kB site. Specificity determined by the 
association of NF-kB subunits provides a mechanism 
for selective regulation of variant kB sites associated 
with different cellular and viral genes. 
NF-kB also functions in concert with the tat-l gene 
product to stimulate HIV transcription. To deter- 
mine whether specific members of the NF-kB family 
contribute to this effect, Dr. Nabel and his col- 
leagues examined the abilities of different NF-kB 
subunits to act with Tat-I to stimulate transcription 
of HFV in Jurkat T leukemia cells. They found that 
the p49(100) DNA-binding subunit, together with 
p65, can act in concert with Tat-I to stimulate the 
expression of HIV-CAT (chloramphenicol acetyl- 
transferase) plasmid. Little effect was observed with 
50-kDa forms of pi 05 NF-kB or rel, in combination 
with p65 or full-length c-rel, which do not stimulate 
the HIV enhancer in these cells. These findings sug- 
gest that the combination of p49(100) and p65 NF- 
kB can act in concert with the tat-l gene product to 
stimulate the synthesis of HIV RNA. 
The chromosomal locations of the human genes 
NFKBl and NFKB2, which encode two alternative 
DNA-binding subunits of the NF-kB complex, pi 05 
and p49/plOO, respectively, have been deter- 
mined. Through Southern blot analysis of panels of 
human-Chinese hamster cell hybrids, pi 05 was as- 
signed to 4q21.1-q24 and p49/pl00 to chromo- 
some 10. The locations were confirmed by fluores- 
cence in situ hybridization and mapped with 
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