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REGULATION OF GENE EXPRESSION IN HIV-1 AND OTHER COMPLEX RETROVIRUSES 
Bryan R. Cullen, Ph.D., Associate Investigator 
The focus of research in Dr. Cullen's laboratory 
continues to be on the regulation of the replication 
of human immunodeficiency virus type 1 (HIV- 1 ) , 
the etiologic agent of acquired immune deficiency 
syndrome (AIDS), and on the regulatory steps that 
control the replication of other complex retrovi- 
ruses that infect human or animal hosts. 
Regulatory Proteins of HIV-1 
A major interest of the laboratory relates to the 
mechanism of action of Tat and Rev, the two essen- 
tial regulatory proteins encoded by HIV-1 . Both are 
critical to HIV-1 replication in culture. The Tat pro- 
tein is a potent trans-activator of transcription di- 
rected by HIV- 1 's long terminal repeat (LTR) pro- 
moter element. The Rev protein, in contrast, acts 
post-transcriptionally to induce the cytoplasmic ex- 
pression of a subset of HIV- 1 mRNA species that en- 
code the viral structural proteins. Both Tat and Rev 
are similar, however, in that they act through struc- 
tured viral RNA target sites, termed TAR in the case 
of Tat and RRE in the case of Rev. 
During the past year, Dr. Cullen's laboratory has 
used both biochemical and genetic approaches to 
define sequences within the HIV- 1 RRE that are criti- 
cal for Rev binding. Modification interference analy- 
sis was used to delineate a small, ~ 1 3-nucleotide 
RNA sequence within the larger, 234-nucleotide 
RRE that serves as the primary binding site for Rev in 
vitro. The physiological relevance of these in vitro 
observations was confirmed by the in vivo demon- 
stration that a fusion protein consisting of Rev at- 
tached to the Tat carboxyl terminus could activate 
gene expression from an HIV-1 LTR promoter in 
which critical TAR sequences had been replaced by 
this minimal Rev-binding site. 
The demonstration that Rev could serve to direct 
the activation domain of Tat to a promoter-proximal 
RRE-derived RNA target has allowed the genetic dis- 
section of sequences within Tat and Rev that are 
required for either RNA binding or its subsequent 
activation. These studies have also demonstrated the 
existence of a cellular Tat-binding protein that is 
required for not only Tat function but also TAR bind- 
ing in vivo. 
The HIV-1 Nef Protein 
The action of the Rev protein divides the temporal 
pattern of HIV-1 gene expression into an early 
phase , marked by the expression of the " regulatory' ' 
proteins Tat, Rev, and Nef, and a late phase charac- 
terized by the high-level expression of the viral 
structural proteins. While Tat and Rev are both well 
characterized, Nef has remained an enigma. It is 
known, however, as a myristoylated, membrane- 
associated cytoplasmic protein that, in contrast to 
Tat and Rev, is dispensable for HIV-1 replication in 
culture. Many isolates of HIV-1 , in fact, seem to ex- 
press Nef proteins that are defective, a problem that 
has made their functional analysis particularly 
difficult. 
Recently the laboratory of Dr. Ron Desrosiers 
(Harvard University) showed that Nef was abso- 
lutely critical for viral replication and pathogenesis 
in rhesus monkeys infected with a cloned isolate of 
simian immunodeficiency virus (SIV) . This observa- 
tion both rekindled interest in the biological role of 
Nef and provided a Nef isolate that was clearly bio- 
logically active. 
Analysis of the SIV nef gene has demonstrated that 
Nef profoundly down-regulates the cell surface ex- 
pression of CD4, the glycoprotein receptor for both 
HIV-1 and SIV. Nef has no effect on either the rate of 
synthesis or the level of expression of CD4 in these 
cells and must, therefore, be blocking cell surface 
CD4 expression via a post-translational mechanism. 
GENETICS 177 
