The main goal of this laboratory is to use retroviruses
as model systems to study the molecular and cellular
biology of higher eukaryotes. Until recently, our
major focus has been on human immunodeficiency virus
type 1 (HIV-1), a virus that is not only a major pathogen
but also a uniquely complex retrovirus with several
unusual molecular attributes. One particularly intriguing
feature of HIV-1 is that it encodes two small yet
essential regulatory proteins, termed Tat and Rev.
The Tat protein is a potent trans-activator of transcription
directed by the HIV-1 long terminal repeat (LTR) promoter
element. The Rev protein, in contrast, acts post-transcriptionally
to induce the nucleocytoplasmic transport of a subset
of HIV-1 mRNA species that encode the viral structural
proteins. Both Tat and Rev are similar, however, in
that they act through structured viral RNA target
sites. These are termed TAR in the case of Tat and
RRE in the case of Rev. We have used a combination
of biochemical and genetic approaches to define the
functional organization of these critical viral regulatory
proteins. In the case of Rev, this work has demonstrated
the existence of an N-terminal protein domain that
mediates not only the direct binding of Rev to a discrete
RNA site within the RRE but also a subsequent multimerization
of Rev on the RRE. A second, leucine-rich motif, located
proximal to the C-terminus of Rev, is dispensable
for RNA binding but essential for Rev function in
vivo. This motif is required for recruitment of an
essential cellular Rev co-factor to the Rev:RRE ribonucleoprotein
complex and functions as an autonomous protein nuclear
export signal (NES) when attached to carrier proteins.
This cellular co-factor has now been identified as
Crm1, a cellular factor that also mediates the nuclear
export of a wide variety of cellular protein substrates,
most of which contain a leucine motif similar to the
one found in HIV-1 Rev. Crm1 binds to this leucine
motif and then recruits the resultant complex to the
nuclear pores by directly binding to nuclear pore
components. In addition to Rev, we have also expended
considerable effort on understanding the viral Tat
protein. Tat has been shown to directly bind to the
viral TAR RNA target in a complex with a cellular
factor, termed cyclin T1, which in turn is bound to
the kinase cdk9. The TAR element forms the first 59
nucleotides of the viral genome and recruitment of
the Tat:cyclin T1:cdk9 complex to TAR results in the
phosphorylation of a critical segment of the RNA polymerase
that transcribes the HIV-1 provirus. This in turn
dramatically increases the efficiency of transcription
of the viral genome. One interesting aspect of Tat
is that it does not function effectively in rodent
cells due to the inability of rodent cyclin T1 molecules
to bind the viral TAR element, although they interact
with the Tat protein itself perfectly well. This restriction
reflects the absence of a critical cysteine residue
that is replaced in rodent cyclin T1 by a tyrosine.
Remarkably, insertion of this cysteine into rodent
cyclin T1 is fully sufficient to rescue Tat function
in rodent cells, thus raising the possibility that
rodents, such as mice, might be able to support HIV-1
replication and pathogenesis if made appropriately
transgenic.
