What Viruses Are Telling Us About Gene Regulation 
in Mammalian Cells 
Steven Lanier McKnight, Ph.D. — Investigator 
Dr. McKnight is also a staff member in the Department of Embryology at the Carnegie Institution of 
Washington, Baltimore, and Adjunct Professor in the Departments of Biology and of Molecular Biology 
and Genetics at the Johns Hopkins University School of Medicine. He earned his Ph.D. degree in biology 
from the University of Virginia and, except for four years with the Fred Hutchinson Cancer Research 
Center in Seattle, has been with the Carnegie Institution ever since. Dr. McKnight was recently elected to 
the National Academy of Sciences. 
VIRUSES that attack mammalian cells rely on 
preexisting enzymes, factors, and cellular 
functions to negotiate their infectious cycle. Mo- 
lecular studies of virus infection have thereby 
provided key insights into normal cellular pro- 
cesses. For example, studies of the processing 
and transport of membrane glycoproteins that 
form the exterior coats of influenza virus and ve- 
sicular stomatitis virus have helped to explain 
\\oy<i proteins are selectively transported to the 
appropriate cellular compartment. 
Studies of viruses have also illuminated com- 
plex phenomena regarding selective gene ex- 
pression. RNA splicing was first discovered in 
studies of human cells infected by adenovirus. 
Likevi'ise, the capacity of DNA segments known as 
enhancers to regulate gene expression from re- 
mote locations was discovered in studies of sim- 
ian virus 40. 
Work from our laboratory has focused on the 
mechanisms of herpesvirus gene regulation. The 
herpesvirus chromosome contains roughly 50- 
100 genes that are expressed in a tightly con- 
trolled temporal cascade. Early during the in- 
fectious cycle, five immediate-early (IE) genes 
are activated. The IE genes encode protein prod- 
ucts, termed transcription factors, that act to regu- 
late subsequent viral gene expression. Several 
hours later about 25 delayed-early (DE) genes are 
activated. Transcription of DE genes is strictly de- 
pendent on the prior production of IE proteins. 
DE genes encode proteins required for replica- 
tion of viral DNA. Following viral DNA replica- 
tion, about 25 late (L) genes are activated. Her- 
pesvirus L genes encode structural proteins that 
form the intact virus particle, including a com- 
plex set of membrane glycoproteins and struc- 
tural proteins of which the viral capsid is 
composed. 
Interestingly, one of the L gene products en- 
capsidated in the mature virus is a potent and 
specific transcription factor dedicated to the acti- 
vation of IE genes in the subsequent infectious 
cycle. Roughly 1,000 molecules of this protein, 
termed viral protein 16 (VP 16), are packaged 
into the mature virus particle. Upon infection of 
an otherwise healthy cell, VP 16 is released from 
the infecting virus and comes to be associated 
with enhancer elements located upstream from 
each IE gene. Thus emplaced, VP 16 acts as a po- 
tent "trigger" for the rapid and prolific expres- 
sion of IE genes. 
Early studies in our laboratory were focused on 
the herpesvirus DE gene encoding thymidine ki- 
nase (TK) . In order to probe the mechanisms of 
activation by IE proteins, the TK gene was system- 
atically mutated with the aim of defining specific 
regulatory switch points within or around the 
gene. A region of 35 base pairs encompassing the 
site of transcription initiation was identified as 
being responsive to activation by the IE transcrip- 
tion factors. Surprisingly, however, such studies 
identified three additional regulatory DNA se- 
quences located upstream of the TK gene. These 
supplementary regulatory elements were identi- 
fied as binding sites for host cell proteins, includ- 
ing the Spl (selectivity protein 1) transcription 
factor discovered by Robert Tjian (HHMI, Univer- 
sity of California, Berkeley) and his colleagues. 
The involvement of host cell transcription fac- 
tors in viral gene expression has since been ob- 
served in numerous cases. An exciting recent dis- 
covery along such lines has come from Joseph 
Nevins (HHMI, Duke University Medical Center). 
Dr. Nevins and his colleagues have identified a 
human transcription factor, termed E2F (early re- 
gion 2 factor) , that plays a pivotal role in the tran- 
scriptional induction of certain adenovirus 
genes. Remarkably, the E2F factor has been 
shown to form a specific complex with the prod- 
uct of a recessive oncogene encoded by the reti- 
noblastoma locus. Such observations are begin- 
ning to provide mechanistic insight into the role 
of the retinoblastoma protein in growth control 
and cancer. 
More recent studies in our laboratory have fo- 
cused on the activation of herpesvirus IE genes 
during the early stage of infection of cultured 
mammalian cells. As mentioned previously, IE 
gene activation is stimulated by VP 16, a virally 
encoded L protein that is encapsidated in mature 
virus particles. Like the soldiers sequestered in 
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