REPLICATION AND PATHOGENESIS OF RNA VIRUSES 
Michael M.-C. Lai, M.D., Ph.D., Investigator 
RNA viruses, which include many common hu- 
man and animal pathogens, provide important in- 
sights into general and unique features of the molec- 
ular biology of normal cells. Dr. Lai's laboratory has 
been studying two different RNA viruses that cover 
two extremes of the whole spectrum of RNA viruses. 
Molecular Biology and Pathogenesis 
of Hepatitis Delta Virus 
Hepatitis delta virus (HDV) is associated with a 
severe form of hepatitis with a high probability of 
developing chronic hepatitis and liver cirrhosis. 
HDV contains the smallest viral genome among ani- 
mal viruses and thus must utilize its genetic infor- 
mation economically and rely on the host cells for 
its own replication. The genome is a single- 
stranded, circular RNA of 1 .7 kb that may have been 
evolved from viroids, a plant pathogen. Dr. Lai and 
his colleagues demonstrated that the HDV RNA con- 
tains a "ribozyme" activity that can autocatalyti- 
cally cleave and ligate itself. They have now charac- 
terized the in vitro and in vivo sequence 
requirement of this catalytic activity, which repre- 
sents a new class of ribozyme. Furthermore, they 
have demonstrated that a cell-specific factor may 
modulate the ribozyme activity, and that the ribo- 
zyme is required for HDV RNA replication, which is 
carried out by cellular RNA polymerase O via a dou- 
ble rolling circle model. 
HDV RNA encodes a single protein, hepatitis delta 
antigen (HDAg), from its antigenomic strand. This 
protein is the signature protein of HDV and is re- 
quired for HDV RNA replication. Dr. Lai's laboratory 
has identified it to be a nuclear phosphoprotein 
with an RNA-binding activity specifically binding 
HDV RNA. The structure of HDAg has been dis- 
sected, and several functional domains have been 
identified. The first is a nuclear localization signal, 
which consists of two discontiguous basic amino 
acid-rich sequences. The second is an RNA-binding 
signal, which is composed of two discontiguous ar- 
ginine-rich motifs (ARMs). The third is a leucine 
zipper-like sequence that promotes the oligomer- 
ization of HDAg. Dr. Lai and his colleagues have 
demonstrated that all of these activities are required 
for the replication of HDV RNA. They have proposed 
a model for the mechanism of action of HDAg, devel- 
oped monoclonal antibodies specific for HDAg, and 
identified a potential domain important for the tran- 
scription function of HDAg. HDAg may be important 
for directing cellular enzymes to carry out RNA- 
dependent RNA synthesis. 
Additional features of HDV biology were studied, 
including the heterogeneity and evolution of HDV 
RNA, which can be correlated with viral pathogenic- 
ity. HDV RNA has a high mutation rate, which may 
be responsible for the fluctuation in the clinical 
course of delta hepatitis. 
Molecular Biology and Pathogenesis 
of Coronaviruses 
Coronaviruses include a group of viruses that 
cause the common cold in humans and a variety of 
severe gastrointestinal and respiratory diseases in 
animals. One type also causes symptoms similar to 
those of multiple sclerosis, thus providing a model 
system for studying that disease. Dr. Lai's laboratory 
has demonstrated that mouse hepatitis virus (MHV), 
one of the prototype coronaviruses, contains an RNA 
genome that is 31,000 nucleotides long, which is 
the longest viral RNA known to exist in nature. This 
RNA contains various genes encoding viral struc- 
tural and nonstructural proteins, whose properties 
and biological activities are currently being studied 
in Dr. Lai's laboratory. 
The laboratory has demonstrated that MHV em- 
ploys a unique discontinuous transcription mecha- 
nism that fuses a leader RNA to genes located some 
distance downstream to synthesize mRNAs. This 
transcription mechanism is an alternative to the con- 
ventional RNA-splicing mechanism seen in most 
mammalian cells. During the past year. Dr. Lai and 
his colleagues have continued to unravel the details 
of this mechanism. They have identified new tran- 
scription initiation sites and characterized the struc- 
ture of template RNAs. This mechanism allows the 
leader sequence to control the expression of various 
viral genes. Thus, by altering the sequence of the 
leader RNA, the composition of viral proteins 
may vary. One most notable example is the 
hemagglutinin-esterase (HE) protein, whose pres- 
ence is variable from virus to virus. Dr. Lai's labora- 
tory has demonstrated that this protein is rapidly lost 
as virus evolves, and as a result the biological prop- 
erties of the virus change. This rapid evolution of a 
viral protein is unique among viruses. 
Dr. Lai's laboratory previously discovered the phe- 
nomenon of RNA recombination in coronavirus. Al- 
though once considered to be rare, genetic recombi- 
nation has in recent years been increasingly 
CELL BIOLOGY AND REGULATION 83 
