STRUCTURE AND RFPLICATION OF INFLUENZA VIRUS AND PARAMYXOVIRUSES 
Roberta. Lamb, Ph.D., Sc.D., Investigator 
A major focus of Dr. Lamb's laboratory is to under- 
stand the structure and function of the integral 
membrane proteins encoded by influenza virus and 
the paramyxovirus SV5 (simian virus 5). In addi- 
tion, the laboratory is investigating the mechanism 
by which these seven integral membrane proteins 
are transported in the exocytotic pathway to the cell 
surface. They provide a diverse group of differing 
prototype membrane proteins that span the mem- 
brane once and are known as type I, type II, and type 
III integral membrane proteins. 
Membrane Protein Orientation Signals 
Dr. Lamb and his colleagues are examining the 
signals necessary to establish the proper orientation 
of integral membrane proteins in a lipid bilayer. The 
paramyxovirus hemagglutinin-neuraminidase (HN) 
polypeptide, a model type II membrane protein, 
contains an internal uncleaved signal/anchor 
(S/A) and is oriented in the membrane with an 
amino-terminal cytoplasmic domain and carboxyl- 
terminal ectodomain. One of the major factors in 
establishing the orientation of this protein is the 
presence of positively charged residues flanking the 
hydrophobic membrane-spanning domain to retain 
this region of the protein in the cell cytoplasm. To 
test further the roles of amino-terminal positively 
charged residues in directing the HN membrane to- 
pology, the three arginine (Arg) residues within the 
1 7-amino acid amino-terminal domain were system- 
atically converted to a glutamine or glutamate, and 
the topology of the mutant proteins was examined 
after expression in CV-1 cells. The data indicate that 
1 ) each of the amino-terminal Arg residues contrib- 
utes to the signal directing proper HN topology, 
since substitutions in any of the three positions re- 
sulted in ~ 13-23% inversion into the N^^^ form; 2) 
substitutions in the Arg directly flanking the S/A do- 
main resulted in slightly more inversion than those 
that were located more distal ly; and 3) substitution 
with a negatively charged glutamate led to more in- 
version than did replacement with an uncharged 
glutamine. 
Virus-mediated Cell-to-Cell Fusion 
Although it has been well established that the par- 
amyxovirus SV5 F protein when expressed in tissue 
culture cells can mediate syncytium formation, 
much evidence reported in the literature indicates 
that for other paramyxoviruses to cause fusion, 
coexpression of the attachment protein (HN) is also 
required. Dr. Lamb and his colleagues have investi- 
gated this further by expressing the SV5 F and HN 
proteins individually or by coexpression in CV-1 
cells with SV40-based vectors and recombinant vac- 
cinia viruses. The extent of detectable fusion in a 
syncytium formation assay was found to be affected 
by the expression system used. In addition, when 
HN was coexpressed with F, the expression vector 
system influenced the contribution of HN in form- 
ing syncytia. When the abilities of the SV5, human 
parainfluenza virus type 3, and Newcastle disease 
virus F proteins to cause fusion (when expressed 
alone or coexpressed with HN) were directly com- 
pared using the SV40-based vector system in CV-1 
cells, the F proteins exhibited various degrees of 
fusion activity independent of HN expression, but 
the formation of syncytia could be enhanced to a 
different extent by the coexpression of the homoty- 
pic HN protein. 
Internalization and Degradation 
of Glycoproteins 
The SV5 HN glycoprotein expressed at the cell 
surface is internalized and degraded in lysosomes. 
HN lacks the typical aromatic amino acid residue in 
its cytoplasmic tail that has been found to be neces- 
sary for the internalization of several well- 
characterized receptor molecules. To investigate 
the nature of the signal encoded within the HN mol- 
ecule that specifies internalization and degradation, 
chimeric molecules have been constructed between 
HN and another type II integral membrane protein 
that is not internalized, influenza virus neuramini- 
dase. The data indicate that the HN transmembrane 
domain specifies internalization and targeting to ly- 
sosomes. Dr. Lamb and his colleagues are attempt- 
ing to analyze the rate of internalization of HN and 
to determine whether it follows the clathrin-coated 
vesicle pathway. The project described above was 
supported by a grant from the National Institute of 
Allergy and Infectious Diseases, National Institutes 
of Health. 
Influenza Virus M2 Protein Has Ion 
Channel Activity 
The influenza virus M2 is a small (97-amino acid) 
integral membrane protein that is minimally a disul- 
fide-linked tetramer. Circumstantial evidence based 
on the sensitivity of influenza virus to the drug 
amantadine hydrochloride, the coupling of antiviral 
action to the transmembrane domain, and the 
premature acid-induced conformational change in 
CELL BIOLOGY AND REGULATION 85 
