Structure and Replication of Influenza Virus and Paramyxoviruses 
duced by various M2 proteins with changes in the 
transmembrane domain suggests that the domain 
forms the channel pore, and that the M2 protein is 
therefore a channel per se. 
Our experiments have also indicated that the 
M2 protein channel activity is activated by lov^ 
pH, suggesting that the channel is only switched 
on in endosomes and the trans Golgi network — 
-intracellular compartments with lowered pH. 
We are currently beginning a detailed structure- 
function analysis of this channel to characterize 
further the residues involved in drug sensitivity 
and ion specificity. 
We have also shown that the influenza B virus 
NB glycoprotein, which has a similar overall 
structure but no obvious amino acid homology to 
the influenza A virus M2 protein, has ion channel 
activity when expressed in oocytes. However, the 
conductance of the NB ion channel activity is spe- 
cific for chloride ions and is regulated by calcium 
ion concentration. 
The proposed pivotal role of ion channels in 
the replicative cycle of the influenza viruses sug- 
gests that the proteins present an important target 
for a point of intervention by drugs (in addition 
to amantadine) in the prophylaxis and therapy of 
virus infections. We are currently testing whether 
other viral proteins with similar structures to M2 
and NB have ion channel activities — e.g., the 
paramyxovirus SH protein, the vpu integral mem- 
brane protein of the human immunodeficiency 
virus I, and the C3 protein of the coronavirus 
avian infectious bronchitis. 
Intracellular Transport of Glycoproteins 
To elucidate the rules that govern protein ori- 
entation in the lipid bilayer, we are examining 
how polypeptides are initially inserted into the 
endoplasmic reticulum (ER) and are determining 
the signals necessary for the protein-bilayer inter- 
action. One of the major factors is the presence of 
positively charged residues flanking the hydro- 
phobic membrane-spanning domain to retain a 
region of the protein in the cytoplasm. We have 
also been focusing on the factors and signals 
needed to fold the primary polypeptide chain 
once it has been translocated across the mem- 
brane of the ER. 
The cellular glucose-regulated protein GRP78- 
BiP is a member of the HSP70 stress family of 
gene products and is a resident component of the 
ER, where it is thought to play a role in the fold- 
ing and oligomerization of secretory and mem- 
brane-bound proteins. GRP78-BiP also binds to 
malfolded proteins and this may be one mecha- 
nism for preventing their intracellular transport. 
During folding, the SV5 hemagglutinin-neuramin- 
idase (HN) glycoprotein specifically and tran- 
siently associates with GRP78-BiP. This complex 
formation can only be detected prior to oligo- 
merization of the immature HN molecules to 
form the native tetramer, suggesting that GRP78- 
BiP acts as a chaperone to promote correct fold- 
ing of the molecule. 
Internalization and Degradation 
of Glycoproteins 
The SV5 HN glycoprotein is extensively inter- 
nalized from the virus-infected cell surface and 
degraded in lysosomes. We are making an exten- 
sive study of the mechanism of HN internaliza- 
tion. This is of considerable interest because HN 
lacks an aromatic amino acid in its cytoplasmic 
tail that has been found necessary for the internal- 
ization of several well-characterized receptor 
molecules internalized by the clathrin-coated ves- 
icle pathway. Examination of chimeric mole- 
cules constructed between HN and another type- 
II integral membrane protein that is not 
internalized, influenza virus neuraminidase, sug- 
gests that the HN transmembrane domain signals 
internalization from the cell surface and specifies 
targeting to lysosomes. 
Some aspects of research in our laboratory on 
influenza virus and paramyxoviruses are sup- 
ported by grants from the National Institutes of 
Health. 
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