Structure and Replication of Influenza Virus and Paramyxoviruses 
capable of interacting with GRP78-BiP and that, 
without the binding of GRP78-BiP to the altered 
HN molecules, an increase in the accumulation 
of GRP78-BiP mRNA does not occur. Thus the 
transduction of the signal from the endoplasmic 
reticulum to the nucleus minimally requires the 
binding of GRPTS-BiP to protein. 
Internalization and Degradation 
of Glycoproteins 
The SV5 HN glycoprotein is internalized exten- 
sively from the virus-infected cell surface and de- 
graded in lysosomes. We are intensively examin- 
ing the mechanism of internalization. This is of 
considerable interest because HN lacks an aro- 
matic amino acid in its cytoplasmic tail that has 
been found necessary' for the internalization of 
several well-characterized receptor molecules 
via the clathrin-coated vesicle pathway. Examina- 
tion of chimeric molecules constructed between 
HN and another t>'pe II integral membrane pro- 
tein that is not internalized, influenza virus neur- 
aminidase, suggests that the HN transmembrane 
domain signals internalization from the cell sur- 
face and species targeting to lysosomes. 
Virus Cation Channels 
The influenza virus M2 protein is a small 
(97-residue) type III integral membrane protein 
that forms a disulfide-linked tetramer. Circum- 
stantial evidence based on the virus's sensitivity 
to the drug amantadine hydrochloride, the cou- 
pling of antiviral action to the M2 transmembrane 
domain, and the premature acid-induced confor- 
mational change in the viral hemagglutinin in the 
presence of the drug has led to the suggestion 
that M2 is an ion channel that alters the intracellu- 
lar pH of the trans-Golgi network. Our direct ex- 
periments (in collaboration with Lawrence 
Pinto) of injecting M2 mRNA into Xenopus oo- 
cytes and using a two-electrode voltage-clamp ap- 
paratus to measure surface currents indicate that, 
in the presence of M2, an amantadine hydrochlo- 
ride-sensitive cation channel conductance can 
be measured that has the characteristics of an H 
current, an electrical response previously ob- 
served in heart and neuronal cells. 
We are beginning a structure-function analysis 
of this cation-specific channel to identify the resi- 
dues involved in drug sensitivity and cation speci- 
ficity. We are also investigating the possibility 
that the influenza virus NB glycoprotein, the 
paramyxovirus SH protein, and the human immu- 
nodeficiency virus type I vpu integral membrane 
proteins, all of which have M2-like structures, 
have cation channel activity. 
Virus Fusion Activity 
We are also interested in the manner in which 
the F protein of paramyxoviruses causes cell fu- 
sion. Considerable evidence has implicated the 
hydrophobic amino terminus of the Fj subunit 
(FRED) as being directly involved in membrane 
fusion, and we have shown that the FRED can in- 
teract stably with a lipid bilayer. Comparison of 
the amino acid sequences of many paramyxovi- 
ruses has revealed a high degree of amino acid 
identity in the FRED. To investigate the role of the 
conserved residues in mediating cell fusion, we 
have constructed many mutant F genes in which 
the invariant residue of FRED has been changed, 
in most cases for a similar residue. 
Unexpectedly, in view of the remarkable 
amino acid conservation between paramyxovi- 
ruses, all of the mutant fusion proteins that are 
expressed at the cell surface are capable of caus- 
ing cell fusion, which suggests that the residues 
serve some other function of F protein activity. 
Interestingly, when the highly conserved glycine 
residues in the FRED are changed to alanine resi- 
dues — a change that can be predicted to make 
the FRED more a-helical in nature — the specific 
activity of the F protein in causing cell-cell fusion 
increases by several orders of magnitude. Thus 
we have engineered an even more potent fuso- 
genic protein than the natural F. 
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