AbstractFollowing the first two Frederick meetings on virus entry in 1997 [Cell 91 (1997) 721]11[1]. and in 2000 [Cell 101 (2000) 697]22[2]., further developments in our understanding of the multifactorial and multistage process of virus entry, and possible biomedical implications were presented and discussed in a lively fashion by leading scientists from around the world at the third Frederick meeting on the Cell Biology of Viral Entry (May 7–10, Frederick, MD) organized by R. Blumenthal (NCI-Frederick, NIH, Frederick) and E. Hunter (University of Alabama, Birmingham). Unlike the previous two meetings, non-enveloped viruses were not discussed this time, and the focus was how envelope glycoproteins (Envs) mediate entry into cells. Major topics included Env structure, virus receptors, entry intermediates, membrane fusion, fusion kinetics, and rafts. Virus envelope structures will be described in more detail here because the other topics are extensively discussed in the other chapters of this volume

Dimitrov, Dimiter S.

Phogat, Sanjay K.

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Cell biology of virus entry: a review of selected topics from the 3rd International Frederick meeting 

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Biochimica et Biophysica Acta 1614 (2003) 85–88Review
Cell biology of virus entry: a review of selected topics from the
3rd International Frederick meeting
Sanjay K. Phogat, Dimiter S. Dimitrov*
Laboratory of Experimental and Computational Biology, CCR, NCI-Frederick, NIH, Building 469, Room 216, P.O. Box B, Miller Drive,
Frederick, MD 21702-1201, USAReceived 28 April 2003; accepted 15 May 2003Abstract
Following the first two Frederick meetings on virus entry in 1997 [Cell 91 (1997) 721]1 and in 2000 [Cell 101 (2000) 697]2, further
developments in our understanding of the multifactorial and multistage process of virus entry, and possible biomedical implications were
presented and discussed in a lively fashion by leading scientists from around the world at the third Frederick meeting on the Cell Biology of
Viral Entry (May 7–10, Frederick, MD) organized by R. Blumenthal (NCI-Frederick, NIH, Frederick) and E. Hunter (University of
Alabama, Birmingham). Unlike the previous two meetings, non-enveloped viruses were not discussed this time, and the focus was how
envelope glycoproteins (Envs) mediate entry into cells. Major topics included Env structure, virus receptors, entry intermediates, membrane
fusion, fusion kinetics, and rafts. Virus envelope structures will be described in more detail here because the other topics are extensively
discussed in the other chapters of this volume.D 2003 Elsevier B.V. All rights reserved.1. Virus envelope structure and function
Binding to receptor(s) and changes in conformation,
which are the ‘‘heart’’ of the entry machinery, are critically
dependent on the virus 3D structure. Virus envelope
structures were discussed at different levels: whole viruses,
oligomeric Envs, Envs containing the receptor binding
site(s), fusion proteins, and peptides mimicking fusion
proteins. Stephen Fuller discussed extensively major fea-
tures of the class II fusion proteins revealed by cryo-
electron microscopy (cEM) that in combination with image
reconstruction has provided three-dimensional structures of
a whole virus (Semliki Forest Virus (SFV)) at 9 A˚. cEM of
SFV in isolation, and in the presence of liposomes of
varying lipid compositions, provided wealth of information
for the series of stages toward productive fusion and the
rearrangements of the fusion protein during these stages.
cEM faithfully preserves the structures of enveloped0005-2736/03/$ - see front matter D 2003 Elsevier B.V. All rights reserved.
doi:10.1016/S0005-2736(03)00165-2
* Corresponding author. Tel.: +1-301-846-1352 (office), or x5628 or
x5629 (lab) or x5532 (secretary); fax: +1-301-846-5598 (secretary).
E-mail addresses: dimitrov@ncifcrf.gov, dimitrov@helix.nih.gov
(D.S. Dimitrov).
1 [1].
2 [2].viruses and allows their visualization and interpretation of
their three-dimensional structure. cEM also preserves their
infectivity. This is important for the use of cEM with
pathogens.
Mike Lawrence discussed the structure of the ectodo-
main of the fusion glycoprotein of Newcastle disease
virus that has been determined to 3.3 A˚ resolution via
X-ray crystallography. The observed molecule is a homo-
trimer with a club-like shape morphologically consistent
with images via negative stain electron microscopy. He
suggested that because the orientation of the central HR-
A coiled coil could be in the opposite direction (with
respect to the viral membrane) to that observed in
influenza virus haemagglutinin (HA), the structural tran-
sitions accompanying fusion might be quite different for
these two Envs.
Richard Wyatt and his collaborators have analyzed the
crystal structure of the HXBc2 ‘‘core’’ gp120 exterior
envelope glycoprotein in ternary complex with the primary
viral receptor, CD4, and a neutralizing antibody, 17b. The
structural analysis revealed a recessed CD4 binding site that
spanned the two major domains of the gp120 glycoprotein.
The CD4 binding site was characterized by several unusual
cavities evident on the gp120 molecular surface. One of the
gp120 cavities possessed critical contacts with the CD4
S.K. Phogat, D.S. Dimitrov / Biochimica et Biophysica Acta 1614 (2003) 85–8886residue Phe 43, which was positioned at the neck of the
cavity. This cavity is highly conserved and has virtually an
identical relationship to the Phe 43 CD4 residue in the
recently solved YU2 core gp120 crystal structure. The
primary isolate YU2 core glycoprotein has essentially the
same fold as the HXBc2 core, with amino acid and
glycosylation variability predominantly localized to the
gp120 outer domain. Trimer modeling of the two structures
indicates that this variable surface is the region of the
envelope spike most exposed to the immune system and
to potential neutralizing antibodies. Biophysical analysis by
titration microcalorimetry indicated that CD4 binding to
gp120 dramatically decreases gp120 entropy and likely
locks gp120 into a distinct conformation. The flexibility
may be a prerequisite for gp120 to perform its functional
role in receptor binding and viral entry. Several structure-
based mutants have been designed to lock gp120 into a state
that mimics the low entropy, CD4-bound conformation. One
such gp120 mutant, S375W, increases CD4 binding yet
eliminates recognition by several CD4 binding site anti-
bodies. The identification of conformationally fixed glyco-
proteins may provide unique molecules useful for the
elucidation of gp120 structure/function, for the design of
gp120-based vaccines and may provide insights for gp120-
directed drug development.
Dennis Burton presented an analysis of the recognition
of viral envelope glycoproteins by human monoclonal
antibodies with an emphasis on how the recently solved
crystal structure of IgG1 b12 helps our understanding of
HIV neutralization. It appears that a major determinant of
the potent and broad neutralizing activity of b12 is the long
protruding finger-like CDR3 of its heavy chain that allows
high affinity binding to native trimeric HIV envelope
glycoproteins. He pointed out that mouse monoclonal
antibodies might not have such long H3 as human mono-
clonal antibodies that could explain the fact that all potent
broadly HIV neutralizing monoclonal antibodies are hu-
man.
Dimiter Dimitrov, who chaired the session on Viral
Envelope Structure and Function, gave an overview of
the history and the current rapid development of this area
of research. For a very long time, the only structure that has
been solved was that of influenza HA in Don Wiley’s
laboratory, and this was the complete trimeric ectodomain
including both the surface (HA1) and transmembrane
(HA2) proteins. The solution of the tick-borne encephalitis
virus (TBEV) E glycoprotein crystal structure in 1995
initiated a new period of explosive development in the
structural biology of envelope virus entry. For the last 6–7
years, a number of structures have been solved including
portions of Envs responsible for binding (Friend MuV Env,
HIV gp120), fusion (low pH influenza HA2, MoMuLV
TM, HIV gp41, Ebola GP2, SV5F F, HRSV F, Newcastle
Disease Virus F) and even whole viruses (SFV, TBEV
particles, Dengue virus). Since 1995, the structure of the
TBEV E glycoprotein stood alone being very differentcompared to that of HA. While numerous studies confirmed
the original model proposed in 1993 by Peter Kim for the
role of coiled coils in fusion for a number of viruses, until
very recently, there were no other Env structures similar to
that of the TBEV E glycoprotein. A major theme of
discussion at the meeting was the recent proposition by
Felix Ray for two classes of fusion proteins—class I based
on HA, and class II based on the TBEV E glycoprotein
structure. This proposition was based on the newly solved
structure of the SFV E1 glycoprotein and more recently the
one of dengue virus that exhibited structures and confor-
mational changes very similar to those for the E glycopro-
tein without involvement of coiled coil structures that are
critical components of the fusion mechanism mediated by
class I proteins. Dimiter Dimitrov also briefly discussed
recent data from his group on identification and character-
ization of broadly cross-reactive HIV neutralizing human
monoclonal antibodies, and the development of tethered
Envs where gp120 and gp41 are joined by flexible linkers
of variable lengths allowing restricted exposure of entry
intermediates.2. Virus envelope glycoproteins and their receptors
Viruses have evolved to utilize cell surface molecules for
gaining access into the cell interior. Receptor-mediated viral
entry has been documented for Paramyxoviruses such as
measles virus, respiratory syncytial virus, parainfluenza
viruses, and retroviruses such as HIV, RD114 group of
gamma retroviruses, and others. Edward Berger gave an
overview of the various receptors involved in entry of
viruses to cells including CCR5/CXCR4 for HIV and
CD46 for Herpesvirus. James Hoxie described Env-core-
ceptor interactions in CD4-independent isolates of HIV-1
and HIV-2 that are able to directly interact with chemokine
receptors as a result of mutations in the Envs including a
highly cytopathic CD4-independent HIV-2 isolate and rep-
lication competent variants of HIV-2 lacking V1/V2 as well
as major region of V3; these novel variants would also be
tested for generating new immune responses to cryptic
epitopes. John Moore discussed disulfide-stabilized HIV-1
Envs mimicking native cleaved Envs and their potential as
vaccine immunogens. David Kabat described the RD114
group of gamma retroviruses utilizing Na+-dependent trans-
porters as receptors and how sequence alteration in their
extracellular loop 2 and variation in N-linked glycosylation
of this region affects the host-range of this viral family.
James Cunningham discussed cooperative mechanisms of
infection by some retroviruses. Dusty Miller reported iden-
tification of a hyaluronidase family member Hyal2 as the
cell-surface receptor for the Bovine retroviruses jaagsiekte
sheep retrovirus (JSRV) and enzootic nasal tumor virus
(ENTV), and its potential role as a tumor suppressor gene.
Mark Goldsmith discussed coreceptor specificity and HIV
pathogenesis.
S.K. Phogat, D.S. Dimitrov / Biochimica et Biophysica Acta 1614 (2003) 85–88 873. Viral envelope proteins and fusion
This session was chaired by Paul Bates, and lectures
were presented by Robert Lamb (Membrane fusion
machines of paramyxoviruses: capture of intermediates of
fusion), Min Lu (Attempts to define conformational transi-
tion in the HIV-1 Env), Yechiel Shai (The HIV-1 gp41 N-
terminal heptad repeats play an essential role in membrane
fusion), Christopher Broder (Membrane fusion tropism and
heterotypic functional activities of the Hendra and Nipah
virus Envs), Judith White (Activation of a model retroviral
fusion glycoprotein), and John Young (Characterizing the
receptor-priming and low pH-dependent mechanism of
ASLV entry into cells).4. Membrane fusion
This session was chaired by Fred Cohen and talks were
given by Barry Lentz (Spontaneous lipid rearrangements
leading to fusion: lessons from model systems), Michael
Kozlov (Interplay of lipids and proteins in membrane
fusion: theoretical analysis), Joe Bentz (Architecture of the
influenza HA fusion pore), Andreas Hermann (Stability of
the ectodomain of influenza virus hemagglutinin—a theo-
retical and experimental study), and Richard Epand (The
thermal denaturation of influenza virus and its relationship
to membrane fusion).5. HIV kinetics and intermediates
This session was chaired by Leonid Chernomordik and
talks presented by Robert Blumenthal (The HIV Env-medi-
ated fusion reaction), Eric Hunter (Sensitivity of HIV-1 to
fusion inhibitors is modulated by coreceptor specificity and
regions of Env outside of HR1), Jacqueline Reeves (Sensi-
tivity of HIV-1 to entry inhibitors correlates with Env:cor-
eceptor affinity and coreceptor density), Gregory Melikyan
(Evidence that folding of HIV-1 gp41 into a six-helix bundle
coincides with or immediately follows the fusion pore
opening), Hana Golding (Dissection of HIV-1 entry with
neutralizing antibodies to gp41 fusion intermediates), and
Anthony DeVico (Antigenic properties of the HIV Env
during cell–cell fusion).6. Endocytosis, rafts, and viral entry
It has been proposed that sphingolipids and/or glyco-
sphingolipids (GSL) plus cholesterol associate laterally to
form membrane microdomains termed rafts. Such rafts have
been suggested to associate with specific proteins while
excluding others, and to be involved in numerous cellular
functions including membrane trafficking, cell morphogen-
esis, and signaling. Recently, the role of rafts for entry hasbeen the subject of extensive studies. The session on rafts
and endocytosis was chaired by Mark Marsh and presenta-
tions were given by Gary Whittaker (Role of protein kinase
C and clathrin in SFV and influenza virus entry), Warner
Greene (HIV Nef enhances viral particle entry into the
cytoplasm contributing to increased viral infectivity and
HIV pathogenesis), Margaret Kielian (Lipid interactions
with the alphavirus membrane fusion protein), Joshua
Zimmerberg (Effect of microdomain lipids and the trans-
membrane domain of influenza hemagglutinin on clustering
in the plane of the membrane and on membrane fusion),
Santos Manes (HIV-1 entry into the cell: rafting on the cell
surface), and James Hildreth (Role of rafts for HIV entry).7. Selected topics of lively discussions
This meeting is well known with the lively discussions
on interesting and in some cases somewhat controversial
issues. This year was not an exception and the discussions
were frequently very animated. The recent proposition by
Felix Ray for existence of a class II fusion proteins (class I
is based on the HA structure) has generated a lot of
excitement in the field. This proposition was based on the
recently solved structures of the SFV E1 glycoprotein and
the dengue virus. Thus, the Envs of these viruses joined the
TBEV E glycoprotein which stood alone since 1995 to form
a group with fundamentally different mechanism of confor-
mational changes leading to entry where coiled coil struc-
tures play no role in contrast to their prominent role for
entry of influenza, HIV, and a number of other viruses.
Whether there will be class III fusion proteins remains to be
seen.
In an interesting further development of our understand-
ing of HIV fusion kinetics, Robert Blumenthal demonstrated
how the slow kinetics of HIV entry could be of critical
importance for the efficacy of some of the new entry
inhibitors. The fusion kinetics itself is dependent on cor-
eceptor density and envelope coreceptor affinities (Jackie
Reeves). It appears that HIV entry is much more stochastic
than, for example, influenza entry, and the time from
binding to the major receptor, CD4, to binding the core-
ceptor could be on the order of minutes that would allow
sufficient time for inhibitors that bind conserved structures
exposed after CD4 binding to inhibit entry. One example is
the CD4i antibodies that act through a post-CD4 binding
mechanism.
At the previous Frederick meeting, John Young proposed
a new mechanism of viral membrane fusion for the avian
sarcoma and leukosis virus (ASLV) based on priming upon
interaction with its receptor, TVA, followed by activation at
low pH. At that meeting, there were interesting discussions
about these possibilities with Paul Bates and Judy White
being the major opponents. At this meeting, John Young
presented new data supporting his original finding. Judy
White showed data suggesting that lipid mixing in the case
S.K. Phogat, D.S. Dimitrov / Biochimica et Biophysica Acta 1614 (2003) 85–8888of ASLV could occur at neutral pH after initial priming by
Tva receptor.
In another lively discussion, Gregory Melikyan and
Hana Golding attempted to resolve the issue whether the
formation of the six-helix bundles (6HB) occurs simulta-
neously or before the formation of the fusion pore. Gregory
Melikyan identified two types of pores, ‘‘labile pore’’ and
‘‘robust pore’’, based on their sensitivity to inhibitory
peptides and relative density of coreceptor. His data sug-
gested that pore creation, maintenance, and perhaps its
growth are all controlled by formation of the 6HB. Hana
Golding presented data that neutralizing antibodies target-
ing fusion intermediates, including those targeting 6HB,
potently arrested fusion suggesting the presence of 6HB,
which form before the pore formation. Kozlov and Cher-
nomordik suggested that there might be other proteins
outside the fusion pore formation region that might also
play a role in fusion reaction. Yechiel Shai added another
role to the N-terminal heptad repeat besides its role in the
formation of coiled coil structure. He showed that it plays
an important role in merging of membranes and works
synergistically with the fusion peptide (FP) to promote the
lipid mixing.
The laterally associated sphingolipids and/or glycosphin-
golipids (GSL) plus cholesterol form rafts, which could play
a role in viral entry and budding. Santos Manes presented an
interesting approach to study the role of rafts in viral entry.
He made mutants of CD4 that partition in non-rafts com-
partment and showed that the mutant although was efficient
in binding to Env, it did not allow any fusion. Similar,
mutations in influenza HA effected raft partitioning and
fusion phenotype. Based on these observations Josh Zim-
merberg proposed the concept of a ring raft that glues
together the transmembrane domains of HA to make the
fusion complex a functioning machine. However, Gregory
Melikyan and Judy White did not observe a correlationbetween HA raft partitioning and HA fusion phenotype.
Depletion of cholesterol affects rafts. James Hildreth
showed data that depletion of cholesterol using methyl beta
cyclodextrin (mBC) or 2 hydroxy propyl-beta-cycodextrin
prevented HIV infection. He proposed to use mBC as a
chemical condom and the initial trials are ongoing. Many
other interesting topics have been also discussed.8. Future perspectives
The future remains bright for studies of virus entry. The
trend for combining structural, molecular biological, bio-
chemical, and biophysical methods will undoubtedly pro-
vide a better understanding of the molecular mechanisms of
virus entry. Late stages of virus entry including uncoating
should be better characterized. New virus receptors will
certainly be identified. Development of novel inhibitors of
virus entry is urgently needed. We will certainly witness a
more complete understanding of cell biology of virus entry
and, hopefully, the development of novel inhibitors, vac-
cines, and delivery tools.Acknowledgments
We thank some of the meeting participants for their help
in preparing this review and for allowing us to use their
unpublished results.References
[1] D.S. Dimitrov, How do viruses enter cells? The HIV-1 coreceptors
teach us a lesson of complexity, Cell 91 (1997) 721–730.
[2] D.S. Dimitrov, Cell biology of virus entry, Cell 101 (2000) 697–702.


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