An alternative pathway for alphavirus entry.

Abstract

The study of alphavirus entry has been complicated by an inability to clearly identify a receptor and by experiments which only tangentially and indirectly examine the process, producing results that are difficult to interpret. The mechanism of entry has been widely accepted to be by endocytosis followed by acidification of the endosome resulting in virus membrane-endosome membrane fusion. This mechanism has come under scrutiny as better purification protocols and improved methods of analysis have been brought to the study. Results have been obtained that suggest alphaviruses infect cells directly at the plasma membrane without the involvement of endocytosis, exposure to acid pH, or membrane fusion. In this review we compare the data which support the two models and make the case for an alternative pathway of entry by alphaviruses.

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REVIEWOpen Access
An alternative pathway for alphavirus entry
Joseph P Kononchik Jr, Raquel Hernandez and Dennis T Brown*
Abstract
The study of alphavirus entry has been complicated by an inability to clearly identify a receptor and by
experiments which only tangentially and indirectly examine the process, producing results that are difficult to
interpret. The mechanism of entry has been widely accepted to be by endocytosis followed by acidification of the
endosome resulting in virus membrane-endosome membrane fusion. This mechanism has come under scrutiny as
better purification protocols and improved methods of analysis have been brought to the study. Results have been
obtained that suggest alphaviruses infect cells directly at the plasma membrane without the involvement of
endocytosis, exposure to acid pH, or membrane fusion. In this review we compare the data which support the two
models and make the case for an alternative pathway of entry by alphaviruses.
Alphaviruses
Alphaviruses are transmitted to vertebrates by hemato-
phagic insects such as mosquitoes and ticks [1]. These
insects are the vectors in the enzootic cycle. Reptiles,
small mammals and birds are primary reservoirs.
Humans and larger mammals are largely a dead end in
the virus life cycle due to the low levels of viremia pro-
duced. Symptoms of alphavirus infection vary from
asymptomatic to encephalitis or arthritis [2]. Of those
that cause disease, Eastern, Western and Venezuelan
encephalitis viruses contribute significantly to disease in
large mammals and humans[3]. Chikungunya is reemer-
ging as a threat to humans and has been gaining ground
in Africa, Asia, the Philippines and Italy with imported
cases in France and in international travelers returning
to the United States and South America [4-8].
Sindbis Virus (SINV), the prototype virus of genus
alphavirus in the family Togaviridae is a group IV mem-
brane containing virus with a positive sense RNA gen-
ome [9]. It is widely used in laboratory studies due to its
non-pathogenic phenotype in humans and biosafety
level 2 containment status. SINV can be grown to high
titer in both mammalian and insect cells [10,11] and has
been shown to infect diverse cell types. Certain strains
of SINV, specifically the heat resistant SINV (SVHR) are
ideal for Alphavirus entry studies. Purified SVHR, in
contrast to other membrane containing viruses, can
have a particle to plaque-forming-unit (pfu) ratio that
approaches unity [11]. Knowing that every particle is
infectious ensures that all observations of cell-virus
interactions are of productive virus infections. These
features of SINV are optimal for the study of entry of
this class of membrane containing viruses including stu-
dies involving direct observation by electron microscopy.
The term virus entry refers specifically to the mechan-
ism by which the virus binds to the host cell receptor,
penetrates the cell membrane barrier and releases the
infectious RNA into the cell initiating the infection.
Alphavirus Structure
Alphaviruses are small 70 nm viruses that have 240 copies
each of three structural proteins, E1, E2 and capsid (C)
assembled in a 1:1:1 stoichiometry. These three proteins
create two nested T = 4 icosahedral shells that sandwich a
host derived lipid bilayer [12] (Figure 1). The outer protein
shell is composed of E1 and E2 heterodimers that assem-
ble into aggregates of three producing the three pronged
spike which protrudes from the virus surface. The inner
protein shell is made of only C, encapsulating the 49S
RNA. This inner protein shell is the shape determining
component of the virus [13,14]. The organization of the
E1/E2 complexes on the cell membrane likely exist in a
two-dimensional 6-fold symmetry sheet prior to capsid
envelopment [15]. When the membrane glycoproteins
begin the process of encapsulation, the nucleocapsid
recruits the E1-E2 trimers into the developing outer shell
by specifically binding the E2 endodomains. Through its
repeated 240 interactions between a hydrophobic cleft on
C and the E2 endodomain this process organizes the
* Correspondence: dennis_brown@ncsu.edu
Department of Molecular and Structural Biochemistry N C State University,
Raleigh, NC 27695, USA
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glycoproteins into the 6-fold and requisite 5-fold rotational
arrays necessary to form a three-dimensional icosahedral
structure [16]. Mutations which disrupt this process result
in structurally misshaped particles [13,17,18] The resulting
virus particle has 80 spikes that are primarily made of E2
(colored blue in Figure 1 pH 7.0) with a protein skirt that
is primarily E1 (colored green in Figure 1 pH 7.0) which
covers the incorporated membrane [19,20]. The two pro-
tein shells with their significant level of lateral and trans-
membrane interactions [21,22] result in a very rigid and
precisely organized particle that is unlike that of other
membrane containing viruses. It has been shown recently,
that the particles of mammalian and insect grown SVHR
are not structurally identical [23]. The cellular response to
infection by insect and mammalian derived virus has also
been shown to be different [24]. Particles produced from
insect cells are more compact, lacking some RNA interca-
lation in the capsid protein shell seen in mammalian
grown virus. The thickness of the membrane of virus pro-
duced from mosquito and mammalian cells does not
solely account for the difference in the structure of the
virus particles; however the outer protein shell seems to
be extended in the mammalian grown virus suggesting
that the protein organization between the two particles
may be in slightly different functional conformations.
The highly organized structure of the alphaviruses with
the many protein-protein interactions stabilizing the
structure and the membrane bilayer occluded by the
outer protein shell have important implications for the
process of entry of the viral genome into host cells.
Whereas many membrane containing viruses such as
influenza can be described as a membranous structure
with embedded proteins the alphaviruses are protein
shells with an associated membrane. This protein shell
must be compromised if the virus is to transfer its RNA
to the cell cytoplasm. The structure of the virus protein
shell may be critical for the mechanism of genome deliv-
ery to work properly. It is hypothesized that the E1 pro-
tein in the mature alphavirus exists in a metastable
configuration poised to use the energy stored during
virus assembly for the entry process upon encountering
an appropriate trigger [25,26]. The energy stored for this
conformational change is the result of folding the E1 pro-
tein though a series of disulfide bonded intermediates as
the protein is compacted into the metastable structure
during the assembly of the spike heterotrimer [25]. The
metastable nature of the spike complex is revealed when
the E1 protein is released from the mature virus using
detergent because the protein reorganizes into several
disulfide bridged configurations which can be separated
on non-denaturing gels[25].
The current molecular model for how the virus trans-
fers its RNA into the cell is hypothesized to be by low
pH mediated membrane fusion, after endocytosis of the
virus attached to its receptor. E2 contains the receptor
binding sequence while E1 is known to contain the
properties necessary for membrane fusion [9]. It is pre-
dicted that as the endosome is acidified, the virus mem-
brane fuses with the endosome and releases the
nucleocapsid into the cell cytoplasm. There are some
reports which implicate ribosomes in the process of
releasing RNA from its association with the capsid pro-
tein [27-29]. The process by which E1 mediates mem-
brane fusion can theoretically be supported by a crystal
structure of E1* (PDB code: 1I9W) which displays the
position and the conformation of the putative fusion
domain [30] and flexible regions within the protein.
This structure was obtained by proteolytic cleavage
from purified Semliki Forest Virus (SFV). A second
structure was obtained by expression of the E1 ectodo-
main in E. coli. [31-33]. This crystal structure of E1 was
subsequently fit into a cryoEM-reconstruction of SINV
(PDB code: 1Z8Y) [34]. The crystal structure of the E1
protein has three primary domains I, II and III. Domain
I is the NH proximal domain that contains the putative
fusion loop. Domain II is the central domain and
domain III is the most distal domain. One important
caveat when interpreting structural data is that the data
represents a native structure or structural intermediate
of the entity in question. This condition would require
that sound biochemical data confirm assumptions about
the structural data without imposing other methods of
confirmation to the analysis. A second condition would
be that the fit of the higher resolution crystal structure
not be distorted in the larger density landscape of the
Figure 1 Cryo-EM reconstruction of Sindbis Virus. Cryo-EM
reconstruction of Sindbis Virus at neutral pH (pH 7.0) and at low pH
(pH 5.3) illustrating the conformational change that occurs at this
threshold pH. The green arrow highlights the protrusion that
appears at the 5-fold vertex at low pH. Reprinted by permission
from Elsevier from: Paredes, A. M., Ferreira, D., Horton, M., Saad, A.,
Tsuruta, H., Johnston, R., Klimstra, W., Ryman, K., Hernandez, R., Chiu,
W., and Brown, D. T. (2004). Conformational changes in Sindbis virions
resulting from exposure to low pH and interactions with cells suggest
that cell penetration may occur at the cell surface in the absence of
membrane fusion. Virology 324(2), 373-86.
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second much more diffuse cryoEM density. One such
analysis put in question the number and location of the
disulfide bonds in the native, infectious form of E1. In
the crystal structure, there are a number of disulfide
bonds identified; supporting the hypothesis that disulfide
bonds play a role in protein assembly [35]. However
some cystines identified as participating in disulfide
bridges in the crystal structure were identified as free
cystines using protein modification and mass spectrome-
try in the intact infectious virus [36]. These conflicts are
not unexpected because the protein crystal structure is
dependent on how the protein is purified, the crystals
are grown, and the structure is refined. It is the nature
of the crystal analysis process that the structure is of a
protein in its lowest energy conformation. This means
that the crystal structure could be any one of the var-
ious intermediate conformations assumed when E1 is
extracted by detergent (described above) or expressed
without its membrane spanning domain in an environ-
ment other than the endoplasmic reticulum.
A second caveat which seriously affects the quality of
the data is that the entity to be studied not be distorted
by the ability of proteins to be manipulated and
expressed in some form in E. coli. There are currently
crystal structures of an E1-E2 fusion protein (PDB codes
3MUU), chikungunya glycoproteins (PDB codes:
3N40;3N41;3N42;3N43;3N44;2XFB;2XFC) and their fit
into the SINV and/or Semliki Forest Virus cryo-recon-
struction (PDB codes: 3MUW;2XFB;2XFC). These struc-
tures were produced by expressing the ectodomains of
the E1 and E2 glycoproteins connected together by a lin-
ker (58). This fusion protein was constructed due to the
inability of expressing E2 in E. coli. While it is common
practice to remove troublesome domains from proteins
to allow for crystallization to occur, how is it determined
when there has been too much manipulation of the
sequence? These structures have the same problems as
E1*. The assumption that the transmembrane domains,
which have been removed in the c DNA clone, and inte-
gration into the ER membrane play no role in the correct
folding and assembly of the spike structure is an unpro-
ven assumption. For virus entry of the macromolecular
SINV particle, structure is critical. The overall alphavirus
icosahedral structure is unique in that it is a membrane
containing virus that does not have the membrane
exposed. The membrane itself is not the form determin-
ing factor as with influenza, and because it is beneath the
outer protein shell is not readily available to fuse with
host membranes. The essential role of the membrane
may be to provide the scaffold upon which the virus is
assembled. To fully explore the mechanism of virus pene-
tration and entry other methods of analysis other than
interpretations of structural fit can provide the missing
events required for SINV infection.
Adsorption and receptor recognition
The process of virus attachment/absorption to the host
cell is probably a multistep event. It is not a new propo-
sal that virus infection begins with scanning of the cell
surface that begins with a general “sticking” to the cell
via one or more proteins (or the cell membrane itself)
followed by “rolling” on the cell surface as it locates the
proper receptor to initiate the penetration step of virus
entry. This type of interaction has been identified
recently using single particle fluorescence resonance
energy transfer (FRET). SINV saturated with a mem-
brane intercalated fluorescent self-quenching dye can be
visualized when the dye is excited with a specific wave-
length light [37]. Fusion of virus with the cell membrane
is detected when lipid mixing (fusion) releases the pro-
teins and dequenching of the dye occurs causing it to
fluoresce. Using this technique it has been shown that
SINV moves on the cell surface in a neutral pH environ-
ment. If the pH of the medium bathing the cells is low-
ered the virus does not fuse with the cell surface as is
the case with influenza, rather the virus “freezes” in
place (K. Wenniger, unpublished observation). These
observations indicate that viruses probe the cell surface
for the proper receptor molecule.
As arboviruses, alphaviruses infect insect and verte-
brate hosts. Since alphaviruses need to infect cells which
provide widely divergent biochemical and genetic envir-
onments, it is likely that they either use a ubiquitous
receptor, or are able to use multiple proteins as a recep-
tor. The receptor/s has not been identified. Many pro-
teins and polysaccharides have been implicated as being
part of the receptor complex. The list includes, heparin
sulfate [38-40], the major histocompatability complex
(MHC) [41,42], the major laminin receptor [43], DC
sign, L sign [44], heat shock 70 protein [45], an uniden-
tified 110 kDa nerve cell protein [46], and a 63 kDa pro-
tein in chicken cells [47]. The length of the list of
possible receptors strongly suggests that there are multi-
ple proteins that alphaviruses can use, and that the spe-
cific receptor is both cell and virus specific. If, however,
there is one widely used receptor it would have to be a
fundamental piece of genetic and biochemical machinery
that has been conserved throughout evolution. The
alphaviruses have evolved to use highly conserved pro-
teins that span the large breath of species that serve as
hosts and/or multiple proteins as a receptors.
The concept that the receptor is, at least in part, pro-
teinaceous comes from a study which showed that pro-
tease treatment of cells prior to adsorption decreased
the number of infected cells. Phospholipase and neura-
minidase treatment did not have an effect on infection
[48]. Using chemical cross-linking, the first candidate as
a receptor was identified by Maassen and Terhorst [42]
as a 90 kDa protein. Following this report there were a
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number of additional studies that employed various
techniques to determine the receptor. One such study
used soluble glycoproteins from Semliki Forest Virus
(SFV) and showed that the MHC bound the glycopro-
teins and that detergent soluble MHC protein was able
to inhibit SFV infection of HeLa cells [41]. Since then, a
number of questions about the validity of this argument
have arisen since cells that lack the MHC complex are
not resistant to SFV infection [49]. Additionally, mos-
quito cells, which fundamentally lack a human immune
system and thus do not express MHC, are also readily
infected by alphaviruses.
The use of anti-idiotypic antibodies as receptor loca-
tors has been used as well to determine the receptor for
alphaviruses. This approach is responsible for the dis-
covery of the 63 kDa chicken protein [47] and even-
tually led to the implication of the high-affinity laminin
receptor [43]. While this is considered a major receptor
for alphaviruses, reexamination of the original experi-
ment that identified the 63 kDa chicken protein revealed
that this protein was not the chicken laminin receptor
[47]. Additionally antibody against the chicken laminin
receptor did not inhibit infection significantly (< 10%).
This suggests that although the laminin receptor is con-
served across many species, it is not the only virus
receptor. It is entirely likely that there are multi-protein
complexes that are not required for, but enhance infec-
tion. Other investigations of the laminin receptor used
anti-idiotype antibodies to examine the laminin receptor
as a possible virus receptor in mosquito cells. In these
studies a 32 kDa protein was discovered in mosquito
cells to which Venezuelan Equine Encephalitis Virus
(VEE) bound as did laminin and SINV [9]. Antibodies
were also used to investigate virus binding with a strain
of SINV which was selected to be a rapid penetrating
virus [50]. Upon binding to the cell at neutral pH assu-
mingly to its receptor, SFV was shown to go through
conformational changes as new antibody epitopes were
exposed on the surface of the virus [51]. This is presum-
ably related to the conformational change that is seen in
reconstructed virus particles which were exposed to low
pH [12].
A measure of successful virus entry
There are numerous techniques that can be used to
examine the interactions between the virus and the cell.
These techniques can generally fall into two categories:
direct and indirect observation. Direct observation uses
familiar techniques such as thin-section microscopy,
cryo-electron microscopy, tomography, and other less
familiar techniques like freeze-fracture immunolabeling
to examine virus-cell interactions. When properly pre-
pared and processed, the interactions between the virus
and cell seen in the microscope are the actual
interactions that occurred at the time of fixation. By
examining different times during the infection process,
the possible mechanism and pathway of virus entry can
be elucidated. Unfortunately, thin-sections and other
direct observation techniques cannot distinguish
between a successful infection process, and that of an
unproductive interaction. This makes microscopy very
subjective and is the primary problem when observing
virus-cell interactions, as even with a relatively low par-
ticle/pfu ratio of 1:10, most observations are of virus-
cell interactions that do not lead to a successful infec-
tion. For SINV, the SVHR strain can be purified to a
particle/pfu ratio of 1:1, (28) and this virus was used in
EM studies of virus infection that led to the direct cell
penetration hypothesis of virus entry [12]. Many of the
previous observations that have been made using EM
are of viruses preparation made with strains or samples
with high or undetermined particle/pfu ratio and there-
fore are difficult to interpret.
When direct observation is not possible, or cannot
answer the question being asked, techniques that take
advantage of indirect or secondary reporters have been
used. Some indirect reporters that have been used as a
measure of successful virus entry include virus RNA
production [52], virus protein synthesis [53] and virus
production [54]. While these tools are useful and have
shed light on many virus-cell interactions, they are at a
disadvantage insomuch that they cannot determine if a
virus has not entered the cell as the events assayed are
not entry. Many of these biochemical reporters are sig-
nificantly downstream of the initial events of virus
adsorption and entry. As a result, each step between
entry and the reporter has the potential to be inhibited
giving a false negative. To give a time-scale to RNA
translation and protein synthesis, super infection inhibi-
tion is detectable 15 minutes post infection [55,56]. This
implies that entry is a very fast process, and that the
genome is quickly unpacked and rapidly available for
processing. The speed of infection, the problems of
indirect reporters, and the limits of direct observation
need to be carefully considered when building an assay
to measure successful virus entry.
Alphavirus entry
Once the virus has bound to its receptor, the virus outer
shell must compromise the plasma membrane barrier.
The classical mechanism of membrane containing
viruses to breach this barrier is by triggering fusion of
the virus membrane with the host cell membrane,
releasing its contents into the cytoplasm [57]. This is
accomplished by a conformational change that can
occur by receptor recognition as with HIV [58,59], or by
environmental changes such as pH or artificially with
heat or reducing agents as seen with SINV [60,61]. The
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conformational change of SINV was visualized using
cyro-electron microscopy reconstruction of particles
exposed to low pH [12]. The change resulted in a pro-
truding spike from the 5-fold axis of the virus, com-
pared to the lack of this density in the untreated
particles (Figure 1, green arrow). The location of the
spike suggests that it is both 5-fold in symmetry, and
that it is likely composed of E1, the glycoprotein that
expresses the putative fusion loop. Interpretation of
these data in the context of the prevailing infection by
fusion model would predict that the virus and cell mem-
branes fuse and the nucleocapsid is delivered to the cell
cytoplasm. However, evident from these reconstructions
is that no lipid is exposed at high pH, or upon return of
the particles to low pH. This observation forms the
basis of the following hypothesis which suggests that
there is no a priori reason to conclude that because a
virus has a membrane, penetration must be by fusion.
Discussed here are two suggested modes of entry for the
alphavirus and the supporting data.
Viral Endocytosis followed by membrane fusion
The most commonly accepted mechanism of entry for
alphaviruses is via endocytosis and the subsequent acidi-
fication of the vesicle leading to membrane fusion. This
is similar to the mechanism accepted for influenza. The
studies in support of this mechanism are extensive, and
rely on the observations that alphaviruses have mem-
brane fusion capabilities, and that inhibition of the acidi-
fication of endosomes inhibits the entry of the virus.
The original fusion assays were done with cultured cells,
and it was shown that treating cells with adsorbed virus
to a brief low pH environment, resulted in cell-cell
fusion (fusion from without) [62]. This suggested the
acid environment of an endosome could provide the
requirements for fusion and penetration. However in
these studies the cells were always returned to neutral
pH before fusion was seen and it was subsequently
shown that this return to neutral pH was required to
induce fusion of the virus and cell membranes[63]. The
pH of the endosome does not return to neutrality. The
analysis of fusion was then moved from a live cell cul-
ture to an in vitro assay including liposomes and other
artificial membranes [64]. In these assays, the fusion
process did not require a return to neutral pH, but
required a significant amount of cholesterol in the target
membrane [65]. This cholesterol requirement was sup-
ported by a report that cells grown in serum treated
with Cab-O-Sil, a silicate that removes cholesterol from
the serum could not be infected as measured by immu-
nofluorescent antibody labeling of newly folded virus
protein [66]. Here a third caveat must be add must be
made, that is how far afield from the native system can
the experimental design be taken before the results are
valid biochemically but cannot reproduce the biological
environment? This should especially be considered in
the context of a biological macromolecule expressing
multiple required functions. There are two specific con-
cerns regarding the cholesterol requirement for infection
that have been neglected. First, the liposome is protein
free and therefore receptor free and second the amount
of cholesterol required to induce fusion of SINV to lipo-
somes is high, up to 50 mol%. However, the require-
ment for such a high concentration of cholesterol is in
direct conflict with the fact that alphaviruses infect and
assemble in mosquitoes, which are cholesterol auxo-
trophs [67,68]. Second, Cab-O-Sil, while removing cho-
lesterol also removes a significant number of other
lipids, which may be required for proper function of the
cells and virus stability [68]. In fact, growing the same
cell type used in the Cab-O-Sil experiment in serum-
free media resulted in no significant change in their sen-
sitivity to infection [68]. Together, these data imply that
a large amount of cholesterol is not required for alpha-
virus infection but is required for fusion with liposomes.
Thus, if cholesterol is not required for infection and
fusion of living cells does not occur at acid pH the lipo-
some model for studying virus penetration may not be
valid.
Treatment of cells with chemicals that inhibit the
acidification of endosomes has been used to assay the
role of acid environment in the infection of mammalian
cells by alphaviruses [69,70]. The assays for successful
penetration were virus RNA or protein production or
the production of progeny virions. It was concluded
from these studies that the mechanism of entry was via
endocytosis and endosome acidification. Drug studies
suffer from the fact that no drug is specific for a single
target and secondary and tertiary effects can affect the
observation made. Because of this inherent problem it is
critical that all possible controls be done and that the
results are interpreted with caution. Drugs such as Bafi-
lomycin A1, chloroquine, monensin and ammonium
chloride (NH4Cl) were used to inhibit endosome acidifi-
cation [69-72]. The initial intent of these experiments
was to demonstrate what the effect of the lack of acidifi-
cation using these chemicals would have on the infec-
tion process. It was found that virus, virus RNA, or
virus protein were not produced when these agents
were present during the period of virus entry and this
was interpreted to indicate that lack of acidification
inhibited the release of the nucleocapsid from the endo-
some into the cell cytoplasm. These early experiments
led to the current model of alphavirus infection by low
pH mediated membrane fusion. However upon more
rigorous analysis it was discovered that these drugs have
various inhibitory effects on virus production not
directly related to virus penetration. Chloroquine was
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