Avian sarcoma and leukosis virus-receptor interactions: From classical
genetics to novel insights into virus–cell membrane fusion
R.J.O. Barnard, D. Elleder, J.A.T. Young*
The Infectious Disease Laboratory, The Salk Institute, La Jolla, San Diego, CA 92037-1099, USA
Received 30 August 2005; accepted 10 September 2005
For over 40 years, avian sarcoma and leukosis virus (ASLV)-receptor interactions have been employed as a useful model system to study the
mechanism of retroviral entry into cells. Pioneering studies on this system focused upon the genetic basis of the differential susceptibilities of
different lines of chickens to infection by distinct subgroups of ASLV. These studies led to the definition of three distinct autosomal recessive
genes that were predicted to encode cellular receptors for different viral subgroups. They also led to the concept of viral interference, i.e. the
mechanism by which infection by one virus can render cells resistant to reinfection by other viruses that use the same cellular receptor. Here, we
review the contributions that analyses of the ASLV-receptor system have made in unraveling the mechanisms of retroviral entry into cells and
focus on key findings such as identification and characterization of the ASLV receptor genes and the subsequent elucidation of an unprecedented
mechanism of virus–cell fusion. Since many of the initial findings on this system were published in the early volumes of Virology, this subject is
especially well suited to this special anniversary issue of the journal.
D 2005 Published by Elsevier Inc.
Keywords: Avian sarcoma leukosis virus; Retroviral entry; Viral receptors; Membrane fusion
ASLV receptors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
ASLV Env and viral fusion proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
ASLV entry. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Concluding remarks and future perspectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Avian sarcoma and leukosis viruses are divided into 10
different viral subgroups (designated A–J) that are based, in
part, on the specific cellular receptors that they use (Weiss,
In the 1960s and early 1970s, cross-breeding experiments
performed with different chicken lines indicated that three
independent genes (denoted tv for tumor virus) control
susceptibility to infection by ASLV viral subgroups A–E
(for a review, see Weiss, 1993). These loci were predicted to
encode different subgroup-specific viral receptors. The tva
and tvc loci were genetically linked and susceptibility alleles
of each locus (tvasand tvcs) conferred susceptibility of viral
subgroups A and C, respectively (Crittenden et al., 1967;
Duff and Vogt, 1969; Hanafusa, 1965; Payne and Biggs,
1964; Payne and Pani, 1971; Vogt and Ishizaki, 1965). The
tvb locus was predicted to encode cellular receptors for
ASLV subgroups-B, -D, and -E (Crittenden et al., 1967;
Payne and Biggs, 1966; Rubin, 1965; Vogt and Ishizaki,
1965). Two functional alleles of tvb were defined, tvbS1
which conferred susceptibility to infection by all three viral
0042-6822/$ - see front matter D 2005 Published by Elsevier Inc.
* Corresponding author. Fax: +1 858 554 0341.
E-mail address: email@example.com (J.A.T. Young).
Virology 344 (2006) 25 – 29
subgroups and tvbS3for viral subgroups-B and -D only
(Crittenden and Motta, 1975; Crittenden et al., 1973).
Moreover, other alleles were defined at each locus (desig-
nated as tvaR, tvbR, tvcR) that were associated with host
resistance to infection by the cognate viral subgroups, and
each of these was recessive in nature.
The gene products of these three putative receptor loci
remained unknown until the development of techniques that
allowed the introduction of whole genomes into populations
of transfected cultured cells. The first of the genes identified
by this method was tva (Bates et al., 1993; Young et al.,
1993). TVA, a low-density lipoprotein (LDL) receptor-
related protein with a single extracellular LDL-A module,
was found to possess the features expected of a bona fide
subgroup A viral receptor. It bound specifically to the
ASLV-A envelope glycoprotein, via residues in the LDL-A
module, and conferred susceptibility solely to ASLV
subgroup A infection when expressed in receptor naive
cells. Two different isoforms of TVA are generated by
alternative splicing, a transmembrane version with a single
membrane-spanning domain, and a form with a glycosylpho-
sphatidylinositol (GPI) anchor (Barnard and Young, 2003).
The second ASLV receptor gene to be identified by this
method was tvb. The chicken tvbs3and tvbs1alleles were
characterized and, as predicted, encoded cellular receptors for
viral subgroups-B, -D, and-E, and for subgroups-B and-D,
respectively (Adkins et al., 2000; Brojatsch et al., 1996). In
addition, the turkey homolog of tvb (designated as tvbt) was
also isolated and was shown to encode a subgroup E-specific
viral receptor (Adkins et al., 1997). TVB proteins are members
of the tumor necrosis factor receptor (TNFR) family and are
most likely the avian homologs of the mammalian TRAIL
receptors. Subgroup-specific viral interaction determinants
have been mapped in the extracellular, TNFR-related, cyste-
ine-rich domains (Barnard and Young, 2003).
More recently, the tvc gene was identified using a
positional cloning approach which took advantage of the
close linkage between the tva and tvc loci. In contrast to
either TVA or TVB, TVC is a member of the immuno-
globulin superfamily that most closely resembles the
mammalian butyrophilins (Elleder et al., 2005). The extra-
cellular region of TVC contains two immunoglobulin-like
domains while the cytoplasmic region contains a B30.2
domain. Therefore, different subgroups of ASLV have
evolved to use very different types of proteins as their
cellular receptor. It is interesting to note that unlike most
other retroviruses, ASLV utilizes receptors with a single
membrane anchor. Because TVC was just described, the
viral interaction determinants of this receptor have not yet
The identification of each of these receptor genes led to
the uncovering of the molecular basis of resistance
associated with tvaR, tvbR, and tvcRalleles. These alleles
exhibit a variety of distinct debilitating mutations including
premature stop codons, a translational frameshift, and an
amino acid substitution that impacts Env binding (Elleder et
al., 2004; Elleder et al., 2005; Klucking et al., 2002).
ASLV Env and viral fusion proteins
Like all retroviral Env proteins, those of ASLV are class I
fusion proteins with an N-terminal surface (SU) subunit
involved in receptor-binding and a C-terminal transmembrane
(TM) subunit that directs membrane fusion. The TM subunit
contains an N-terminal hydrophobic fusion peptide, a C-
terminal transmembrane domain that anchors the protein to
the viral membrane, and N- and C-terminal heptad repeats that
juxtapose the fusion peptide and C-terminal transmembrane,
respectively (for a review, see Barnard and Young, 2003). The
overall architecture of ASLV Env is characteristic of other
prototypical class I fusion proteins such as influenza HA,
human immunodeficiency virus (HIV) Env, and Ebola Gp2
(Eckert and Kim, 2001).
Class I viral fusion proteins exist as a metastable complex on
the viral surface and upon fusion activation they are converted
to a more stable, lower-energy form. The fusion activities of
these proteins are usually triggered either by receptor- (and in
some cases also coreceptor-) induced conformational changes,
as is the case with HIV, or instead by structural changes caused
by the low pH environment of intracellular acidic endosomes.
The first fusion-activating changes result in exposure of the
N-terminal hydrophobic fusion peptide which then inserts into
the target membrane (Eckert and Kim, 2001). Further
conformational changes then reorient the protein so that the
fusion peptide and transmembrane domains lie in close
proximity at the same end of the transmembrane protein. This
step is thought to lead to the merger of the contacting lipid
membranes, i.e. hemifusion (Eckert and Kim, 2001). The N-
and C-terminal heptad repeats then associate into a six-helical
bundle (6HB) that facilitates the completion of the fusion
reaction by leading to the formation and expansion of a fusion
pore (Barnard and Young, 2003; Eckert and Kim, 2001). The
6HB form of these viral glycoproteins is highly thermostable
and is resistant to denaturation in SDS-containing buffers.
Broad interest in ASLV entry was stimulated following the
unexpected finding that ASLV Env utilized an unprecedented
two-step fusion activation mechanism that borrowed features
from both pH-independent, and pH-dependent, viruses (Mothes
et al., 2000). The first step involves receptor-induced confor-
mational changes in Env at neutral pH that lead to exposure of
the fusion peptide so that it inserts into the cell surface
membrane. The next step involves low pH activation resulting
in completion of the fusion reaction in an acidic endosomal
compartment, following virus uptake and endosomal traffick-
ing. These events were recently reviewed in detail elsewhere
(Barnard and Young, 2003).
The existence of a stable receptor-primed intermediate form
of ASLV Env, with an extended conformation, is supported by
studies performed using a C-terminal inhibitory heptad repeat
peptide (termed R99) which can inhibit ASLV infection when
added at the cell surface (Barnard et al., 2004; Earp et al., 2003;
Netter et al., 2004). These results suggest that the N- and C-
R.J.O. Barnard et al. / Virology 344 (2006) 25–29
terminal heptad repeats of ASLV TM are already exposed at
the plasma membrane following the receptor interaction.
Consistent with this idea, after receptor-priming, ASLV Env
becomes competent to bind to the R99 inhibitory peptide
(Netter et al., 2004). Since this peptide can access its target at
the plasma membrane, i.e. the N-terminal heptad repeat, this
suggests that the receptor-primed form of ASLV Env is likely
to be a pre-6HB intermediate form (Fig. 1, Step 1).
Unlike other low pH-dependent viruses, ASLV can remain
stable for many hours in cells that are transiently treated with
lysosomotropic agents to elevate endosomal pH (Mothes et al.,
2000; Narayan et al., 2003). This result also points toward the
remarkable stability of the receptor-primed intermediate of
ASLV Env. Thus, unlike all other class I fusion proteins, ASLV
Env seems capable of pausing for long periods of time in a pre-
the target membrane and before acid pH triggering. Subsequent
low pH-triggering of the receptor-primed ASLV Env results in
formation of a tight SDS-resistant 6HB (Smith et al., 2004).
How far does fusion progress at the receptor-priming stage?
Studies performed with pyrene-labeled viruses suggested that
receptor-primed ASLV is capable of reaching the hemifusion
stage at neutral pH (Earp et al., 2003). However, cell–cell
fusion experiments indicated that ASLV Env-dependent hemi-
fusion and fusion occur only after receptor-priming and low pH
triggering (Melikyan et al., 2004). Consistently, the receptor-
primed form of ASLV Env can undergo acid pH-dependent
triggering at low temperatures generating a restricted hemifu-
sion intermediate (Fig. 1, Steps 2 and 3). Subsequent fusion
pore formation and expansion steps occur in a pH-independent
manner at physiological temperature (Melikyan et al., 2004)
(Fig. 1, Steps 4 and 5).
The imaging of individual retroviral fusion events has also
added support for the model that the receptor-primed, pre-6HB
form of ASLV Env is arrested at a stage prior to hemifusion.
Using a novel virus–cell fusion assay, which monitored both
lipid and content mixing of DiD/yellow fluorescent protein
(YFP) labeled virions, it was found that ASLV Env-dependent
nascent fusion pores formed only after receptor and low pH
treatment (Melikyan et al., 2005). As both hemifusion and
content mixing were inhibited by the R99 peptide, it is likely
that formation of a tight 6HB is required to drive fusion pore
formation and/or expansion. More importantly, these experi-
ments also provided direct evidence that a hemifusion
intermediate, that exists for up to several minutes, is a bona
fide intermediate in the membrane fusion reaction mediated by
ASLV Env (Melikyan et al., 2005).
Fig. 1. ASLV Env-membrane fusion driven by receptor and low pH. Following
receptor interaction at the cell surface, the fusion peptide of ASLV Env is
exposed and inserted into the target membrane (Step 1). The receptor-primed
form of ASLV Env then forms a stable pre-6HB intermediate that can bind
inhibitory C-terminal heptad repeat peptides (Steps 1 and 2). In this state,
ASLV Env can be triggered by low pH at non-physiological temperatures (Step
2). Upon addition of low pH, ASLV Env folds into its tight 6HB promoting first
a restricted hemifusion intermediate (Step 3), an unrestricted hemifusion
intermediate (Step 4), and finally formation and expansion of the fusion pore
(Step 5) that allow delivery of the viral core into the cell cytoplasm.
R.J.O. Barnard et al. / Virology 344 (2006) 25–29
Concluding remarks and future perspectives
The pioneering studies on the ASLV-receptor system that
were initiated over 40 years ago set the foundation for the
isolation and characterization of ASLV receptor genes and for
the detailed molecular and biophysical analyses of the viral
entry pathway. These studies revealed that ASLV uses an
unanticipated mechanism of viral entry that represents a hybrid
of the more common pH-independent and pH-dependent viral
entry mechanisms. These findings are probably of broader
significance since we already know of other viruses that exhibit
a similar two-step entry mechanism (Nurani et al., 2003; Seth
et al., 2003).
The studies on the ASLV-receptor system have also revealed
two novel intermediates in class I viral glycoprotein-driven
fusion. The first is the highly stable receptor-primed interme-
diate which can exist presumably in an extended, pre-6HB,
conformation for many hours. This property of ASLV Env is
unique among the class I viral fusion proteins and a better
understanding of the structure of this intermediate is likely to
add significantly to our knowledge about the step-wise
conformational changes in class I viral fusion proteins that
lead to membrane fusion. Moreover, this property of ASLV
was recently exploited in the development of a cell-free
retroviral fusion and uncoating system that is being used to
examine the ill-defined series of uncoating events that occur
after viral fusion and lead to formation of an active viral reverse
transcription complex (Narayan and Young, 2004). The second
intermediate is arrested transiently at a stage that lies between
hemifusion and fusion pore opening/expansion. The existence
of this intermediate provides direct evidence that hemifusion is
a bona fide intermediate in the virus–cell membrane fusion
reaction. Since this intermediate stage likely exists for other
class I fusion proteins, such as influenza and Ebola viruses, it
may represent a viable target for future antiviral development.
Future studies in the ASLV-receptor system are likely to
contribute additional insights into the steps involved in viral
entry as well as those that immediately follow virus–cell
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