GENETIC AND MOLECULAR BIOLOGICAL
ANALYSIS OF PROTEIN-PROTEIN INTERACTIONS IN
Paul S. Masters, Lili Kuo, Rong Ye, Kelley R. Hurst, Cheri A. Koetzner,
and Bilan Hsue*
Virions of coronaviruses (CoVs) are pleiomorphic, with a roughly spherical structure
brought about by cooperation among a relatively small set of structural proteins and a
membranous envelope acquired from the endoplasmic reticulum–Golgi intermediate
compartment (ERGIC) (Fig. 1). Three integral membrane proteins reside in the envelope.
The most salient of these is the spike glycoprotein (S), which mediates receptor attach-
ment and fusion of the viral and host cell membranes. The membrane protein (M) is the
most abundant virion component and gives the envelope its shape. The third constituent
is the envelope protein (E), which, although minor in both size and quantity, plays a
decisive role is envelope formation. In some group 2 CoVs, an additional protein, the
hemagglutinin-esterase (HE), appears in the viral envelope. Finally, interior to the
envelope, monomers of the nucleocapsid protein (N) wrap the genome into a helical
A number of approaches have been taken to elucidate the network of interactions,
among the canonical structural proteins S, M, E, and N, and the genomic RNA, that lead
to assembly of virions. The earliest efforts employed the fractionation and reassociation
of components of purified virions. These studies were followed by molecular genetic and
co-immunoprecipitation analyses of expressed proteins or proteins from virus-infected
cells. More recently, reverse-genetic techniques have become available. This chapter will
briefly review the current understanding of CoV assembly, highlighting some recent
results from our laboratory in the context of work that has been done by numerous other
groups in this field.
From a large body of work extending over two decades, the main principle that has
emerged is that M is the central organizer of CoV assembly. The M protein (~25 kDa)
* Paul S. Masters, Lili Kuo, Rong Ye, Kelley R. Hurst, Cheri A. Koetzner, New York State Department of
Health, Albany, New York. Bilan Hsue, Stratagene, La Jolla, California.
P. S. MASTERS ET AL.
has a small, amino-terminal ectodomain that is either O-glycosylated (group 2 CoVs) or
N-glycosylated (groups 1 and 3 CoVs) (Fig. 1). This domain is followed by three trans-
membrane segments and a large carboxy-terminal endodomain.1–4 For the group 1 CoV
TGEV, it has been shown that roughly one-third of M protein assumes a topology in
which part of the endodomain constitutes a fourth transmembrane segment, thereby
positioning the carboxy terminus on the exterior of the virion5; however, this has not yet
been demonstrated for other CoV family members.
The dominant role of M was, in part, deduced by the process of elimination. Early
experiments with tunicamycin-treated MHV-infected cells showed that (noninfectious)
virions could assemble without S protein.6, 7 This finding was also consistent with the
properties of an S gene ts mutant, which failed to incorporate spikes into virions at the
nonpermissive temperature.8 Careful co-immunoprecipitation studies subsequently dem-
onstrated that M selects both the S and HE proteins for assembly.9, 10 However, it was
apparent that M could not act on its own: expression of M protein alone does not lead to
formation of virion-like structures. In addition, M, expressed in the absence of other viral
proteins, travels to the Golgi, whereas CoVs bud into the ERGIC.11–15 This paradox was
resolved by the landmark demonstration that co-expression of MHV M protein and a
previously overlooked structural protein, E, resulted in the formation of virus-like
particles (VLPs).16, 17 That just the M and E proteins are necessary and sufficient for the
formation and release of VLPs has since been shown for CoVs of all three groups:
BCoV18 and SARS-CoV19 (group 2), TGEV18(group 1), and IBV20, 21(group 3). To date,
the only apparent exception is a report that, for SARS-CoV, M protein and N protein
were necessary and sufficient for VLP formation.22 It remains to be seen whether this
finding indicates a unique aspect of SARS-CoV virion morphogenesis, or whether it
reflects a singular characteristic of the cell line or the expression system that was used.
Figure 1. Structure of the CoV virion (left), and a model of the M protein (right).
CORONAVIRUS PROTEIN-PROTEIN ASSEMBLY INTERACTIONS
2. INTERACTIONS OF S WITH M
Experiments with VLPs made possible the systematic manipulation of individual
constituents of the virion envelope, leading to the first glimpses of the function of E
protein and the partial localization of M-S intermolecular interactions.23, 24 The S protein
is a large (~180 kDa) type I transmembrane protein that assembles into trimers to form
the distinctive CoV spikes. Although it is not required for VLP formation, S protein, if
present, becomes incorporated into VLPs. The heavily N-glycosylated amino-terminal
ectodomain of S, which makes up more than 95% of the mass of the molecule, has been
found to be essentially inert in the assembly process. Construction of chimeric MHV-
FIPV S proteins with swapped ectodomains showed that the 61-amino-acid trans-
membrane domain and endodomain determined the incorporation of the S protein into
VLPs formed by the homologous M and E proteins.24
When this principle – the functional separation of the domains of S for receptor
binding and for virion assembly – was extended to whole viruses, it allowed the
development of host range-based selective systems for the reverse genetics of CoVs
through targeted RNA recombination. A mutant of MHV (designated fMHV) was
constructed, in which the ectodomain of the MHV S protein was replaced by that of the
FIPV S protein. This chimeric virus had the host cell-restricted growth pattern that was
predicted from its precursors: it had simultaneously lost the ability to grow in murine
cells and gained the ability to grow in feline cells. The use of fMHV as the recipient virus
in targeted RNA recombination enabled us to efficiently carry out reverse genetics on
MHV, by restoring the MHV S ectodomain (in conjunction with mutations of interest)
and selecting for the reacquisition of the ability to grow in murine cells.27 Selections of
even greater stringency were subsequently made possible by the rearrangement of genes
downstream of S, in fMHV.v2; this rearrangement effectively precluded the possibility
of unwanted secondary crossover events during targeted RNA recombination.28
Among the many problems to which this system has been applied was the genetic
dissection of the transmembrane domain and endodomain of the MHV S protein, in order
to localize the determinants of S incorporation into virions.29 We used two strategies for
this investigation (Fig. 2). First, the S protein transmembrane and endodomains were
attached to a heterologous ectodomain to produce a surrogate virion structural protein
(named Hook), which could be mutated without consequence to viral infectivity. Second,
significant mutations from Hook were transferred to the S protein (in the absence of
Hook), to enable examination of their effects on viral phenotype. We found that
assembly competence mapped to the endodomain of S, which was sufficient to target
Hook for incorporation into virions. Further mutational analysis indicated a major role
for the charge-rich carboxy-terminal region of the endodomain. Additionally, we found
that the adjacent, membrane-proximal, cysteine-rich region of the endodomain is critical
for cell-cell fusion during infection, thus confirming results previously obtained with S
protein expression systems.30, 31 A separate study32 came to the same fundamental
conclusion that virion incorporation was determined by the endodomain of S, but in the
latter work the major role in assembly was ascribed to the cysteine-rich region of the
endodomain. The differences among the detailed conclusions of these two studies may
have been due to the relative importances of particular endodomain residues that were
ablated in the differently constructed deletion mutants.
P. S. MASTERS ET AL.
Figure 2. Genetic dissection of the determinants for incorporation of the S protein into virions.
3. INTERACTIONS OF N WITH M
We have also examined the association between the M protein and the N protein of
MHV. This was initially accomplished through the construction of a highly impaired
mutant, M∆2, containing a two-amino-acid truncation of the M protein.27 This mutant
formed tiny plaques and grew to maximal titers that were three orders of magnitude
lower than those of the wild type. Analysis of multiple second-site revertants of M∆2
revealed a number of changes in either the M protein or the N protein that could
individually compensate for the lesion in M∆2 (Fig. 3). The latter set of suppressors
provided the first genetic evidence for a structural interaction between the M and N
proteins, and they allowed that interaction to be localized to the carboxy termini of both
The MHV N protein (~50 kDa) comprises three conserved domains that are
separated by two highly divergent spacer regions.33 Domains 1 and 2, which make up
most of the molecule, are very basic, and the RNA-binding capability of N maps to
domain 2.34, 35 In contrast, domain 3, the carboxy-terminal 45 amino acids of N, has an
excess of acidic over basic residues. To complement the results obtained with the M∆2
mutant, we recently created a complete set of clustered charged-to-alanine mutants in
domain 3 of N.36 One of these mutants, CCA4, was extremely defective, thereby
implicating a pair of aspartate residues (D440 and D441) as making a major contribution
by N protein to the N-M interaction. Moreover, independent second-site reverting
mutations of CCA4 were found to map in the carboxy-terminal region of either the N or
the M protein (Fig. 3), thereby displaying genetic cross-talk reciprocal to that uncovered
with the M∆2 mutant. Indeed, one particular mutation in N domain 3 (Q437L) was
isolated multiple times, either as a suppressor of the M∆2 mutation or as a suppressor of
the N CCA4 mutation. Additionally, we showed that the transfer of N protein domain 3
CORONAVIRUS PROTEIN-PROTEIN ASSEMBLY INTERACTIONS
to a heterologous protein (GFP) was sufficient to allow incorporation of GFP into MHV
It is not yet clear how this genetically defined N-M interaction is related to
connections that have been uncovered by molecular biological and biochemical means.
For TGEV, the interaction between N and M was assayed by binding of in vitro–
translated M protein to immobilized nucleocapsid purified from virions.37 Deletion
mapping was used to localize this binding to a region of the TGEV M protein, the MHV
counterpart of which partially overlaps with critical residues that we have identified in
the MHV M protein by suppressor mapping. A key early study of MHV, which used
biochemical procedures to fractionate the components of purified virions, found a
temperature-dependent association between nonionic detergent-solubilized M protein and
the viral nucleocapsid.38 More recently, it was shown that N protein could be co-
immunoprecipitated from MHV-infected cells by mAbs specific for M protein.
Significantly, although N was shown to be intracellularly associated with all viral RNAs,
both subgenomic and genomic,39–41 the M protein bound only to those complexes of N
molecules that were, in turn, bound to genomic RNA.41 Such selectivity was determined
to depend upon the presence of the genomic RNA packaging signal; this signal, if
transferred to a heterologous RNA, was sufficient to allow its packaging into virions.42
Surprisingly, further work with co-expressed MHV proteins and RNAs attributed the
selection of packaging signal RNA to the M protein.43 Thus, VLPs composed of M and
E, but not N protein, were found to incorporate an RNA molecule only if it contained the
MHV packaging signal. Although the N-M interaction that we have localized genetically
appears to be independent of RNA, it is conceivable that the accessibility of N protein
domain 3 is modulated by the binding of N to particular RNA substrates.
cross-talk and by transfer of domain 3 of the N protein to a heterologous protein (GFP).
Figure 3. Intermolecular assembly interactions between the MHV N and M proteins revealed by genetic
P. S. MASTERS ET AL.
Figure 4. Alignment of the E proteins of various CoVs (top) and summary of the relative abilities of
heterologous E proteins to functionally replace the E protein of MHV (bottom).
4. THE ROLE OF E PROTEIN
The CoV E protein is a small polypeptide (~10 kDa) that is only a minor constituent
of virions. Nevertheless, it profoundly affects both VLP and virus assembly. E protein
sequences diverge widely across the three CoV groups, but all CoV E proteins have the
same architecture: a short hydrophilic amino terminus, followed by a large hydrophobic
region, and a hydrophilic carboxy-terminal tail that constitutes one-half to two-thirds of
the molecule (Fig. 4). Investigations with both the MHV and IBV E proteins are in
agreement that E is an integral membrane protein and that its carboxy-terminal tail is
cytoplasmic (corresponding to the interior of the virion).
carboxy-terminal tail alone can specify targeting to the budding compartment.45 The
disposition of the amino terminus is less clear, however. A lumenal (or virion-exterior)
topology has been inferred for the IBV E protein amino terminus, based on its inaccessibility
to antibodies at the cytoplasmic face of the Golgi membrane.20 Such a single transit
across the membrane would be consistent with the transmembrane oligomers of E
predicted by molecular dynamics simulations.46 Conversely, for MHV, the E protein
amino terminus has been proposed to be buried within the membrane near the
cytoplasmic face, based on the reactivity of an engineered amino-terminal epitope tag at
the cytoplasmic face.47 This orientation would require that the E protein hydrophobic
domain form a hairpin looping back through the membrane, as envisioned in a recent
biophysical analysis of the SARS-CoV E protein transmembrane domain.48
For MHV, we previously showed that particular clustered charged-to-alanine mutations
constructed in the E gene rendered the virus defective in growth: assembled virions of
one such mutant were found to have strikingly aberrant morphology, exhibiting pinched
and elongated shapes that were rarely seen among wild-type virions.49 This finding
clearly demonstrated an important role for E in virion assembly, as shown earlier for
Moreover, for IBV E, the
CORONAVIRUS PROTEIN-PROTEIN ASSEMBLY INTERACTIONS
viable, albeit highly defective, MHV recombinant (∆E) in which the E gene, as well as
genes 4 and 5a, were entirely deleted from the viral genome.50 This indicated that the
MHV E protein is a critical, but not essential, participant in virion assembly. To more
specifically focus on the E protein, we have very recently generated an additional
recombinant virus (E-KO), in which E protein expression has been ablated by mutation
of the initiation codon and placement of stop codons in all three reading frames. The E-KO
mutant exhibits the same tiny plaque phenotype and extremely defective growth as does
the ∆E mutant. This confirms that the phenotype observed for the ∆E mutant was a direct
result of the E gene deletion.
Similarly, it has recently been found that knockout of SARS-CoV E protein
expression results in a virus that is viable in tissue culture (Almazen, DeDiego, Alvarez,
and Enjuanes, this volume). By contrast, for TGEV it has been shown by two distinct
reverse genetic methods that if the E gene is knocked out, then no viable virus can be
recovered; the resulting defect can only be rescued by E protein provided in trans.
This may indicate that basic morphogenic differences exist between the CoVs of group 2
(MHV and SARS-CoV) and group 1 (TGEV). Alternatively, it may suggest that E
protein has multiple activities, one of which is essential for group 1 CoVs but
unnecessary for group 2 CoVs.
To learn more about the constraints on E protein sequence, relative to the specificity
of this protein’s interaction with M protein, we investigated whether E proteins from
different CoVs could functionally replace that of MHV. Toward this end, we exchanged
the MHV E gene with that from viruses of each of the three CoV groups. In every case,
exact ORF-for-ORF substitutions were made, so that each heterologous E gene was
expressed in the same context as MHV E (i.e., as the second ORF in a message whose
unique region is bicistronic). The results of this work revealed an unexpected flexibility
in the sequence requirements of the E protein (Fig. 4). As predicted, the relatively closely
related E protein of BCoV (group 2) could fully substitute for the MHV E protein.
Replacement of MHV E with the more phylogenetically distant group 2 SARS-CoV E
protein resulted in a virus with a slightly smaller plaque size than wild-type MHV. Very
surprisingly, the group 3 IBV E protein, which is extremely divergent from MHV E in
both size and sequence, was completely functional in MHV infection and assembly. This
could indicate that E protein does not need to directly contact M protein in order to carry
out its role in virion budding. By contrast, the E protein of TGEV (group 1) was not
functional in MHV; the TGEV E substitution mutant had a phenotype indistinguishable
from that of the ∆E mutant. These results lend further support to the notion that there are
differences between the assembly mechanisms of group 1 and group 2 CoVs. We have
been able to isolate multiple independent gain-of-function mutants from the TGEV E
substitution recombinant, and we have found that these viruses have mutations clustering
in two small regions of the TGEV E gene. Systematic analysis of these chimeric viruses
should help to further elucidate the functions of E protein.
We were thus surprised to find that we could successfully generate a
P. S. MASTERS ET AL.
A great deal remains to be learned about the rules governing CoV assembly. One
particularly intriguing question raised by the work discussed above is: does the E protein
need to directly physically interact with the M protein, or does E act at a distance? These
two possibilities are not necessarily mutually exclusive. A direct E-M interaction is
suggested by the observation that there are certain unallowed interspecies combinations
of M and E with respect to VLP assembly18 or virus assembly (see above). The close
physical proximity of the two proteins is also supported by the demonstration that IBV E
and M can be cross-linked to one another in infected or transfected cells.21 Conversely,
some results appear to argue that E acts independently of M. The individual expression
of MHV or IBV E protein results in vesicles that are exported from cells.
been found that the expression of MHV E protein alone leads to the formation of clusters
of convoluted membranous structures highly similar to those seen in CoV-infected
cells,44 suggesting that one role of E is to induce membrane curvature in the ERGIC. The
functional replacement of the MHV E protein by the highly divergent IBV E protein (see
above) also suggests that a specific interaction with M is not necessary for viral
assembly. Moreover, in multiple revertant searches, we have yet to find a suppressor of
an E gene mutant that maps in M or in any gene other than E.49 Similarly, we have never
found intergenic suppressors of the M∆2 mutant or the N CCA4 mutant that map in E.27,
36 A mechanism for the independent action(s) of E in CoV assembly may be found in the
recent demonstration that the SARS-CoV E protein is a cation-selective ion channel.54
A second pressing question arising from the roles of M protein discussed above is:
what is the structural basis for the central position of M in the network of interactions
that determine viral assembly? M associates with other monomers of M,23 with the
endodomain of S,24, 29, 30 with domain 3 of N36 and, possibly, with E and with genomic
RNA.43 We have noted that the viral M protein is extremely sensitive to mutations. This
sensitivity would be consistent with the constraints imposed by M needing to maintain
simultaneous contacts with multiple structural partners. On the other hand, M appears able
to accommodate some radically altered versions of either the S endodomain
protein domain 3,36 suggesting that M offers a variety of surfaces with which interacting
polypeptides can establish alternative binding sites, if their primary interactions have been
abolished by mutation. This versatility of M protein may be a component of the forces that
drive CoV evolution, allowing the incorporation of altered or new proteins into virion
envelopes. Such considerations clearly point to the necessity to obtain structural information
about this crucial virion component.
We acknowledge a very productive collaboration with the laboratory of Peter Rottier
on a number of the subjects discussed here. We are also grateful to Kathryn Holmes,
David Brian, Carolyn Machamer, John Fleming, and Lawrence Sturman for the provision
of viruses, clones, and antisera, and for very insightful discussions and advice. This work
was supported by Public Health Service grants AI 39544, AI 45695, and AI 060755 from
the National Institutes of Health.
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