viral capsids

8/3/2019 Viral Capsids

1/12

proteinsSTRUCTURE O FUNCTION O BIOINFORMATICS

A novel method to map and compareproteinprotein interactions in

spherical viral capsids

Mauricio Carrillo-Tripp, Charles L. Brooks III, and Vijay S. Reddy*

Department of Molecular Biology, The Scripps Research Institute, La Jolla, California 92037

INTRODUCTION

Virus particles are assembled from multiple copies of one ora few chemically distinct coat protein (CP) subunits packagingviral genome and relevant accessory proteins in vivo. Some ofthe viral capsid proteins spontaneously form empty capsids

when expressed in heterologous expression systems orassembled in vitro.15 Whether the virus particles assemblein vivo or in vitro, the inter subunit proteinprotein interac-

tions are critical for the formation, stability, and integrity ofvirus particles. The extent of interactions and the type of

resulting capsids are encoded into individual capsid proteinsubunits as they yield mostly uniform capsids of a single kind(T5 1, T5 3, etc.). Analysis of proteinprotein interactionsin viral capsids within and across members of virus familiesand capsid types will provide the locations of the hotspots andweak links of the assembled capsids and highlight the extent,distribution, and similarity of these interactions.69

Here, we report a new approach to map proteinprotein

interactions onto azimuthal polar orthographic projection dia-grams using spherical coordinates. Its only natural to represent

the quaternary interactions of spherical viral capsids using aspherical coordinate system. In addition, spherical polar projections provide a normalized way to map and compare inter-actions as opposed to projections in Cartesian space, where thecoordinates have to be scaled up or down with respect to thesize of the reference particle. The method described in thiswork involves mapping all the unique residues in the icosahe-dral asymmetric unit that interact at the intersubunit interfacesas azimuthal orthographic projections. These diagrams repre-

sent the structural fingerprints of quaternary interactions ofthe respective capsids. Hence, they can be used as roadmaps to

visualize the distribution and the extent of proteinproteininteractions with respect to the icosahedral symmetry axes,irrespective of the size and the type (T-number) of spherical

Grant sponsor: National Institutes of Health (NCRR center for Multiscale Modeling Tools

for Structural Biology (MMTSB)); Grant number: RR12255.*Correspondence to: Vijay S. Reddy, Department of Molecular Biology, TPC-06, The Scripps

Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037.E-mail: [email protected]

Received 15 October 2007; Revised 29 February 2008; Accepted 9 March 2008Published online 19 May 2008 in Wiley InterScience (www.interscience.wiley.com).

DOI: 10.1002/prot.22088

ABSTRACT

Viral capsids are composed of multiple copies of one or a

few chemically distinct capsid proteins and are mostly sta-

bilized by inter subunit proteinprotein interactions.

There have been efforts to identify and analyze these

proteinprotein interactions, in terms of their extent andsimilarity, between the subunit interfaces related by quasi-

and icosahedral symmetry. Here, we describe a new

method to map quaternary interactions in spherical virus

capsids onto polar angle space with respect to the icosahe-

dral symmetry axes using azimuthal orthographic dia-

grams. This approach enables one to map the nonredun-

dant interactions in a spherical virus capsid, irrespective

of its size or triangulation number (T), onto the reference

icosahedral asymmetric unit space. The resultant diagrams

represent characteristic fingerprints of quaternary interac-

tions of the respective capsids. Hence, they can be used as

road maps of the proteinprotein interactions to visualize

the distribution and the density of the interactions. In

addition, unlike the previous studies, the fingerprints ofdifferent capsids, when represented in a matrix form, can

be compared with one another to quantitatively evaluate

the similarity (S-score) in the subunit environments and

the associated proteinprotein interactions. The S-score

selectively distinguishes the similarity, or lack of it, in the

locations of the quaternary interactions as opposed to

other well-known structural similarity metrics (e.g.,

RMSD, TM-score). Application of this method on a subset

of T5 1 and T5 3 capsids suggests that S-score values

range between 1 and 0.6 for capsids that belong to the

same virus family/genus; 0.60.3 for capsids from different

families with the same T-number and similar subunit fold;

and


2/12

virus capsids. Furthermore, these fingerprints can bereadily compared and contrasted with one another andcan be used to evaluate quantitatively the extent of simi-larity of proteinprotein interactions in different capsidsby representing the projections in a matrix form.

MATERIALS AND METHODS

Azimuthal polar orthographic diagrams

(APODs)

In this method, spherical coordinates (r,F,w) of all theresidues interacting at the intra and inter icosahedral asym-

metric unit interfaces were obtained. The amino acids con-tacting at the subunit interfaces were identified by the resi-due-pair specific distance cut-off criteria between the cen-ters of mass (COMs) of the side-chain atoms.7 Cartesiancoordinates of the COMs of all the interface residues wereextracted from VIPERdb (http://viperdb.scripps.edu), wheretables of contacting pairs of residues are readily available.10

The COMs were then transformed into unit vectors andprojected onto (F,w) space as an azimuthal polar ortho-

graphic diagram (APOD), which resembles projection of asphere (globe) from one of its poles (North Pole). Figure1(a) shows a point in Cartesian space, P(x,y,z), and its

transformation to spherical space representation P(r,F,w),where F is the angle between the projection of the vector ~r

onto the XY plane and the X axis, and w is the anglebetween the vector ~r and the Z axis. The vector~r is nor-malized so that the point P will lie in the surface of aunit sphere. Figure 1(b) shows the point P represented

on an APOD. The F angle grows counterclockwise goingall around from 08 to 3608. The w angle grows radiallystarting with 08 at the center of the diagram and ending

at 908 at the edge of the diagram (outermost circumfer-ence; equator of the unit sphere).

Each symbol on an APOD represents the location of thecenter of mass of side chain atoms of an interface residue ior j, where (i,j) is a specific interacting pair, in Fw space.Both residues of the interacting pairs were mapped. Such arepresentation is unambiguous and better than locating aninteraction as the mid point of the interacting pair, partic-ularly when the APODs are being compared to evaluate S-

scores (see below). Two types of symbols were used to dis-tinguish intra (circles) and inter (triangles) asymmetric

unit interactions. Locations of the icosahedral symmetryaxes comprising the central icosahedral asymmetric unit(CIAU) were highlighted on the APODs as fiducials.

Similarity (S) score, a measure of similarity

in quaternary interactions

The proteinprotein interactions in the polar ortho-graphic diagrams can also be represented as an M 3 L

matrix, N (polar orthographic matrix, or POM), wheredimension M represents the range of the F angle (08

3608), and dimension L corresponds to the range of the

w angle (08908). M and L are constant for all sphericalviral capsids. Each matrix element in N (POM) can beconsidered as a cell with a small uniform surface area inthe Fw space. The differential of the surface area inspherical coordinates is given by dS5 r2 sin(w) dw dF5 r2 dcos(w) dF; r2 5 1 for an unit sphere. To obtaincells of constant surface area of the sphere, the range of

Figure 1

(a) Representation of coordinates of a point P in Cartesian space andits corresponding coordinates in spherical polar space. (b) Projection ofthe point P onto an azimuthal polar orthographic diagram (APOD).The four relevant icosahedral symmetry axes in the VIPERconvention11 are shown in bold. These include two threefold axes(triangles) at [08, 218] and [1808, 218], fivefold axis (pentagon) at [908,328], and twofold axis (oval) at [08, 08]. The central icosahedralasymmetrical unit, or CIAU, is represented by the black linesconnecting the four icosahedral symmetry elements. The correspondinglocations of both the X and YCartesian axes are also shown. The Z-axiswould be perpendicular to the plane of the diagram at the center,growing in positive values above the page towards the reader.

Protein Interactions in Spherical Viral Capsids

PROTEINS 645


3/12

F (083608) was divided into regular intervals as well asthe range of cos(w) (01). The F angle was divided into360 bins, with a dF 5 18 and the cos(w) is divided into180 bins, which amounts to dcos(w) of 0.0056. Then, foreach value of cos(w), the corresponding value of w can

be obtained by applying the acos() function. The area ofeach cell (dS) will be 0.97 3 1024 A2 for a unit sphere.

This translates into $1.0 A2

for a sphere of 100 A in ra-dius (200 A in diameter). The value of each element(cell) in the matrix N is equal to the number of interac-tions found in the corresponding cell in Fw space, i.e.,an element (or cell) of the matrix will be zero if no inter-actions were found in that cell or a positive integer equalto the number of interactions present. Since the average

resolution of all the X-ray structures found at VIPERdbis $3 A, COM of a residue in Cartesian space was con-sidered to be anywhere inside a cubic box of the size of 3

A (XCOM 1.5 A, YCOM 1.5 A, ZCOM 1.5 A), thusgiving rise to a range of possible Fw values dictated bythe volume of the box. This smearing of the residue

positions was achieved by turning on the eight adjacentelements in the N matrix. Another way to consider thesmearing of residue locations would be to increase thearea of the cells in the Fw space. The former approach,however, proved to be more sensitive than the latter.

S score 2P

i

Pj N

aijN

bijP

i

Pj N

aij

2 Nbij

2h i

Similarity (S) score corresponds to a fraction of com-mon locations of quaternary interactions between a pair

of spherical virus capsids with respect to the total num-

ber of interactions found in the respective capsids. Thedot (inner) product of two POMs (Na and Nb) provides

the number of common contacts between the two capsidsa and b. Therefore, the S-score is defined as twice the ra-tio of common locations of interactions normalized bythe total number of points of interaction in both thecapsids being compared. The S-score for identical capsidswill be 1, and


4/12

locations of the sequence-conserved interface residuespresent in the spatially conserved fingerprint of the fourmembers of the Nodaviridae family were identified. Wesuggest that these residues could be of importance forthe self-assembly of Nodavirus capsids.

APODs for the capsids of STNV,15 SBMV,16 andTBSV17 are shown in Figure 2. The diagrams for SBMV

and TBSV capsids look very similar. However, there aredifferences near the threefold axis at [1808, 218] arisingdue to the differences in the extent of the ordered pep-tide arm of the C-subunit forming the b-annulus. Inaddition, the diagram for TBSV shows an extra set ofinteractions near the middle of each of the three sides ofthe triangular central icosahedral asymmetrical unit

(CIAU), in comparison to that of SBMV. These contacts(residues) protruding out of the CIAU correspond to theinteractions resulting from the P domains of the TBSV

subunits, which are absent in SBMV. Locations of theinteractions at the intraunit (inside the CIAU), interunitinterfaces and their relative extents at these interfaces are

readily discernible from these diagrams. For example, theP domains of the TBSV capsid participate only in theinterunit interactions while the S domains are involvedin the contacts at intraunit as well as interunit interfaces.The P domain of the TBSV CP contributes on average 33more interactions per subunit compared with that of the

SBMV subunit.

Comparison of interactions in STNV,

TBSV, and SBMV capsids

The APODs of different capsids can be compared withone another to assess the degree of similarity in the loca-tions of their proteinprotein interactions using S-scoresas well as to obtain a common characteristic structuralfingerprint of quaternary proteinprotein interactions

among a group of capsids.Similarity in quaternary interactions (S-scores) were

calculated between the three viruses studied by Rossmannet al.14 An S-score of 0.60 between TBSV and SBMVcapsids suggests that there is moderate similarity betweenthe locations of interface residues in both viruses, eventhough the capsids have only 26% overall sequence iden-tity and belong to different virus families. As noted ear-lier, STNV forms T5 1 capsids, which are composed of

60 subunits, while TBSV and SBMV form T5 3 capsidscontaining 180 subunits. Not surprisingly, comparison ofthe APOD of STNV with those of TBSV and SBMVresulted in lower S-scores; 0.16 and 0.17, respectively. Totest whether or not the poor scores are simply due tocomparing the interactions of the T5 1 capsid of STNVcapsid having 1 subunit in the CIAU with those of theT 5 3 capsids of TBSV and SBMV with three subunitsoccupying the CIAU, we analyzed the environments of

the individual subunits. The interface residues corre-sponding to the individual subunits (A/B/C) of TBSV

and SBMV capsids were identified and transformedaccording to the structural superposition of the respective(T 5 3) subunits on the STNV subunit. The resultanttransformed APODs of the individual subunits ofSBMV and TBSV capsids were compared with the APOD

of the STNV subunit (see Fig. 3). Interestingly, the S-scores calculated for each individual subunits of SBMV

and TBSV with respect to STNV were 0.21, 0.24, and0.22 for the SBMVA, SBMVB and SBMVC respectively,and 0.20, 0.21, and 0.17 for the TBSVA, TBSVB andTBSVC, respectively. The subunit wise S-scores are onlyslightly better than the S-scores (0.16 and 0.17) for com-paring the conventional APODs of the CIAUs of theTBSV and SBMV capsids. Taken together, this suggests

that the individual subunit environments of the SBMVand TBSV capsids are indeed different compared withthat of the STNV subunit.

Additionally, environments of the individual A, B, andC subunits of the TBSV and SBMV capsids were com-pared with one another within each capsid and between

the capsids (Table I). The S-scores for all inter-subunitpairs within the individual CIAUs of TBSV and SBMVare listed in Table I, respectively. In both cases, the Csubunits display distinct environments (S-score: h0.7i) incomparison to that of the A or B subunits, while theenvironments of the A and B subunits compare well (S-

score: h0.85i) with each other. This distinction in theenvironments of the C-subunits is likely due to selectiveordering of a segment of the N-terminal peptide arm re-sponsible for forming the b-annulus structure. Further-more, S-scores for the intersubunit pairs between theSBMV and TBSV capsids showed a similar pattern (TableI). This provides a quantitative assessment of the extentof variation in the environments of the C-subunits com-pared to the A and B-subunits both in intra- or inter-capsid analysis of the two T5 3 capsids.

Analysis of parvo, sobemo, and

nodavirus capsids

In order to gain further insights into the relationshipbetween the S-scores and the structural similarity withinand across virus families, the analysis was extended to afew selected groups of virus capsids. The intrafamily sim-ilarity scores for capsid pairs in three different virus fam-

ilies, Parvoviridae (T5 1), Sobemoviridae (T5 3), andNodaviridae (T 5 3), are shown in Table II. Startingwith the Parvoviridae family, there is a clear differencebetween the densovirus capsid (PDB-ID:1dnv, genus:densovirus; S-score 5 h0.33i)18 and the other membersof the family, which primarily belong to the parvovirusgenus (S-score 5 h0.88i). This difference was clearlyhighlighted in the orthographic diagrams (not shown),which have a lower density of interactions around the

threefold axis at [08, 218] of capsid 1dnv compared tothe other member capsids in the family. These differences

PROTEINS 647



5/12

Figure 2

Schematic illustrations and APODs for STNV (a), SBMV (b), and TBSV (c) capsids. Illustrated on the left are schematic representation of therespective capsids with the subunits that occupy the CIAU are shown in color and the symmetry related copies are shown in gray. Icosahedrallattices are shown as black lines surrounding the individual capsids. Shown on the right are the corresponding APODs. Two types of symbols wereused to distinguish intra (circles) and inter (triangles) asymmetric unit interactions. The locations of the interacting residues are color codedaccording to the corresponding subunits: A-subunit in blue, B-subunit in red, and C-subunit in green. Each subunit is surrounded by a borderapproximately delimiting the spatial extent of the subunits. The CIAU is depicted as a triangle.

648 PROTEINS

M. Carrillo-Tripp et al.


6/12

seen in the APODs of parvo and densovirus capsidshighlight the characteristic differences in their quaternaryinteractions even though they belong to the same virusfamily. This suggests that there could be significant dif-

ferences in the extent of quaternary interactions evenamong members of the same family. This finding is con-sistent with the differences in the estimated intersubunit

Figure 3

Comparison of APODs of STNV (T5 1) (a) with those of the transformed subunits of SBMV: A-subunit ( b), B-subunit (c), and C-subunit (d),which were oriented with respect to the STNV subunit.

Table IPairwise Inter-Subunit S-Scores for TBSV and SBMV Capsids

TBSV17

A0 B0 C0

A0 1.00 0.86 0.75

B0 0.86 1.00 0.68

C0

0.75 0.68 1.00SBMV16

A B C

A 1.00 0.85 0.76

B 0.85 1.00 0.65C 0.76 0.65 1.00

SBMV/TBSVa

A B C

A0 0.54 0.49 0.37

B0 0.54 0.51 0.48C0 0.50 0.48 0.57

aA0, B0 C0 indicate A, B and C subunits in the TBSV capsid.

Table IIPairwise Intra-Family S-Scores for Three Families of Virus Capsids

Parvoviridae1dnv18 1c8d19 1c8h19 1k3v20

1dnv 1.00 0.33 0.33 0.34

1c8d 0.33 1.00 0.92 0.83

1c8h 0.33 0.92 1.00 0.831k3v 0.34 0.83 0.83 1.00

Sobemoviridae1smv21 1x3522 1f2n23 4sbv16 1ng024

1smv 1.00 0.92 0.69 0.88 0.69

1x35 0.92 1.00 0.69 0.87 0.691f2n 0.69 0.69 1.00 0.73 0.76

4sbv 0.88 0.87 0.73 1.00 0.711ng0 0.69 0.69 0.76 0.71 1.00

Nodaviridae

1nov25 2bbv26 1f8v27 fhv28

1nov 1.00 0.86 0.81 0.862bbv 0.86 1.00 0.80 0.90

1f8v 0.81 0.80 1.00 0.78

fhv 0.86 0.90 0.78 1.00


PROTEINS 649


7/12

association energies for these capsids, which are availableat the VIPERdb website.10

The orthographic diagrams of Sobemoviridae showthat they all have very similar densities of interacting res-idues except for the differences occurring due to selective

ordering of the peptide arm of the C subunit; the com-parison of S-scores of the members of the Sobemoviridae

family (Table II) show that they fall into two groups: ses-bania mosaic virus21 (1smv, 1x35) and southern beanmosaic virus16 (4sbv) correspond well with each other(S-score 5 h0.90i) as they share a common configurationof the peptide arm of the C-subunit that makes a sharpU turn near the twofold axis and extending outtowards the threefold axis at [1808, 218]. Whereas the

rice yellow mottle virus23 (1f2n) and cocksfoot mottlevirus24 (1ng0) capsids (S-score 5 0.76) are distinct fromthe first group with the peptide arm adopting a different

configuration from those of the SBMV, SMV, and TBSVcapsids by extending towards to the opposite threefoldaxis at [08, 218] without making the U turn.

In a similar fashion, members of the Nodaviridae fam-ily share the same subunit topology and organizationwith respect to the icosahedral symmetry axes and showhigher S-score values (h0.82i) for the comparisons withone another. Differences arising mainly due to the N-ter-minal arms of the A subunits, which were considered

earlier to be part of the C-subunits. Pariacotovirus27

(1f8v), however, shows a higher density of interactions inthe intraunit interfaces and has 30% more interactionsthan the other three members mainly due to ordering ofthe nearly complete peptide arm of the A-subunit.

As mentioned earlier, Sobemoviridae and Nodaviridaefamilies share the same triangulation number (T 5 3),while Parvoviridae form T5 1 capsids. As expected, thedifferences in quaternary architectures are reflected in thelow values of the S-scores (h0.22i) between capsids ofParvoviridae and Sobemoviridae or Nodaviridae (TableIII). On the other hand, comparison of the members ofthe T 5 3 capsids display higher S-scores (h0.60i) asshown in Table III.

On the basis of the range (01) of S-scores, sphericalcapsids can be clustered into three groups reflecting thesimilarity or lack of it in the quaternary interactions. Thefirst group has S-scores in the range of (1.000.6) and iscomposed of capsids that display the same quaternary

architecture (T-number) and similar subunit topologywith minor variations at the termini and in the loops(Table II). These capsids are mostly structural homologsand belong to the same family and/or genus. The secondgroup consists of capsids that exhibit the same quater-nary architecture and contain CPs with minor differencesin their topology and/or with large insertions relative toone another (e.g., TBSV vs. SBMV). In this group ofcapsids, the subunit interfaces remain at similar locations

with respect to the CIAU. In addition, individual capsidsin this group belong to different virus families and will

have S-scores in the range of (0.600.3) (Table III).

Finally, the third group will have S-scores below 0.30.This group contains capsids that display different trian-gulation numbers and belong to different virus familieswith similar or dissimilar subunit topologies (e.g., Parvo-viridae vs. Sobemoridae/Nodaviridae, Table III).

S-score versus other structural

similarity metrics

Here, we describe the correlation of the S-score withother metrics of structural similarity, namely, root meansquare deviation (RMSD), TM-score12,13 and sequenceidentity. The pair wise TM-score analysis for intra-family

structures suggest that capsids with S-scores in the rangeof 1.00 to 0.6 display the same topology in the coreregions of the CP subunit [Fig. 4(b)]. TM-scores greaterthan 0.8 were observed for most of the intra-family cap-sid pairs except for the comparisons with the Densovirus(1dnv) of Parvoviridae. Not surprisingly, the intra-familycomparisons have RMSD values 2 A [Fig. 4(a)]. Thereis, however, a clear dependence of the S-score on thesequence identity as well as on RMSD, shown by the

intra-family analysis [Fig. 4(a,c)].Even though there is a strong correlation between theS-score and the other metrics mentioned above, only theS-score estimates the similarity in the quaternary (struc-ture) interactions, while the other metrics primarily sug-gest the similarity in the CPs primary and tertiary struc-ture. Inter-family pairwise analysis of TM-score andRMSD versus S-score clearly illustrates the lack of simi-larity in the proteinprotein interactions (S-score), even

though there is a good deal of structural similarity in thetertiary structure of the CP subunits [Fig. 4(d,e)]. How-

Table IIIPairwise Inter-Family S-Scores for Three Families of Virus Capsidsa

P vs. SS1 S2 S3 S4 S5

P1 0.18 0.17 0.17 0.17 0.16

P2 0.23 0.23 0.22 0.23 0.23

P3 0.23 0.23 0.22 0.24 0.23

P4 0.23 0.23 0.23 0.23 0.23

P vs. NN1 N2 N3 N4

P1 0.19 0.20 0.22 0.20

P2 0.21 0.23 0.24 0.24P3 0.22 0.22 0.24 0.24

P4 0.21 0.22 0.24 0.24

N vs. SS1 S2 S3 S4 S5

N1 0.64 0.64 0.60 0.66 0.55N2 0.59 0.60 0.59 0.62 0.55

N3 0.61 0.62 0.60 0.64 0.55N4 0.62 0.62 0.61 0.63 0.56

aParvoviridae (P): 1dnv (P1), 1c8d (P2), 1c8h (P3) and 1k3v (P4), Sobemoviridae(S): 1smv (S1), 1x35 (S2), 1f2n (S3), 4sbv (S4) and 1ng0 (S5), Nodaviridae (N):1nov (N1), 2bbv (N2), 1f8v (N3) and fhv (N4).


650 PROTEINS


8/12

ever, CPs forming the same T number capsids displaygreater S-scores, in agreement with the similarity in the

quaternary organization as opposed to capsids with dif-ferent T numbers [Fig. 4(f)]. Taken together this suggeststhat greater structural similarity in the tertiary structureof CPs is necessary but not sufficient to form similarquaternary structures.

Identification and role of sequence and

space conserved residues

As an immediate application of the APODs, we identi-fied the conserved interface residues in the Fw spaceamong the capsids within a single family. This wasachieved by calculating the matrix product NSij

Qn N

kij,

where Nkij is a polar orthographic matrix (POM) repre-senting the APOD of capsid k in a family (set) of n cap-sids. The resulting matrix, NS, represents spatially con-served locations of the quaternary interactions in all the

capsids being considered. Only the nonzero matrix ele-ments ij common to all n POMs will also be nonzero in

NS. Another way of looking at this is as if all the chosenAPODs were stacked in register as layers and only the

(Fw) locations common to all the layers within a cer-tain cutoff interval were selected as the quaternary finger-print.

The above procedure was employed on structurallycharacterized members of the Nodaviridae family: 2BBV,1NOV, 1F8V, and FHV. Figure 5(a-d) shows the APODsof the individual members in the stack. Figure 5(e) showsthe nonzero cells (locations) that are common to all theAPODs in the stack representing spatially conserved

interface residues, a total of 58, among the four Nodavi-ruses. A subset (18) of these residues is also sequenceconserved and remarkably, the majority of these sequenceand space conserved residues (SSCs) surround the icosa-hedral symmetry axes [Fig. 5(f)].

Figure 6 shows an alignment of the amino acidsequence of the four CPs highlighting the locations ofthe 58 space conserved and the 18 sequence spaceconserved residues (SSCs). Only six of the 18 SSCs are

present in all the A, B and C subunits (Fig. 6; stars withgray background). The remaining (12) residues are present

Figure 4

Comparison of S-scores with other structural similarity metrics. S-score versus RMSD (left column), versus TM-score (middle column), and versussequence identity percentage (right column) between pairs of spherical virus capsids from three families. Results from the intra family analysis areshown in the first row: Parvoviridae (diamonds), Sobemoviridae (squares), and Nodaviridae (asterisk). The results of the inter-family analysis areshown in the second row: Sobemoviridae vs Parvoviridae (triangles), Nodaviridae vs Parvoviridae (Xs), and Sobemoviridae vs Nodaviridae (circles).A-subunits of the T5 3 capsids were used in the structural superpositions and sequence comparisons with the reference subunit of the T5 1capsids. Structural alignments were done using TM-align.12


PROTEINS 651


9/12

in either two out of the three subunits or only in a singlesubunit. We hypothesize that the six SSCs common to allthe three subunits might be of importance for self-assem-bly of Nodavirus capsids. Currently, there is no experi-mental data available on the role of these residues in the

assembly of Nodaviruses. Figure 7 shows the locations ofall (34) SSCs (11 in A-subunit, 13 in B-subunit and 10

in C-subunit) found in the CIAU of the Nodavirusesmapped on to the structure of the Black beetle virus. Itis interesting to note that all the SSCs found in the icosa-hedral asymmetric unit lie nearly on a single plane andthe majority of them are clustered near the icosahedral

symmetry axes, particularly at the icosahedral fivefoldand threefold axes [Fig. 7(a)].

Figure 5

Illustration of the APODs of the four Nodaviruses and the locations of the sequence and spatially conserved interface residues. 2BBV (a), 1NOV(b), 1F8V (c), and FHV (d) capsids of the Nodaviridae family. (e) Spatially conserved locations of the interface residues, which are common to allthe four members. (f) A subset of residue locations in the panel (e), which are also sequence conserved among the four Nodaviruses.


652 PROTEINS


10/12

Figure 6

Sequence alignment of the four Nodaviruses, highlighting the space conserved residues (gray and black stripes) and sequence and space conservedresidues (SSCs) (black stripes). The asterisks, semicolons and dots correspond to identical, similar and homologous residues in the alignment. Thesix SSCs that are common in all the three distinct (A/B/C) subunits are identified by the asterisk with the gray background.


PROTEINS 653


11/12

CONCLUSIONS

APODs of viral capsids provide a normalized finger-print of protein-protein interactions irrespective of theparticle size or the triangulation (T) number. Hence,they can be used as road maps of quaternary interactionsin spherical viral capsids to visualize the distribution andthe relative extent of interactions with respect to the ico-

sahedral symmetry axis. Furthermore, these fingerprintswhen represented in a matrix form facilitate the correla-tion of the fingerprints with one another. The similitudein these fingerprints represented as the S-score provides ameasure of overall similarity in the inter subunit (quater-nary) interactions between pairs or a group of capsids.The S-score accurately identifies the quaternary structuresimilarity or lack of it, which the other metrics (RMSD,TM-score, %Sequence Identity) fail to discern. A limited

analysis of viral capsids in terms of the APODs and S-scores suggests that the intra family/genus comparisons

will have S-scores mostly in the range of 1.0 to 0.6.Inter-family capsid pairs, which exhibit similar subunit-fold and the same capsid architecture (T-number) willhave S-scores in the range of 0.6 to 0.3 and the capsid

pairs with different T numbers and/or subunit tertiarystructures display S-scores below 0.3. Sequence-conservedresidues with the spatially conserved locations within afamily of virus capsids may play an important role in the

self-assembly of the member capsids. Exhaustive inter-and intra-family wide analysis of the other structurallyknown capsids is underway. The resultant APODs andtheir correlations will soon be available at the VIPERdbwebsite (http://viperdb.scripps.edu).

ACKNOWLEDGMENTS

We thank Ian Borelli for help in devising some of the

algorithms used in this work and maintaining VIPERdbup to date.

Figure 7

Illustrations highlighting the locations of sequence and space conserved interface residues (SSCs) in the Nodaviridae family mapped on to the 3D-structure of black beetle virus (2BBV). (a) A tube representation of the icosahedral asymmetric unit of black beetle virus (PDB-ID: 2BBV), a viewdown the quasi threefold axis, showing the traces of the A(blue), B(red), and C(green) subunits and C-alpha locations of the SSCs (spheres of all

colors) in the respective subunits. Residue (SSC) locations in the A, B, and C subunits are shown as blue, red and green spheres respectively whilethe orange spheres correspond to the six SSCs common to all the three distinct subunits. Perpendicular views showing the locations of the SSCs atthe (b) AB, (c) BC, and (d) CA interfaces.


654 PROTEINS


12/12

REFERENCES

1. Choi YG, Dreher TW, Rao AL. tRNA elements mediate the assembly of

an icosahedral RNA virus. Proc Natl Acad Sci USA 2002;99:655660.

2. Fox JM, Wang G, Speir JA, Olson NH, Johnson JE, Baker TS,

Young MJ. Comparison of the native CCMV virion with in vitro

assembled CCMV virions by cryoelectron microscopy and image

reconstruction. Virology 1998;244:212218.

3. Hsu C, Singh P, Ochoa W, Manayani DJ, Manchester M, Schnee-mann A, Reddy VS. Characterization of polymorphism displayed by

the coat protein mutants of tomato bushy stunt virus. Virology

2006;349:222229.

4. Sorger PK, Stockley PG, Harrison SC. Structure and assembly of

turnip crinkle virus. II. Mechanism of reassembly in vitro. J Mol

Biol 1986;191:639658.

5. Zhao X, Fox JM, Olson NH, Baker TS, Young MJ. In vitro assembly

of cowpea chlorotic mottle virus from coat protein expressed in

Escherichia coli and in vitro-transcribed viral cDNA. Virology

1995;207:486494.

6. Reddy VS, Giesing HA, Morton RT, Kumar A, Post CB, Brooks CL,

III, Johnson JE. Energetics of quasiequivalence: computational anal-

ysis of protein-protein interactions in icosahedral viruses. Biophys J

1998;74:546558.

7. Damodaran KV, Reddy VS, Johnson JE, Brooks CL, III. A general

method to quantify quasi-equivalence in icosahedral viruses. J MolBiol 2002;324:723737.

8. Shepherd CM, Reddy VS. Extent of protein-protein interactions

and quasi-equivalence in viral capsids. Proteins 2005;58:472477.

9. Bahadur RP, Rodier F, Janin J. A dissection of the protein-protein

interfaces in icosahedral virus capsids. J Mol Biol 2007;367:574590.

10. Shepherd CM, Borelli IA, Lander G, Natarajan P, Siddavanahalli V,

Bajaj C, Johnson JE, Brooks CL, III, Reddy VS. VIPERdb: a rela-

tional database for structural virology. Nucleic Acids Res

2006;34(Database issue):D386D389.

11. Reddy VS, Natarajan P, Okerberg B, Li K, Damodaran KV, Morton

RT, Brooks CL, III, Johnson JE. Virus particle explorer (VIPER), a

website for virus capsid structures and their computational analyses.

J Virol 2001;75:1194311947.

12. Zhang Y, Skolnick J. TM-align: a protein structure alignment algo-

rithm based on the TM-score. Nucleic Acids Res 2005;33:23022309.13. Zhang Y, Skolnick J. Scoring function for automated assessment of

protein structure template quality. Proteins 2004;57:702710.

14. Rossmann MG, Abad-Zapatero C, Murthy MR, Liljas L, Jones TA,

Strandberg B. Structural comparisons of some small spherical plant

viruses. J Mol Biol 1983;165:711736.

15. Liljas L, Unge T, Jones TA, Fridborg K, Lovgren S, Skoglund U,

Strandberg B. Structure of satellite tobacco necrosis virus at 3.0 A

resolution. J Mol Biol 1982;159:93108.

16. Abad-Zapatero C, Abdel-Meguid SS, Johnson JE, Leslie AGW, Ray-

ment I, Rossmann MG, Suck D, Tsukihara T. Structure of southern

bean mosaic virus at 2.8 A resolution. Nature (London)

1980;286:3339.

17. Harrison SC, Olson AJ, Schutt CE, Winkler FK. Tomato bushy

stunt virus at 2.9 A resolution. Nature 1978;276:368373.

18. Simpson AA, Chipman PR, Baker TS, Tijssen P, Rossmann MG.

The structure of an insect parvovirus (Galleria mellonella densovi-

rus) at 3.7 A resolution. Structure 1998;6:13551367.

19. Simpson AA, Chandrasekar V, Hebert B, Sullivan GM, Rossmann

MG, Parrish CR. Host range and variability of calcium binding by

surface loops in the capsids of canine and feline parvoviruses. J

Mol Biol 2000;300:597610.

20. Simpson AA, Hebert B, Sullivan GM, Parrish CR, Zadori Z, Tijssen

P, Rossmann MG. The structure of porcine parvovirus: comparison

with related viruses. J Mol Biol 2002;315:11891198.

21. Bhuvaneshwari M, Subramanya HS, Gopinath K, Savithri HS,

Nayudu MV, Murthy MR. Structure of sesbania mosaic virus at 3 A

resolution. Structure 1995;3:10211030.

22. Sangita V, Lokesh GL, Satheshkumar PS, Saravanan V, Vijay CS,

Savithri HS, Murthy MR. Structural studies on recombinant T 5 3

capsids of sesbania mosaic virus coat protein mutants. Acta Crystal-logr D Biol Crystallogr 2005;61:14021405.

23. Qu C, Liljas L, Opalka N, Brugidou C, Yeager M, Beachy RN, Fau-

quet CM, Johnson JE, Lin T. 3D domain swapping modulates the

stability of members of an icosahedral virus group. Structure

2000;8:10951103.

24. Tars K, Zeltins A, Liljas L. The three-dimensional structure of

cocksfoot mottle virus at 2.7 A resolution. Virology 2003;310:287

297.

25. Zlotnick A, Natarajan P, Munshi S, Johnson JE. Resolution of

space-group ambiguity and structure determination of nodamura

virus to 3.3 A resolution from pseudo-R32 (monoclinic) crystals.

Acta Crystallogr D Biol Crystallogr 1997;53:738746.

26. Wery JP, Reddy VS, Hosur MV, Johnson JE. The refined three-

dimensional structure of an insect virus at 2.8 A resolution. J Mol

Biol 1994;235:565586.27. Tang L, Johnson KN, Ball LA, Lin T, Yeager M, Johnson JE. The

structure of pariacoto virus reveals a dodecahedral cage of duplex

RNA. Nat Struct Biol 2001;8:7783.

28. Fisher AJ, Johnson JE. Ordered duplex RNA controls capsid archi-

tecture in an icosahedral animal virus. Nature 1993;361:176179.


PROTEINS 655

viral capsids

Documents