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Li et al. Retrovirology 2013, 10:126http://www.retrovirology.com/content/10/1/126

SHORT REPORT Open Access

Functional conservation of HIV-1 Gag: implicationsfor rational drug designGuangdi Li1, Jens Verheyen2, Soo-Yon Rhee1,3, Arnout Voet4, Anne-Mieke Vandamme1,5 and Kristof Theys1*

Abstract

Background: HIV-1 replication can be successfully blocked by targeting gag gene products, offering a promisingstrategy for new drug classes that complement current HIV-1 treatment options. However, naturally occurringpolymorphisms at drug binding sites can severely compromise HIV-1 susceptibility to gag inhibitors in clinical andexperimental studies. Therefore, a comprehensive understanding of gag natural diversity is needed.

Findings: We analyzed the degree of functional conservation in 10862 full-length gag sequences across 8 major HIV-1subtypes and identified the impact of natural variation on known drug binding positions targeted by more than 20gag inhibitors published to date. Complete conservation across all subtypes was detected in 147 (29%) out of 500gag positions, with the highest level of conservation observed in capsid protein. Almost half (41%) of the 136 knowndrug binding positions were completely conserved, but all inhibitors were confronted with naturally occurringpolymorphisms in their binding sites, some of which correlated with HIV-1 subtype. Integration of sequence andstructural information revealed one drug binding pocket with minimal genetic variability, which is situated at theN-terminal domain of the capsid protein.

Conclusions: This first large-scale analysis of full-length HIV-1 gag provided a detailed mapping of natural diversityacross major subtypes and highlighted the considerable variation in current drug binding sites. Our results contributeto the optimization of gag inhibitors in rational drug design, given that drug binding sites should ideally be conservedacross all HIV-1 subtypes.

Keywords: HIV subtype, Gag inhibitor, Matrix, Capsid, Nucleocapsid, Drug binding site, Natural polymorphism, Aminoacid conservation

IntroductionA curative therapy or preventive vaccine for HIV-1 infectedpatients remains elusive to date. Standard HIV treatment isconfronted with the emergence of viral resistance to exist-ing drug classes, necessitating the development of inhibi-tors with new mechanisms of action [1]. The gagpolyprotein, essential for HIV-1 morphogenesis, comprisesfour major domains (matrix, capsid, nucleocapsid, p6) andtwo small spacer peptides (p1, p2) [2]. Recently, HIV-1 in-hibitors that target different stages of virion morphogenesisdemonstrated promising antiviral activity, mainly by inhi-biting capsid assembly, disrupting nucleocapsid bindingwith viral RNA/DNA or blocking proteolytic processing ofpolyproteins during maturation [2-5].

* Correspondence: [email protected] Institute for Medical Research, Department of Microbiology andImmunology, KU Leuven, Leuven, BelgiumFull list of author information is available at the end of the article

© 2013 Li et al.; licensee BioMed Central Ltd. TCommons Attribution License (http://creativecreproduction in any medium, provided the or

HIV-1 subtype B isolates were predominantly used forthe in vitro experiments. Non-B subtypes however ac-count for 90% of HIV-1 infections worldwide [6] andamino acid (AA) compositions can differ up to 30% be-tween subtypes [7]. Recently, treatment failure of pa-tients in a phase II clinical study of the maturationinhibitor bevirimat was attributed to natural polymor-phisms at drug binding positions, showing up insubtype-specific patterns [8]. Studies that extensively in-vestigate the implications of HIV-1 diversity for gag-directed drug development are lacking to date. In thislarge-scale analysis, we examined the distribution of nat-urally occurring sequence variability in full-length gagsequences of major HIV-1 subtypes. Moreover, we evalu-ated the impact of HIV-1 subtypes on the conservationof gag drug binding positions and multisite bindingpockets published to date.

his is an open access article distributed under the terms of the Creativeommons.org/licenses/by/2.0), which permits unrestricted use, distribution, andiginal work is properly cited.

mailto:[email protected]

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Capsid

p2 Nucleocapsid

p1 p6* * * * * * * * * * * *G H Q M K D

Figure 1 Distribution of natural variations at 500 gag positions of HIV-1 group M (subtypes: A1, B, C, D, F1, G and CRF01_AE,CRF02_AG). The first position of each protein region is labeled with its protein name in a box. Annotated protein regions are indicated ascolored bars: light-green for matrix (positions 1–132), light-blue for capsid (133–363), dark-green for p2 (364–377) and p1 (433–448), dark-blue fornucleocapsid (378–432) and grey for p6 (449–500). HXB2 indices for both full-length gag and individual proteins are shown on top of the coloredbars (e.g. ‘180|48’ indicates the gag position 180 and the capsid position 48). Known drug binding positions are marked with red stars. Consensussubtype B amino acid for each position is shown directly under the bar, and is highlighted green when the consensus AA differed in one or moresubtypes. Natural polymorphisms are shown below the consensus subtype B amino acids; proportions (%) are colored blue for proportion ≥ 5%;orange otherwise. Figure S1 in Additional file 2 provides the distribution of natural polymorphisms within each individual subtype.

Table 1 Natural polymorphism proportions in gag domains and drug binding positions across 8 HIV-1 subtypes andCRFs (%)

B A1 C D F1 G 01_AE 02_AG Mean

Matrix[132/13] 57.6/38.5 62.1/46.2 59.1/46.2 64.4/53.8 52.3/46.2 66.7/61.5 61.4/46.2 56.1/30.8 59.9/46.2

Capsid[231/98] 27.3/30.6 34.2/33.7 29.4/29.6 27.7/27.6 31.2/37.8 30.3/28.6 28.1/28.6 27.3/27.6 29.4/30.5

p2[14/8] 71.4/62.5 64.3/62.5 64.3/62.5 64.3/62.5 57.1/62.5 71.4/62.5 64.3/62.5 50.0/50.0 63.4/60.9

NC[55/17] 56.4/58.8 41.8/35.3 38.2/41.2 36.4/29.4 34.5/23.5 54.5/58.8 43.6/35.3 34.5/41.2 42.5/40.4

p1[16/0] 37.5/- 25.0/- 31.2/- 43.8/- 25.0/- 31.2/- 31.2/- 12.5/- 29.7/-

p6[52/0] 76.9/- 69.2/- 69.2/- 55.8/- 65.4/- 61.5/- 69.2/- 57.7/- 65.6/-

Mean 45.2/36.8 46.6/36.8 43.4/34.6 42.8/32.4 41.2/38.2 47.0/37.5 44.0/33.1 39.0/30.9 43.6/35.0

Each gag protein is demonstrated with its total AA and drug binding positions (e.g. Matrix [132/13]: 132 AA positions/13 drug binding positions). Naturalpolymorphism proportion (%) is indicated for each protein and subtype with respect to the total number of AAs and to the number of drug binding positions(e.g. 57.6/38.5 shows that for subtype B matrix, 57.6% of all 132 positions and 38.5% of 13 drug binding positions are polymorphic). Mean values across gagdomains and across HIV-1 subtypes are indicated in the last row and column respectively.

Li et al. Retrovirology 2013, 10:126 of 11http://www.retrovirology.com/content/10/1/126

Table 2 The inter- and intra-subtype diversity of gag AA sequences in 8 HIV-1 subtypes and CRFs (%)

Subtype B A1 C D F1 G 01_AE 02_AG

Intra-subtype 8.96 8.34 9.89 8.91 9.45 10.90 7.58 8.26

Inter-subtype B 17.54 18.38 12.70 16.52 18.46 17.22 18.61

A1 17.65 16.73 16.93 17.27 12.71 14.69

C 16.67 17.21 18.02 18.22 19.59

D 16.55 17.56 16.85 18.72

F1 15.93 16.48 17.68

G 17.70 18.81

01_AE 14.92


ResultsWe analyzed 10862 full-length gag sequences that ful-filled the quality criteria, encompassing 8 HIV-1 groupM subtypes and CRFs: A1 (n = 1648), B (n = 4131), C(n = 2780), D (n = 443), F1 (n = 35), G (n = 49),CRF01_AE (n = 1714) and CRF02_AG (n = 62). Se-quences were sampled from 61 countries between 1981and 2012. Additional file 1: Table S1 summarizes morethan 50 gag inhibitors including their binding sites, tar-get protein, mechanism of action, HIV-1 subtypes andPDB data. These candidate inhibitors were either smallorganic molecules or peptides and primarily targeted thecapsid or nucleocapsid proteins. A total of 136 gag posi-tions were reported as drug binding positions, of which53 interacted with more than one inhibitor.The AA distribution at 500 gag positions among HIV-

1 group M sequences is shown in Figure 1 and subtype-specific distributions are also visualized (Additional file 2:Figure S1). Heterogeneity in consensus sequences was ob-served at 142 (28.4%) positions across subtypes, whilepairwise comparisons of consensus sequences showed anaverage of 11.6% difference between subtypes. On average,43.6 ± 2.7% of positions harbored at least one polymorph-ism relative to its subtype consensus residue (Table 1).The capsid protein (29.4%) contained the lowest numberof polymorphic positions followed by nucleocapsid(42.5%), matrix (59.9%), and p6 (65.6%). Moreover, of 147

Table 3 The pairwise AA diversity of gag domains in 8 HIV-1

Matrix Capsid p2

B 12.36 4.56 20.65

A1 10.69 5.46 13.97

C 14.77 5.77 23.79

D 12.74 4.87 25.62

F1 12.71 5.37 29.24

G 17.51 6.15 16.06

01_AE 11.36 3.44 24.36

02_AG 13.67 4.91 18.50

Mean 13.17±2.01 5.04±0.79 21.49±4.83

conserved positions in gag, 67.8% were in capsid, 11.2% innucleocapsid, 10.5% in matrix and 4.6% in p6. PairwiseAA diversity (Additional file 3) of full-length gagsequences decreased from 17.0 ± 1.6% between subtypesto 9.0 ± 1.0% within subtypes (Table 2). The mean AAdiversity was significantly lower for capsid (5.0 ± 0.8%)than for nucleocapsid (7.9 ± 2.8%), matrix (13.2 ± 2.0%) orp6 (14.7 ± 2.0%) (p-value < 0.05) (Table 3). The CI distri-butions of full-length gag characterized three conservedregions located at the nucleocapsid zinc-finger domains,the capsid N-terminal domain (NTD) and C-terminaldomain (CTD) (Figure 2).Subtype-specific AA prevalence at the 136 drug bind-

ing positions is shown in Figure 3. Most positions werelocated within capsid (72.1%) followed by nucleocapsid(12.5%), matrix (9.6%) and p2 (5.9%). Of these positions,41.2% were conserved across all subtypes, while 20.6%showed a different consensus AA in one or more sub-types. On average, 33.8% of drug binding positions har-bored at least one polymorphism and 16.3% had at leastone polymorphism above 5% prevalence. Non-B sub-types displayed 32 polymorphisms at 20 binding posi-tions that were absent in subtype B. Every inhibitor hadat least one polymorphic binding position and 15 inhibi-tors had more than 50% of drug binding positions show-ing natural polymorphisms. Among all inhibitors,PF-3450074 [9] targeted the most conserved binding

subtypes and CRFs (%)

NC p1 p6 Gag

10.31 4.77 15.74 8.84

4.75 5.60 17.17 8.18

9.79 4.80 15.83 9.96

9.43 8.89 10.17 8.68

9.00 6.71 15.32 9.32

10.53 8.42 13.90 10.71

4.95 7.74 14.07 7.46

3.31 5.39 14.39 8.35

7.92±2.77 6.39±1.60 14.70±1.99 9.02±1.09

0

0.05

0.1

0.15

Matrix Capsid p2 NC p1 p6

50 100 132 150 200 250 300 363 377 400 432 448 500

BA1CDF1G01_AE02_AG

The

con

serv

atio

n in

dex

Matrix1 HAGPIA Capsid1 H8 NYAD Gagp6Peptide-inhibitor-derived regions

Drug binding positions

Conserved positions

p6

p1

NC

p2

Capsid

Matrix

B

A

Conserved positionsDrug binding positionsPeptide-inhibitor-derived regions- Conservation index of HIV-1

subtypes B, A1, C, D, F1, G, CRF01_AE and CRF02_AG.

HXB2 reference index

1

5

4

32

10

11

12

8

9

6

7

4

123

12

11

1

2

3

Figure 2 (See legend on next page.)


(See figure on previous page.)Figure 2 Amino acid conservation in HIV-1 full-length gag. (A) Density plots of CI values are shown for 8 HIV-1 subtypes. Secondary struc-tures are indicated for each protein region, with thick lines for helices and thin lines for coiled-coil structures. Positions conserved in all subtypesare colored blue (layer 1 in a small circle), known drug binding positions are colored red (layer 2) and regions where HIV-1 peptide inhibitors havebeen derived are colored green (layer 3). (B) Distributions of CI values at 500 gag positions across 8 HIV-1 subtypes and CRFs. Visualization soft-ware: Circos v0.64 (http://circos.ca/).


positions at the capsid N-terminal domain, with onlyone being polymorphic (T107A/S, ≤ 6.2%) (Additionalfile 1: Table S2).Finally, we analyzed known crystal structures of 9

protein-inhibitor complexes, with 8 inhibitors targetinga total of 75 positions (binding pockets 1–4) in capsidand one targeting 23 positions in nucleocapsid (bindingpocket 5) (Figure 4, Additional file 2). Natural polymor-phisms with prevalence ≥ 5% were observed in 28 posi-tions of the binding pockets. Conserved positions wereobserved in 56% of the capsid binding pockets and 43%of the nucleocapsid binding pocket. Pocket 1 (0.0024)had the lowest average CI values compared to pocket 2(0.008), 3 (0.0216), 4 (0.0337) or 5 (0.0369).

Discussion and conclusionsTo our knowledge, our large-scale analysis provided thefirst detailed mapping of functional conservation of gagacross major HIV-1 subtypes, with implications for the ra-tional design of gag inhibitors. With more than 50 gag in-hibitors published to date, targeting virion morphogenesisis considered a potential new drug class for HIV-1 treat-ment [2]. A clinical proof-of-concept was demonstrated ina phase II clinical trial of the maturation inhibitor bevirimat[10], which blocks proteolytic processing at the capsid-p2cleavage site [11]. Lack of response was observed in 50% ofpatients and attributed to naturally occurring polymor-phisms in the p2 region [8]. A single polymorphism V370Ais sufficient for a 40-fold reduction in bevirimat drug sus-ceptibility [12], with A370 representing the consensusamino acid in several non-B subtypes. Natural diversity wasalso observed to affect drug effectiveness of other experi-mental gag inhibitors [13-15]. Polymorphisms T190I,E230D and I256V, for instance, reduced drug susceptibilityto the benzodiazepine and benzimidazole compounds [13].Moreover, known HIV vaccine candidates containing sub-type B gag gene in HIV-derived vectors did not show suffi-cient protective efficacies in several large-scale clinical trials[16]. The high diversity of gag and env genes within and be-tween subtypes can contribute to the challenges of design-ing a global HIV vaccine neutralizing all HIV-1 subtypes[17]. For the development of HIV vaccine and a potentialnew drug class targeting virion morphogenesis [2], an as-sessment of gag functional conservation and polymor-phisms at known drug binding positions is warranted.We found that 23.4% of drug binding positions in the

full-length gag showed natural polymorphisms in non-B

subtypes which could not be detected in subtype B.More importantly, all gag inhibitors had at least onepolymorphic binding position irrespective of subtype.We also found levels of gag intra- and inter-subtype di-versity (9.04% and 17.0%) that exceeded diversity esti-mates of key viral enzymes (< 7% and < 11%) targeted bystandard HIV-1 treatment [18]. However, the most con-served gag protein capsid has the same level of intra-subtype diversity as integrase (~5%) [18], favoring it as aconserved drug target.The capsid protein targeted by most candidate inhibi-

tors accounted for 67.7% of conserved gag positionsand contained 72.1% of the 136 binding positions previ-ously reported. Our sequence analysis identified twoconserved capsid regions (Figure 2) located at the inter-action interfaces between N-terminal domains (NTD-NTD) as well as between N-terminal and C-terminaldomains (NTD-CTD) (Figure 5). These interactioninterfaces, crucial for the assembly and stabilization ofpentamer and hexamer lattices [19], provide potentialconserved drug targets. To reveal the ideal drug target,we described 4 crystalized drug binding pockets incapsid (Figure 4, Additional file 2: Figure S4). Inhibitorsthat target pockets 1–3 have shown promising antiviralactivity against capsid multimerization in different subtypestrains by altering NTD-CTD interaction (pockets 1 and 3)or NTD-NTD interaction (pocket 2) [15,20,21]. Pocket 4 isless conserved and its polymorphic residues make directcontact with inhibitors, hindering the development of in-hibitors that target this pocket [13].Another potential drug target is the nucleocapsid pro-

tein, containing two critical zinc-finger domains forbinding with viral RNA genomes [2]. Our conservationanalysis mapped the conserved nucleocapsid regions tozinc-finger domains (Figures 2 and 5) and confirmedprevious findings of absolute conservation of CCHC mo-tifs at zinc-coordinating positions [22]. However, we de-tected considerable variation at other positions, whichmay alter drug binding and affect antiviral activity. Fur-thermore, nucleocapsid inhibitors tend to suffer fromlimited specificity and high toxicity due to the ubiqui-tous presence of zinc finger domains in many humanproteins [4].Matrix inhibitors with broad spectrum antiviral activ-

ities were recently reported, but mutations at drug bind-ing positions significantly reduced their effectiveness[23,24]. We also observed many natural variants at their

http://circos.ca/

Figure 3 Natural polymorphisms at 136 drug binding positions in 8 HIV-1 subtypes and CRFs. For each gag position, the HXB2 index isshown at the top, followed by the consensus amino acid and natural polymorphisms. Polymorphisms with proportions ≥ 5% are indicated withblue superscripts; orange otherwise.


Figure 4 (See legend on next page.)


(See figure on previous page.)Figure 4 Mapping of drug binding positions and binding pockets to HIV-1 gag protein monomers. The surface spectrum colors indicatethe most to the least conserved positions in subtype B from blue CI = 0 to pink CI ≥ 0.1. (A) Secondary structures of 4 gag proteins and 2 spacerpeptides, annotated with five drug binding pocket locations. Gag proteins in cartoon representation are colored olive for matrix, blue for capsid, yellowfor nucleocapsid, grey for p6, gold for p1 and p2. Bound inhibitors are represented in green sticks. (B) Mapping of drug binding positions to a surfacerepresentation of gag structure, with front and back views. Hypothesized binding positions of bevirimat are also annotated; known drug bindingpositions are colored red. (C) Surface representation of gag conservation in HIV-1 subtype B (Figure S3 in Additional file 2 illustrates other subtypes). (D)Surface representations of five drug binding pockets in HIV-1 subtype B (Figure S2 in Additional file 2 shows other subtypes). Inhibitor names are anno-tated according to publication (Additional file 1: Table S1). PDB entries of gag proteins: matrix, 1HIW; capsid, 3NTE; p2, 1U57; nucleocapsid, 2M3Z; p6,2C55. PDB data of capsid inhibitors: 2BUO, 2L6E, 2XDE, 4E91, 4E92, 2JPR and 4INB, each of which was superimposed to 3H4E using PDBs of 5 drug bind-ing pockets: pocket 1, 2XDE; pocket 2, 4INB; pocket 3, 2BUO; pocket 4, 4E91; pocket 5, 2M3Z. PyMOL V1.5 (http://www.pymol.org/).


drug binding sites (Additional file 1: Table S2), suggest-ing that further optimization of matrix inhibitors isneeded.Studies that analyzed genetic variability and drug bind-

ing site heterogeneity in gag using large-scale sequencepopulations are lacking. Previously, small subtype B se-quence datasets were used to characterize gag conserva-tion (n = 125) [25] or positive selective pressure (n =635) [26]. Polymorphisms at drug binding sites of capsidinhibitor PF-3450074 [9] and conservation of nucleocap-sid zinc-finger domains [22] were also reported usingfewer than 200 sequences. The only large-scale analysisthat we found [27] quantified the drug binding site con-servation of a single matrix inhibitor and lacked infor-mation on subtype-specific variations. By contrast, wepresented here a large-scale and integrative analysisusing 10862 full-length gag sequences, 136 gag inhibitordrug binding positions and 14 PDB structures. Naturalpolymorphisms of full-length gag were detected across 8major HIV-1 subtypes and a robust estimation of func-tional conservation was performed using CI analysis,which incorporated biochemical similarities betweenamino acids (Additional file 3). This sequence analysispredicted three conserved drug targets in gag (Figure 2)which were confirmed by existing structural knowledge(Figure 5).This study is limited in that it neither addressed how

to optimize known gag inhibitors nor quantified the im-pact of newly identified polymorphisms on antiviral ac-tivities of investigated inhibitors. We collected allavailable PDBs of gag-inhibitor structures from theRCSB protein data bank, but more crystallized com-plexes are needed to reveal novel mechanisms of action.Moreover, the limited number of available gag sequencesfor subtypes F1, G and CRF02_AG (n < 100) may haveaffected the identification of polymorphic positions, butconsistent conservation patterns were observed in gagregardless of HIV-1 subtype (Figure 2). While weattempted to be as comprehensive as possible, additionalinhibitors may have been reported. Conservation of theirbinding positions can nevertheless be deduced from ourfull-length gag analysis. Future studies are also neededto address whether interactions between gag and

protease can affect gag drug binding sites, leading tocompromised drug activities of gag inhibitors [28].In conclusion, our study presented a comprehensive

mapping of functional conservation in gag and strength-ened the idea of capsid as a potential target for HIV-1therapeutics. Increased knowledge on HIV-1 natural di-versity in drug binding pockets contributes to rationaldesign of gag inhibitors and it remains a challenge to de-sign gag inhibitors with drug binding sites conservedacross HIV-1 subtypes.

MethodsWe retrieved 12543 gag sequences spanning all 1500base pairs from the HIV Los Alamos database(http://www.hiv.lanl.gov). Sequences were aligned againstthe HXB2 reference and manually curated using Seaview4.3 [29]. Hypermutated sequences were detected usingthe Los Alamos hypermut tool [30]. HIV-1 subtype wasdetermined by the Rega [31] and COMET subtyping tools(http://comet.retrovirology.lu/). Sequence quality was en-sured by excluding duplicates and sequences withinternal stop-codons, hypermutations, more than 1% am-biguous nucleotides, discordant subtype classification oran identical combination of patient code, sampling yearand country. The analysis was restricted to the majorsubtypes and circulating recombinant forms (CRFs) char-acterizing the global HIV-1 subtype distribution [6]. Foreach individual subtype, amino acids that differed fromthe corresponding consensus AA and with prevalence ≥0.5% were defined as polymorphisms [18]. PDB data ofprotein-inhibitor complexes were collected from the RCSBProtein Data Bank [32], summarized in Additional file 1.The AA sequences in each PDB were aligned against theHXB2 reference. Drug binding pockets were defined byprotein positions within a minimum Euclidean distance ofless than 5Å between atoms of inhibitors and non-hydrogen atoms of residues [33]. Information on knowngag candidate inhibitors and binding positions was re-trieved from more than 50 publications, summarized inAdditional file 1.To quantify the degree of positional conservation, a

conservation index (CI) was calculated for each posi-tion by averaging pairwise scores between all AAs

http://www.hiv.lanl.gov/

http://comet.retrovirology.lu/

http://www.pymol.org/

Figure 5 Visualization of conserved regions in capsid and nucleocapsid. The capsid hexamer structure (PDB: 3H4E) is shown in top (A) andside (B) views, with the 6 capsid units (pink, blue), conserved NTD-NTD interaction domains (yellow) and conserved NTD-CTD interaction domains(red). Figure (C) shows the structural complex of nucleocapsid and RNA (left, PDB: 1A1T) and the structural complex of nucleocapsid and inhibitorCAA (right, PDB: 2M3Z). The first zinc-finger domain (nucleocapsid positions: 14–29, gag positions: 389–404) and the second zinc-finger domain(nucleocapsid positions: 35–50, gag positions: 410–425) are colored red and orange, respectively. Figures S5 and S6 in Additional file 2 provide de-tailed structures of conserved gag regions.


using the BLOSUM62 substitution matrix. Adaptedfrom Karlin and Brocchieri [34], the conservationindex (CI) of position x is calculated as:

CI xð Þ ¼ 1− 2N N−1ð Þ

XNi¼1

XNj¼iþ1

S xi; xj� �

=ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiS xi; xið ÞS xj; xj

� �q� �, where

xi is the amino acid at position x in the ith sequence ofthe multiple sequence alignment (MSA), N is the num-ber of sequences in the MSA and S(xi, xj) is the

substitution score of BLOSUM62 between amino acidsxi and xi. Given that denominators cannot be zero, a lin-ear transformation was applied to S(xi, xj) by adding theabsolute value of the minimum score | min(S)| + 1. CImeasures were scaled between 0 and 1, with a CI valueof 0 indicating that AA variation was absent at that pos-ition. A highly conserved position was identified if its CIis below 0.01 for each HIV-1 subtype, a cutoff which


corresponds approximately to a cumulative polymo-rphism prevalence below 1% (Additional file 3). TheMann–Whitney U test was performed to compare CIdistributions. Performance of the CI method is evaluatedin Additional file 3 and our Matlab toolbox for sequenceanalysis is available in Additional file 4.

Additional files

Additional file 1: Table S1. The summary of gag candidate inhibitorspublished in literature. Table S2. The prevalence of natural polymorphismsat known drug binding sites in 8 HIV-1 subtypes and CFRs.

Additional file 2: Figure S1. The distribution of natural variations offull-length gag in 8 HIV-1 subtypes. Figure S2. The surface representationof full-length gag in 8 HIV-1 subtypes and CRFs. Figure S3. The surfacerepresentation of five drug binding pockets in 8 HIV-1 subtypes. Figure S3.The surface representation of full-length gag in 8 HIV-1 subtypes and CRFs.Figure S4. The structure of capsid hexamer superimposed with 8 crystalizedinhibitors. Figure S5. The surface representation of conserved regions inHIV-1 capsid Figure S6. The surface representation of conserved regions inHIV-1 nucleocapsid.

Additional file 3: Note S1. The mathematical model of conservationindex. Note S2. The mathematical model of inter- and intra-subtypediversity.

Additional file 4: The Matlab toolbox developed for conservationanalysis, inter- and intra-subtype diversity analysis. The full-lengthgag sequence datasets are also included.

AbbreviationsAA: amino acid; CI: conservation index; CTD: C-terminal domain;CRF: circulating recombinant form; MSA: multiple sequence alignment;NC: nucleocapsid; NTD: N-terminal domain; PDB: protein data bank.

Competing interestsThe authors declare that they have no competing interests.

Authors’ contributionsGL and KT conceived and designed the study. All authors have participatedin the discussion and interpretation of the results and writing of themanuscript. All authors read and approved the final manuscript.

AcknowledgementsWe thank Supinya Piampongsant, Fossie Ferreira, Andrea Clemencia Pineda,Ricardo Khouri, Mónica Eusébio, Soraya Maria Menezes and Dan Clements fortechnical assistance and valuable contributions to the analysis. The researchleading to these results has been supported in part by the EuropeanCommunity's Seventh Framework Programme (FP7/2007-2013) under theproject "Collaborative HIV and Anti-HIV Drug Resistance Network (CHAIN)"grant agreement n° 223131; by the Fonds voor Wetenschappelijk Onderzoek– Flanders (FWO) grant G.0611.09. GL acknowledged his funding from ChinaScholarship Council. AV acknowledged his JSPS funding. KT received fundingfrom the Fonds voor Wetenschappelijk Onderzoek – Flanders (FWO).

Author details1Rega Institute for Medical Research, Department of Microbiology andImmunology, KU Leuven, Leuven, Belgium. 2Institute of Virology, Universityhospital, University Duisburg-Essen, Essen, Germany. 3Division of InfectiousDiseases, Department of Medicine, Stanford University, Stanford, CA, USA.4Zhang IRU, RIKEN Institute Laboratories, Hirosawa 2-1, Wako-shi, Saitama,Japan. 5Centro de Malária e Outras Doenças Tropicais and Unidade deMicrobiologia, Instituto de Higiene e Medicina Tropical, Universidade Novade Lisboa, Lisbon, Portugal.

Received: 14 July 2013 Accepted: 21 October 2013Published: 31 October 2013

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doi:10.1186/1742-4690-10-126Cite this article as: Li et al.: Functional conservation of HIV-1 gag:implications for rational drug design. Retrovirology 2013 10:126.

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