Retroviruses are a major concern for public health in humans but also in animals. The feline immunodeficiency virus (FIV) is the causative agent of an acquired immunodeficiency syndrome (AIDS) in felines [1
] with a prevalence rate of up to 30% of domestic cats in some areas [2
]. Feline immunodeficiency virus is a member of the genus Lentivirus from the Retroviridae family [4
], which also contains human immunodeficiency virus (HIV), equine infectious anemia virus (EIAV), and simian immunodeficiency virus (SIV), among others. Due to their common biological characteristics such as virion morphology, physiology, and pathogenesis, FIV has been described as a useful non-primate model for HIV infection, antiretroviral therapy and vaccine development. Feline immunodeficiency virus could also be used as a simple model for a rational drug design for HIV [5
Like all infectious retroviruses, the FIV genome contains the three genes—Gag
, and Env
—encoding for the structural proteins, the viral enzymes, and the envelope proteins, respectively [8
]. The Gag polyprotein is involved in the architecture of the viral particle [9
]. As for HIV-1 and SIV (but not EIAV), the FIV Gag protein is myristoylated at its N-terminus [11
], which allows its targeting of the plasma membrane at the time of particle assembly [15
]. During the maturation step of the virus life cycle, the FIV Gag polyprotein is cleaved by the viral protease into different subunits—the matrix protein (MA), the capsid protein (CA), and the nucleocapsid protein (NC)—with a spacer peptide p1 between the CA and NC domains and a C-terminal peptide p2 [16
]. The MA protein forms a layer that underlies the viral envelope of the virion, which contains the CA cone-shaped core and the complex formed by the NC and the RNA viral genome [5
Retrovirus assembly is a critical step in the viral replication cycle and this process is driven by the CA protein [17
]. Indeed, CA protein forms a protective coat around the viral genome and its assembly into a cone-shaped core is characteristic of a mature lentivirus. Moreover, a virus particle without a properly assembled cone-shaped core appears to be non-infectious [20
]. Based on this observation, CA protein is a promising therapeutic target for antiretroviral therapy against HIV-1 [21
Previous crystallographic studies have determined the structure of full-length retroviral CA units as a dimer for EIAV [22
] and dimer, pentamer, or hexamer for HIV-1 [23
]. Such high-order oligomers are necessary for the formation of a mature capsid core in the viral particle [27
]. Cross-linking agents and antibody fragments have often been used to stabilize CA for crystallographic studies [29
] and CryoEM studies have deciphered supramolecular assemblies of tubular and cone-shaped CAs [30
]. Despite low sequence homologies, retroviral CA proteins harbor a rather similar α-helical topology, with two domains—the amino-terminal domain (NTD) and the carboxy-terminal domain (CTD)—connected by a flexible linker [22
]. This linker plays an important role in the relative flexibility of the CANTD
during the assembly of CA proteins into pentamers or hexamers [25
]. A highly-conserved sequence is observed amongst all retroviral CA proteins, called the major homology region (MHR), while the rest of the sequence is less conserved between retroviral species. This MHR is required for the correct folding and stability of the CACTD
domain and, thus, is essential for viral replication [31
Thus far, only the structure of the CTD domain of FIV CA has been described [33
]. To better understand the molecular and structural specificities of this protein, its crystal structure was determined at 1.67 Å resolution and original features when compared with other lentiviral CAs such as HIV-1 and EIAV were observed. Functional consequences will be discussed.
2. Materials and Methods
2.1. Construction of Recombinant Plasmid Encoding the FIV Capsid Protein
The full-length native FIV capsid protein was amplified by a polymerase chain reaction on the plasmid p34TF10 (Petaluma strain) as described [34
]. A truncated form of the CA protein in its C-terminal end of 9 amino acid residues with a mutation of Pro1 in Thr1 (p24EΔCP-T) was then constructed by PCR using the same protocol and a pair of primers, 5′-AGGATCCAATAGAAGGACGAACT
ATTCAAACAGT-3′ and 5′-TGAATTCTCA
TATTTCTTGACAAGCCCTCAAC-3′, where the Pro1Thr mutation and the introduced stop codon are shown in bold and underlined, respectively. The Pro1Thr mutation was introduced to allow the removal of the 6 × His tag by the Factor Xa protease, which removes all the amino acid of the cleavage site, allowing an intact N-terminus of the protein of interest. However, Factor Xa was not active when the first amino acid after the cleavage site was a proline residue. The product was digested with BamHI and EcoRI and then ligated into the BamHI/EcoRI sites of the vector pRSET-B (Invitrogen, Thermo Fisher Scientific, Villebon-sur-Yvette, France) to form the recombinant plasmid pRSET-p24EΔCP-T encoding the FIV CA protein with a 6 × His tag at its N-terminal end.
2.2. Expression and Purification of FIV CA Protein
Escherichia coli cells (BL2I (DE3) pLysS, Lucigen, Middleton, WI, USA) transformed with pRSET-p24EΔCP-T were grown in Lysogenic broth medium (Sigma-Aldrich, Saint-Quentin-Fallavier, France) supplemented with 50 mg/mL of ampicillin, at 37 °C. Cell density was monitored by measuring the optical density at 600 nm (OD600). When cells reached an OD600 value between 0.3 and 0.4, the expression of CA protein was induced by the addition of IPTG (Isopropyl-β-d-1-thiogalactopyranoside, Euromedex, Souffelweyersheim, France) to a final concentration of 1 mM. Expression was carried on for an additional 20 h at 25 °C, then cells were harvested by centrifugation and the pellets were stored overnight at −20 °C.
Purification of CA protein was performed by nickel affinity chromatography, as described for the native CA protein [34
]. Briefly, the lysate was clarified by centrifugation at 10,000× g
for 45 min, and the supernatant was filtered through a 0.45 μm membrane. Purification of the protein from the supernatant was done by batch incubation Ni2+
-TED resin (Macherey-Nagel, Hoerdt, France) followed by loading onto a gravity column. The column was washed three times with LEW buffer (50 mM NaH2
, 300 mM NaCl, pH 8.5), and the elution was then performed with LEW buffer containing 50 mM of imidazole.
The concentration of CA protein was quantified by spectrophotometry at 280 nm, using a Nanodrop (Thermo Fisher). The purity of the protein was evaluated by SDS-PAGE analysis. Buffer exchange, using Vivaspin ultrafiltration devices (10 kD MWCO, Sartorius, Aubagne, France), was performed against HEPES/NaCl Buffer (50 mM HEPES pH 6.5, 100 mM NaCl).
2.3. Removal of the 6 × His Tag
To remove the 6 × His tag, purified CA protein in HEPES/NaCl buffer was digested overnight with 16U of Factor Xa (Qiagen, Courtaboeuf, France) per mg of CA protein, at 19 °C. After proteolysis, the tag-free protein was obtained by loading the sample on a Ni-Nitrilotriacetic acid (NTA) centrifugation column (Proteus, Cliniscience, Nanterre, France) according to the manufacturer’s protocol, and collecting the flowthrough. Purified CA proteins were then concentrated to 7 mg/mL using a Vivaspin centrifugal concentrator (10 kD MWCO, Sartorius).
2.4. Crystallization of the FIV CA Protein
Screening of crystallization conditions was performed in 96-well plates using a mosquito nanopipette and commercial crystallization screening kits (Hampton Research, Aliso Viejo, CA, USA and Qiagen) with the sitting drop procedure. The FIV CA protein at 7 mg/mL in HEPES/NaCl buffer crystallized in the presence of an equal volume of 0.2 M magnesium sulfate, 20% PEG 4000, 10% glycerol (condition E11 of the Qiagen PEGs II Suite) supplemented with 10% DMSO final. Using these conditions with the hanging drop technique and drops of 1 μL of protein with 1 μL of crystallization conditions, plate-shaped crystals grew within 15 days. Due to the presence of 10% glycerol in the crystallization solution, the cryoprotection step was dispensable and crystals were directly flash frozen in liquid nitrogen prior to data collection.
2.5. X-ray Data Collection and Structure Determination
X-ray data were collected at best to 1.67 Å resolution at the European Synchrotron Research Facility (ESRF) beamline ID30-B (Grenoble, France) at 100 K with a wavelength of 0.99187 Å and a PILATUS 6M-F detector. Crystals belonged to a monoclinic space group C2 with cell dimensions a = 122.2 Å, b = 74.6 Å, c = 77.0 Å, α = γ = 90.0°, β = 128.7°. Indexation and scaling were performed using XDS and XSCALE programs [35
]. The structure of FIV CA protein was determined by molecular replacement using the program MrBUMP [36
] of the CCP4 program suite [37
] and the structures of RELIK (Rabbit Endogenous Lentivirus) CANTD
fragment (PDB ID: 2XGU) [38
] and FIV CACTD
fragment (PDB ID: 5DCK) [33
] as search models. One solution was found with two monomers in the asymmetric unit and an R-factor of 48%. The crystallographic refinement was performed with PHENIX (version 1.12-2829) [39
]. A few residues in the β-hairpin, the cyclophilin binding loop and the C-terminal end were built manually using WinCOOT [40
] and six molecules of glycerol were positioned in the electron density maps. The structure was refined to a final Rwork
of 19.7% and Rfree
of 24.1%, respectively, and statistics of the X-ray data are showed in Table 1
. It showed a good geometry with 98.6% in preferred regions, 1.4% in allowed regions, and no Ramachandran outliers. The omit map around the cis
-peptide of the CypBL loop was generated using the PHENIX software with the annealing method on residues 88–92 from chain B. Figures were generated using PyMol (Schrödinger, New York, NY, USA) [41
To define the specificity of the molecular mechanisms underneath FIV assembly, investigation of the structure of the FIV full-length CA protein was performed and compared with structures of other retrovirus CA proteins. Feline immunodeficiency virus CA protein is mostly composed of α-helices, like the CA protein of other retroviruses, confirming that the overall α-helical fold of CA protein is highly conserved among retroviruses.
During the assembly, retroviral CA proteins assemble into pentamers and hexamers [44
] to form a cone-shaped core, but no pentameric or hexameric assemblies were observed for this FIV CA structure. Nevertheless, six monomeric FIV CAs could be superimposed on a HIV-1 native hexamer (Figure S3
), without requiring strong conformational changes. This superposition does not generate any steric clash between FIV monomers. In this superposition, the C-terminal domain of FIV CTD are not completely superimposed to that of HIV-1 in hexamers. This could come from the fact that the dimeric interface of our structure has set the flexible linker between NTD and CTD of FIV in a position which results in a different orientation of the CTD than the one observed in HIV-1 hexamers. However, as isolated CTDs of FIV and HIV-1 can be superimposed with a RMSD of less than 1 Å on Cα pairs, this structure of monomeric FIV CA is compatible with the formation of hexamers as functional units for capsid assembly.
The crystal structure of FIV CA contains one dimer of CA protein (chains A and B) in the asymmetric unit. This dimer is probably not functional since the N-terminal ends are oriented in opposite directions while they should be oriented in the same way for proper interaction with the FIV matrix protein (MA). Dimerization of HIV-1 CACTD
has been described as involving a tryptophan residue at position 184 [46
]. Notably, no tryptophan is observed in the CTD of FIV CA. Specific identification of dimeric interfaces will be necessary to understand the specific mechanisms of FIV oligomerization [46
This study’s structure finds that the FIV CA dimer is covalently linked by a disulfide bridge between the Cys61 of each monomer. It showed that this disulfide bridge is a crystallization artifact, as it is absent from the protein solution used for crystallogenesis. This is consistent with the observation that Cys61 is not involved in disulfide bridges in functional FIV CA [19
]. This crystallization artifact is likely due to the presence of dimethyl-sulfoxide (DMSO) in the crystallization condition. Indeed, DMSO has been reported to promote oxidation of thiol into disulfide at low pH and room temperature [47
]. The presence of this artifactual disulfide bridge might have helped stabilize the CA dimer in the asymmetric unit during the crystallization process, resulting in the formation of FIV CA crystals which were not obtained in the absence of DMSO (data not shown). However, as a drawback, the formation of this bridge might also have stabilized the FIV CA dimers in non-relevant interfaces and/or impaired the formation of high order oligomers (pentamers, hexamers) which are necessary for the formation of the retroviral capsid. Crystallogenesis experiments in the absence of DMSO are therefore currently pursued to unambiguously identify the functional oligomeric interfaces.
In addition to this interchain artifactual disulfide bond between Cys61 to each monomer, it was observed that Cys190 and Cys210 form an intramolecular disulfide bridge in 75% of the proteins in the crystal for both chains of the asymmetric unit. Although it was absent from the isolated FIV CACTD
]. This cysteine bond agrees with biochemical studies who reported that cysteines Cys190 and Cys210 are involved in an intramolecular disulfide bond which is necessary for FIV capsid assembly and FIV infectivity [19
]. Moreover, this cysteine bond is highly conserved across several retroviral CA proteins, from HIV-1 to EIAV [22
]. Thus, this structural feature is probably relevant for the biology of FIV CA.
As expected from biochemical data [19
], the last free cysteine of FIV CA—Cys121 (from α7′)—is not be involved in any cysteine bond. Interestingly, the sulfur atom of this cysteine participates as a cluster with sulfur atoms of Met51 (from α4) and Met100 (from α6). These three sulfur atoms show an intriguing feature, as they are aligned and distributed at 4 Å one after the other (data not shown). This distribution is unique to FIV CA as other retroviral do not harbor a cysteine residue homologous to FIV Cys121, but its function (if any) remains to be determined.
However, the FIV CA monomeric structure that was obtained harbors important features to understand FIV assembly. An example is that FIV CA contains at its amino-terminal end a β-hairpin motif (Figure 5
). This motif could be expected as it has been shown to be required for the formation of the HIV-1 capsid core particle since it participates directly in intermolecular CA–CA interactions [23
]. This study demonstrates that this β-hairpin seems to adopt a conformation which corresponds to the “open” conformation described for HIV-1 CA protein [26
] which might be important for the import of dNTP in the virus core during reverse-transcription.
The Pro1 of CA has been described as essential for the formation of this N-terminal β-hairpin in HIV-1 CA [50
]. Interestingly, this proline is conserved in most lentiviral CA proteins such as HIV-1, SIV, and EIAV, which probably reflects a key function for viral replication. Thus far, the functional role of this proline has been attributed to the formation of the β-hairpin. However, the Pro1 into Thr1 mutation that this study has introduced in FIV CA for practical reasons (described in the Materials and Methods section) did not impair the formation of the β-hairpin nor the assembly of FIV CA in vitro (Figure S2
). Moreover, the salt bridge between the terminal NH2+
group of Pro1 and the side chain carboxyl group of Asp51, which stabilizes this motif in HIV-1 [50
], has an equivalent in this FIV CA structure. Indeed, we could observe a salt bridge between the terminal NH2+
group of Thr1 and the side chain carboxyl group of Asp50 (Figure 5
), with about the same bond length than that observed for the Pro–Asp salt bridge in HIV-1 CA (2.6 Å versus 2.8 Å, respectively). Studying the functionality of this Pro1Thr CA mutant for FIV or HIV-1 replication is beyond the scope of this article, but could help understanding if the key function of this proline indeed is to induce the formation of the β-hairpin, (which is not suggested by our data) or if its main role is for the Gag precursor to adopt the best conformation at the MA-CA junction for efficient protease cleavage during maturation.
Additionally, amino-acid His12 of HIV-1 CA has been shown to be important for the stabilization of the β-hairpin in an “open” or “closed” position, since it is involved in a salt bridge with Asp51 of helix α3 [26
]. The equivalent of His12 in FIV CA structure is a tyrosine (Tyr11), which is not able to form a salt bridge with Asp50 (Figure 5
). However, in this study’s structure, a salt bridge exists between the hydroxyl group of this Tyr11 and the terminal NH2+
group of Thr1 (3.7 Å, Figure 5
). This salt bridge between the extremities of the two strands of the β-hairpin in FIV CA might contribute to enhance its stability.
The CypA-BL present in lentiviral capsid protein is also observed in the FIV CANTD
. Interestingly, as for RELIK CA [38
] but not HIV-1 CA, the presence of a cis
Arg89–Pro90 peptide bond (Figure 4
) in FIV CA CypA-BL could be detected. Remarkably, among the five prolines present in FIV CypA-BL, only this Pro90 residue is in a cis
-conformation. The CypA is a cis
peptidylprolyl isomerase with a stronger specificity for natural substrates containing cis
]. This could explain why, among the proline residues of FIV CypA-BL, Pro90 is the critical target for CypA binding to FIV CA [52
The last two residues of the CACTD
domain are not defined in the electron density. This confirms the high flexibility of the C-terminal end of FIV CA protein, which had already been truncated by nine residues to avoid problems with crystallogenesis. This C-terminal end might be flexible to allow the correct conformation of the CA–NC cleavage site of the Gag polyprotein, as was hypothesized for the flexibility of the FIV MA C-terminus in the MA–CA cleavage process [53
Altogether, these results show that the various domains which have been involved in key functions of retroviral CA, or which have been observed as important for FIV replication, are present in this FIV CA structure, although with their own specificities.