The pH stability of foot-and-mouth disease virus

Non-enveloped FMDV is a spherical and smooth virion without the feature of “canyon” or “pit” structure on the coat [10]. Capsid and RNA element are the determinants of acid sensitivity of FMDV, especially the capsid. The pseudo T = 3 icosahedral capsid composed by protein VP1-VP4 can protect RNA from degradation and induce host cell to produce protective immune response. The core structure of protein VP1-VP3 is broadly similar, which contains eight β-barrel folds named alphabetically B to I. Moreover, VP1, VP2, and VP3 are interactively connected by many loops inside or outside capsid, showing that their N-terminus are always located in the inner face and C-terminus are situated in the outer surface [10, 19]. In the case of FMDV, VP1 is smaller than the corresponding protein of other picornaviruses. Part of β-barrel folds of VP1 distributes around the five-fold axes, delimiting the periphery of pore at the five-fold axes of symmetry. The C-terminus of VP1 traverses the external of virion to make the capsid flat. The neighboring N-terminus of VP2 surrounds the three-fold axes of symmetry, forming a tight C structure which may be the calcium-binding site and plays a considerably vital function on keeping capsid stable. Protein VP3 arranged around the three-fold axes is crucial for the capsid stability. Its N-terminus joints together to build a β-annulus, generating a channel around the five-fold axes [10]. Protein VP4, a small, highly hydrophobic protein located in the interior of capsid with a myristoylated N-terminal ends, has been supposed to facilitate the formation of the ion channel and membrane permeability of endosome. The N terminus of VP4 is close to the five-fold axes and the C-terminus is adjacent to the three-fold axes [7, 10]. As referred to the RNA molecule of FMDV, it has been investigated that the RNA genome is more than the genetic material of FMDV, but also involved in the destabilization of virus capsids. One research reports that empty capsid is more pH stable than the homologous virus by 0.5 pH [20]. Some studies also indicate that RNA molecule has the ability to influence the assembly of capsid protein and the capsid protein could assist RNA molecule to fold into a relatively loose and flexible structure [21, 22].

The assembly and stability of viral capsid depend on establishment of a variety of interactions between the capsid subunits and nucleic acid, including hydrophobic interactions, hydrogen bonds, salt bridges, van der Waals forces, covalent bonds, electrovalent bonds, disulfide bonds [23‐25]. However, those electrostatic interactions are easily affected when the charged status of amino acid residues and RNA are slightly altered along with the changing pH in the environment. One of the key factors regulating capsid stability-instability balance is the interactions nearby the interpentameric interface which is formed by two neighboring protomers and related to the residues in VP2 and VP3 proteins close to the two-fold axis of icosahedron [7, 23, 26, 27]. It has been proved that altogether 61 residues in a protomer dispersing at interpentameric interface participate in the direct interpentameric interaction, of which only 42 residues have the ability to form noncovalent interactions. Furthermore, truncation of side chains forming buried salt bridges causes genotypic reversion, and truncation of side chains forming buried hydrogen bonds leads to the largely decrease in viral titer [24]. Some other reports demonstrate that a cluster of histidine residues is another significant inducement of the instability of interpentameric interface for the approximation between the pKa of histidine and the pH value at which FMDV disassembles. Seven histidine residues (H21, H65, H87, and H157 in VP2, and H141, H144, and H191 in VP3) are closely located at interpentameric interface according to the 3D structure of O serotype FMDV. They could establish effective electrostatic interactions with charged residues in adjacent pentamers. However, the amount of positive charges only around residue VP3H141 and VP3H144 is much more than that of negative charges. The enhancement of electrostatic repulsions between these two protonated histidine residues and nearby protonated His, Arg, and Lys residues in acid surroundings would greatly weaken the capsid stability [13, 28‐30].

Virus uncoating is a stepwise procedure spatially and temporally controlled within cells, and two committed events should be concerned: (i) The conformation alteration and exhibition of internal proteins to the surface; (ii) Avoidance of premature exposure of virus RNA which could be identified by the sensors of intracellular defense system and evasion of uncovered RNA from harmful substances in the endocytic vesicle [31]. Uncoating program is linked to the breakage of interactions between capsid subunits by diverse host factors. Thus, the viruses employ a great many methods to guide the uncoating, for example, the conformational arrangements induced by the cell receptors, the proteases, chemical elements including low endosome pH and oxidoreductases, and mechanical forces offered by molecular motor [31].

FMDV particles display high sensitivity to acidic environment and easily disassemble into pentameric subunits at a pH close to neutrality [12, 14‐16, 20, 32]. In vivo, capsid disassembly of FMDV is related to the viral uncoating, while the FMDV uncoating is triggered by the acidification in the early endosome of host cell. With the pH in the early endosome continually dropping due to the pumping of H+ in the endocytic vesicle, the pH sensors in virus particles are activated and the viral uncoating is triggered [33‐35]. The pH sensors detecting subtle variation of pH value are lots of special amino acid residues which change their charged states closely correlative with the pH in the endosome compartment. The pKa of Histidine is 6.4 and the imidazole ring of it could be positively charged in the pH scope of early endosome, all laying the foundation for being an important sensor [13, 29, 36‐38]. Owing to the gradual protonation of a wide variety of pH sensors in the capsid, the electrostatic repulsions between different protein subunits in the capsid surface strengthen and the acid instability of FMDV capsid increase. As a consequence, a few internal proteins and active sites in FMDV are exposed, which facilitate the release of RNA genome into the cytosol [10, 39, 40]. Even so, in contrast to the uncoating research of other picornaviruses, the uncoating mechanism of FMDV remains poorly understood.

The unique capsid construction of FMDV is a result of progressive evolution for the best survival in nature. In vivo, the FMDV capsid is flexible and metastable for the conformational transformation and function implement [23]. But, in vitro, the intact protein shell has a limited ability to deal with the damage of toxic substances [41]. In practice, plenty of factors in the production, storage, and application will influence the pH in the medium and vaccine products, resulting in the degradation of integrated virus particles, damage of antigenic structure, and reduction of immunogenicity. From the above, the capsid of FMDV should hold a delicate balance between acid instability to achieve rapid proliferation in infected cells and the acid stability to resist the aggression in the external environment [42]. This equilibrium is also extraordinarily important for the production of VLPs. One work reporting the introduction of some acid-stable amino acid residues into the empty capsid-like particles suggests that acid-resistant transformation could improve the stability of assembled capsid expressed in inset cells, but the expression yield is seriously suppressed [43].

Interestingly, the compensation effects restoring viability are occasionally detected in the researche of acid sensitivity of FMDV [12, 14, 16, 18, 44]. The FMDV C-S8c1 with substitution VP3 A118V exhibits similar acid stability to the variant containing double mutations VP3 A118V/VP1 N47D, whereas the virus with single mutation displays smaller plaque phenotype and lower titer than the one with double mutations, demonstrating that the substitution VP1 N47D could compensate the negative impact of the substitution VP3 A118V on the virus growth [12]. Another substitution of VP3 D9V located in the outside surface close to the holes around five-fold axes is observed to accompany the mutation VP1 N17D in the same virus, and is speculated to recover the impaired biological characteristics caused by substitution VP1 N17D [14]. While the compensatory substitutions arising frequently at interpentameric and/or nearby the interpentameric interface during virus evolution enable the toleration to the deleterious effects on the virus replication, infectivity, and virulence exerted by the mutations which alter capsid stability [24, 45], it may be served as a strategy to alleviate the conflict between the increase of pH stability and the decrease of fitness.

Asingle amino acid residue substitution in the capsid of FMDV is found to be sufficient to resist the acid dissociation, but its ability is greatly different according to the FMDV serotype, strain, and position in the capsid. Since the more acid-stable FMDV in nature is not easy to be separated, the isolation of virion with acid stability principally depends on the artificial selection. To date, the popular method used to study acid stability of FMDV is that the mutants with increased acid resistance are isolated by several cycles of acid treatment and serially passages. Then, the complete capsid coding regions of the mutants and the parental virus are sequenced and compared to identify the genotypic changes responsible for the acid sensitivity of FMDV. Finally, the recombinant FMDV containing the amino acid replacement found in the acid-resistant FMDV is rescued by using reverse genetics technology. The judgement on whether the selected amino acid residues are responsible for the acid resistance of FMDV is based on the analysis of remaining infectivity of recombinant virus in PBS solutions of different pHs [14, 15, 18, 44]. However, the increased acid stability tends to restrain the endosome acidification of virus, leading to the reduced fitness of FMDV.

There are some researche on the relationship between the increase in acid resistance and the sensitivity to drugs that change the pH in the endosome. According to those researche, the method of endosome acidification blockage is used to select the FMDV mutants with acid instability [12, 16]. Some drugs, such as weak base NH₄Cl, bafilomycin, concanamycin, and monesin, could weaken endosome acidification by preventing vacuolar ATPase or neutralizing endosome pH using protonophore, causing the impairment of membrane fusion or membrane permeability [46‐48]. NH₄Cl availably raising the pH in the endosome is frequently employed to select FMDV with enhanced acid instability. This drug distinctly influences the early steps of FMDV infection without interfering in the binding of FMDV with cell receptors or internalization pathway. The selection process is as follows: FMDV is continuously inoculated in the BHK-21 cells treated by the NH₄Cl in advance, then the mutant FMDVs with improved acid sensitivity are obtained and the amino acid residues responsible for the acid lability are discovered [12, 16]. This selection approach is an opposite strategy compared to the first method above. It is reported that the selection frequency of FMDV mutants with acid instability by NH₄Cl treatment is 10^-1, while the selection frequency of acid-resistant FMDV mutants is 2.9 × 10^-5, suggesting that endosome acidification blockage using NH₄Cl could largely select amino acid residues increasing the acid lability of FMDV. The higher selection frequency with NH₄Cl will be helpful to locate the regions where plenty of acid-labile residues distribute and provide molecular mechanism for uncoating [12, 49].

Nearly 20 years ago, the pH stability research of FMDV capsid was conducted from the perspective of chemical calculations [29]. The finite difference Poisson-Boltzmann method which is utilized to compute the macromolecular electrostatic free energy is the main principle to calculate the protein pKa and predict the influence of pH on the capsid stability [50‐54]. At first, the titration curves of separate protomer and dimer at different pH values are determined. Secondly, the free energy difference of corresponding values in the two curves is calculated. Eventually, the average charge difference is obtained to confirm the relative pH stability. In their research, the residues within 15 Å of the interface are showed to determine the pH sensitivity of capsid subunits, particularly the two residues VP3 H142 and VP3 H145. However, this method is simply involved in the influence of the titratable residues on the capsid stability, such as Asp, Glu, and His, and fails to analyze the effect of other residues which could be not titrated [29].