Foot-and-mouth disease virus (FMDV) is the prototypic member of the aphthovirus genus within the family Picornaviridae
) and the etiological agent of a devastating disease of livestock (34
). The viral particle is composed of a protein capsid that contains a positive-sense RNA molecule of about 8,500 nucleotides that is infectious and encodes a single polyprotein, which is processed in infected cells by cis
- and trans
-acting viral proteases (55
) to yield different polypeptide precursors and the mature viral proteins (9
). The viral genome encodes four structural capsid proteins (VP1 to VP4) and seven nonstructural (NS) proteins, the leader Lb/ab protease, and proteins encoded in the P2 (2B and 2C) and P3 (3A, 3B, 3C, and 3D) regions (9
Replication of picornaviruses occurs associated to cell endomembranes that are recruited during viral infection (25
). NS proteins are involved in crucial aspects of the viral cycle and pathogenesis, such as rearrangements of intracellular membranes required for endomembrane recruitment and the lysis of host cells (1
). Protein 3A is an example of this multifunctional role; in poliovirus (PV), the interaction between the RNA replication complex and intracellular membranes appears to be accomplished by proteins 3A and 2C, which have membrane-binding properties (11
). When expressed as a recombinant protein in transfected cells, PV 3A cofractionates with endoplasmic reticulum markers (66
), and its single transient expression can disrupt the secretory apparatus (23
) and decrease major histocompatibility complex (MHC) class I expression (22
). On the other hand, 3AB presumably anchors 3B in intracellular membranes originated de novo
during the early steps of RNA replication, where uridylylated 3B primes the synthesis of nascent viral RNAs (2
). PV 3AB has a nonspecific RNA-binding activity and associates with the cloverleaf structure in the 5′ end of viral RNA and with the 3CD precursor to form a ribonucleoprotein complex required for PV RNA synthesis (32
While FMDV shows considerable functional and structural analogies with PV and other picornaviruses, some differences have been reported, such as its resistance to the Golgi disruption exerted by brefeldin A, the different pattern of membrane reorganization induced in infected cells (39
), and the ability of 2B and 2BC, instead of 3A, to inhibit the secretory pathway in cultured cells (42
), likely contributing to the evasion of innate and acquired immune responses (58
). In addition, FMDV is the only picornavirus encoding 3 copies of 3B protein, required for both optimal replication in cell culture (26
) and for virulence in natural hosts (50
). All 3 copies have been shown to undergo uridylylation in vitro
). In FMDV, 3A protein has been reported to play a role on viral host range, as a single amino acid replacement (Q44R) conferred FMDV the ability to cause vesicular lesions in guinea pigs (48
). As 2B and 2C, FMDV nonstructural protein 3A contains a hydrophobic domain that in other picornaviruses mediates its targeting to intracellular membranes (19
), which could be responsible for the location of the replication complex within a membrane context (20
). The C-terminal fragment of 3A (from the C terminus of the hydrophobic domain) is considerably longer in FMDV (77 amino acids [aa]) than in other picornaviruses (i.e., 7 aa for PV), and deletions and mutations in 3A are known to contribute both to viral attenuation in cattle (8
) and to decreased replication rates in bovine epithelial cells (49
). However, little is known on the interactions of 3A with other viral and cellular proteins, and no structural data are available for this protein.
Homodimer formation has been revealed by the NMR structure determined for a soluble version lacking the trans-membrane domain (aa 1 to 59) of PV 3A (65
). Each monomer has a structured region consisting of two amphipathic α-helices (aa 23 to 29 and 32 to 41) separated by a 180° loop that forms a helical hairpin, flanked by nonstructured N and C regions. The N terminus of 3A from PV also contains a conserved patch of anionic residues at the top of the dimer structure, in the loop between the two α-helices, as well as three solvent-exposed charged residues (E38, K39, and K40) that may be important for viral replication (65
). A role for 3A homodimer formation in both RNA replication and inhibition of cellular protein transport has also been reported for coxsackievirus (CV) B3. In this case, while the general organization of the CVB3 dimer was similar to that of PV, the establishment of salt bridges between residues D24 and K41 was found critical for dimer stability; using an optimized PV 3A structure, these salt bridges were also found in equivalent PV residues (D23 and K40) (72
To gain insight into the structure-function relationship of FMDV 3A protein, we devised a molecular model for the N-terminal region of this protein, using as the template the structure reported for PV 3A. This model predicted hydrophobic interactions between residues at two α-helices in each monomer as the main homodimerization determinant. Here, we show that amino acid replacements L38E and L41E, located at the predicted hydrophobic dimerization interface, and expected to contribute to dimer stability, decrease 3A dimerization in cells transiently expressing 3A, and abolish dimer/multimer formation in peptides reproducing the N terminus of 3A. Replacements L38E and L41E significantly reduced the homodimerization signal detected for transiently expressed 3A by means of an in situ
proximity ligation assay (63
). In addition, replacements L38E and L41E were detrimental for virus growth, leading to selection of viruses that for mutants L38E and L41E restored the hydrophobicity of the residues, suggesting that 3A dimer formation plays a relevant role in FMDV replication. On the other hand, replacement Q44R that favors or replacement Q44E that impairs the polar interactions that, according to the model, Q44 could establish with residue D32 of the opposite monomer did not abolish dimer formation of transiently expressed 3A, indicating that these polar interactions are not critical for 3A dimerization. Nevertheless, while Q44R led to infectious virus recovery, Q44D resulted in the selection of infective viruses with substitution D44E with acidic charge but with structural features similar to those of the parental virus, suggesting that residue Q44, despite not being essential for 3A dimerization, is involved in biological functions relevant for virus multiplication.
The self-association of proteins to form dimers and higher-order oligomers is a common biological phenomenon. Recent structural and biophysical studies show that protein dimerization or oligomerization is a key factor in the regulation of different protein functions (38
), including proteins relevant for virus replication (36
). In this line, dimerization/multimerization has been shown to be relevant for the biological role of nonstructural proteins of different picornaviruses (15
), including FMDV (67
). In this study, we describe experiments aimed to gain insight on the structure-function relationship of FMDV 3A protein, a nonstructural protein relevant for virus replication, virulence, and host range for which the molecular mechanisms that mediate its biological activity are poorly understood. Previous attempts to obtain structural data from E. coli
-expressed 3A and peptides corresponding to the N terminus of 3A showed an aggregation tendency that impaired subsequent analyses. In this work, using the NMR structure of PV 3A as a template, a molecular model for the N-terminal 94 residues of FMDV 3A was derived. The model shows that 3A contains two α helices (α1, residues 25 to 33, and α2, residues 37 to 44) and that, as in PV and CVB3 3A structures, a number of hydrophobic contacts in helices α1 and α2 could provide physical stability to the dimer. In addition, in FMDV 3A, the two alpha-helices are connected by a 3-residue loop (I34, K35, and E36) that conforms a patch of charged residues. In the equivalent loop positions, a patch of negatively charged residues is present in PV (D29 and E32) (65
) and in CVB3 (D30 and E32) (72
) 3A proteins.
According to the model, the analysis of the transiently expressed FMDV 3A protein suggested that 3A forms dimers/multimers as shown by Western blotting analyses (Fig. 2A
). Protein oligomerization even in the presence of SDS, has been previously described for PV 3AB (35
). This homodimerization of 3A was also evidenced by an in situ
protein ligation assay designed to visualize protein-protein interactions in the cell by fluorescence microscopy (Fig. 3
). The contribution of the N-terminal fragment of 3A to the dimerization was detected using the N-wt synthetic peptide, spanning residues 1 to 52 of 3A, whose mobility in SDS-PAGE revealed by mass staining showed a major band of a size about that corresponding to the monomeric peptide, as well as bands corresponding to higher-order oligomers (Fig. 2B
Based on the model and on sequence conservation among FMDV isolates, hydrophobic interactions between residues at the helical regions of both monomers were expected to be the main dimerization determinant. Indeed, replacements L38E, L41E, and M29R, involving charge acquisition at residues predicted to contribute to the hydrophobic interface, abolished formation of dimer bands in transiently expressed 3A (Fig. 2A
). Moreover, the single replacements L38E and L41E and, to a higher extent, the double replacement L38EL41E showed a reduced fluorescence signal in the proximity ligation assay (Fig. 2B
), while expression of 3A was evidenced by conventional immunofluorescence with a different anti-3A antibody (Ab 346). A similar decrease in the dimerization signal in a double hybrid system has been reported for CVB3 3A carrying a double mutation (by alanine replacement) at residues L25 and L26 (72
). On the other hand, peptides from the N terminus of 3A with substitutions L38E, L41E, M29R, or L38EL41E showed a dramatic reduction of the electrophoretic bands of a size higher than that corresponding to their monomeric forms. Overall, these results suggest that, as reported for CVB3, preservation of a cluster of hydrophobic interactions in this region is essential for 3A dimer stability.
When FMDV RNA with replacement L38E was transfected in BHK-21 cells, a delay, relative to cells transfected with parental C-S8c1 RNA, in both the emergence of cytopathic effect and the recovery of infectious virus was observed in three independent experiments. Viruses recovered from the second passage in cultured cells of the transfection medium displayed replacement E38V that resembled the nonpolar (L) residue of the parental C-S8c1 virus. On the other hand, infectious virus could be recovered in two of the four independent transfection experiments performed with RNA carrying replacement L41E, and the sequencing of the viral populations obtained displayed the replacement E41A, which again restored the nonpolar nature of this position. The lower viral titers recovered from transfections with L41E RNA, relative to those produced by L38E RNA, as well as the lack of recovery of infectious virus from two of the transfections with RNA L41E, suggest that this replacement could affect FMDV replication more severely than L38E. While direct reversion to the parental residue L at E38 and E44 residues requires at least 2 nucleotide substitutions, single transversions mediate replacements E38V (A5411T) and E41A (A5420C) (Table 3
), making their selection more likely. These results indicate that the presence of nonpolar hydrophobic residues at positions 38 and 41 of 3A is essential for virus replication and that no second-site suppressor mutations at other 3A residues, unlike as reported for coxsackievirus RNA with replacements at analogous 3A hydrophobic residues (72
), are frequently selected during the limited replication of RNA mutants L38E and L41E. Thus, taken together, our results suggest that the conservation of hydrophobic interactions at the predicted dimerization interface is required for efficient FMDV replication in cultured cells.
In CVB3, polar interactions, other than the hydrophobic contacts such as those contributed by residues L25 and L26, have been shown to participate in 3A dimer stability; thus, the establishment of salt bridges between residues D24 and K41 was found critical for dimer stability, RNA replication, and inhibition of protein transport (72
). K41 is part of a cluster of charged residues located at the C terminus of CVB 3A α-helix 2. A similar cluster of charged, solvent exposed residues is found in PV 3A α-helix 2, and mutations at these residues (E38, K39, and K40) yield nonviable viruses, indicating the biological relevance of these polar, charged residues located at the α-helix 2 (75
). In contrast, the equivalent positions at the C terminus of FMDV 3A α-helix 2 are occupied by a cluster of polar but not charged residues: Q43, Q44, and T45. According to our model, residues D32 and Q44 of FMDV 3A, located flanking the C-terminal extremes of helices α1 and α2 and exposed to the solvent, could establish polar interactions between both monomers. Replacement Q44R, which mediates adaptation to the guinea pig (48
), could allow formation of an intermolecular salt bridge with D32 with the consequent dimer stabilization. Conversely, replacement Q44D would impair polar interactions with D32. Despite these predictions, neither of these substitutions produced a marked effect on the dimer formation of transiently expressed 3A, suggesting that the polar interactions between Q44 and D32 are not critical for 3A dimerization, albeit they could modulate dimer stability. A similar observation was reported for CVB3 3A in which replacements to A at polar residues S28 and Y37, predicted to establish an intermolecular hydrogen bond, did not affect dimerization and protein transport, while only replacement Y37A impaired virus replication, leading to recovery of a second-site suppressor mutation (72
When the effect of electrostatic charge acquisition at position 44 on the infectiveness of FMDV RNA was analyzed, transfected RNA with replacement Q44R produced a cytopathic effect and infectious virus in a manner similar to that of the parental RNA pMT28, and the viruses recovered maintained the substitution over 3 additional cell passages, indicating that this change is not detrimental for virus replication in cultured cells. This result is consistent with the ability of the guinea pig-adapted virus carrying replacement Q44R to kill suckling mice and to cause acute disease in the pig (47
). On the contrary, in cells transfected with RNA carrying the replacement Q44D, a delay in the production of infectious virus was observed and the recovered virus displayed replacement D44E. In this case, substitution of D by E did not restore a polar, uncharged side chain, maintaining a milder negative charge and structural features similar to those of the parental Q. Direct reversion to the parental residue Q requires 2 nucleotide substitutions, while a single transversion mediates replacement D44E (T5430A) (Table 3
). Residue Q44 is rather conserved among FMDVs, and replacements at these residue have been found in guinea pig-adapted isolates (16
). Our results suggest that, despite the fact that replacements at Q44 do not substantially affect the capacity of transiently expressed 3A to form dimers, this residue is relevant for virus replication. Thus, while replacement Q44R retains infectivity in cultured cells at levels similar to that of the parental C-S8c1, Q44D leads to the recovery of the D44E mutant.
Despite the fact that replication impairment introduced by mutations L38E, L41E, and Q44D permitted selection of revertant mutants able to grow in cultured cells, RNAs carrying these substitutions caused no death or clinical signs when inoculated in suckling mice, opposite to what was observed for RNA from C-S8c1 (pMT28) and from mutant Q44R. These results indicate that, as previously reported (7
), in vivo
multiplication frequently imposes different constrains for virus replication and disease emergence compared to those found in cultured cells.
Although further work is required to assess the contribution of dimerization inhibition on the detrimental effect of the mutants studied, our results suggest that, despite the unique characteristics of FMDV 3A protein among picornaviruses, FMDV requires 3A dimerization for efficient replication. Our data support that the hydrophobic interactions established between the α-helices of both monomers are the main determinant for dimerization and its impairment is detrimental for virus multiplication. On the other hand, mutations affecting polar interactions between residues at the α-helices can affect FMDV replication, without abolishing 3A dimerization.