Dual role of the foot-and-mouth disease virus 3B1 protein in the replication complex: As protein primer and as an essential component to recruit 3Dpol to membranes.
Picornavirus genome replication takes place in specialized intracellular membrane compartments that concentrate viral RNA and proteins as well as a number of host factors that also participate in the process. The core enzyme in the replication machinery is the viral RNA-dependent RNA polymerase (RdR...
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| Format: | Article |
| Language: | English |
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Public Library of Science (PLoS)
2023-05-01
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| Series: | PLoS Pathogens |
| Online Access: | https://doi.org/10.1371/journal.ppat.1011373 |
| _version_ | 1797822026633707520 |
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| author | Cristina Ferrer-Orta Diego S Ferrero Nuria Verdaguer |
| author_facet | Cristina Ferrer-Orta Diego S Ferrero Nuria Verdaguer |
| author_sort | Cristina Ferrer-Orta |
| collection | DOAJ |
| description | Picornavirus genome replication takes place in specialized intracellular membrane compartments that concentrate viral RNA and proteins as well as a number of host factors that also participate in the process. The core enzyme in the replication machinery is the viral RNA-dependent RNA polymerase (RdRP) 3Dpol. Replication requires the primer protein 3B (or VPg) attached to two uridine molecules. 3B uridylylation is also catalysed by 3Dpol. Another critical interaction in picornavirus replication is that between 3Dpol and the precursor 3AB, a membrane-binding protein responsible for the localization of 3Dpol to the membranous compartments at which replication occurs. Unlike other picornaviruses, the animal pathogen foot-and-mouth disease virus (FMDV), encodes three non-identical copies of the 3B (3B1, 3B2, and 3B3) that could be specialized in different functions within the replication complex. Here, we have used a combination of biophysics, molecular and structural biology approaches to characterize the functional binding of FMDV 3B1 to the base of the palm of 3Dpol. The 1.7 Å resolution crystal structure of the FMDV 3Dpol -3B1 complex shows that 3B1 simultaneously links two 3Dpol molecules by binding at the bottom of their palm subdomains in an almost symmetric way. The two 3B1 contact surfaces involve a combination of hydrophobic and basic residues at the N- (G5-P6, R9; Region I) and C-terminus (R16, L19-P20; Region II) of this small protein. Enzyme-Linked Immunosorbent Assays (ELISA) show that the two 3B1 binding sites play a role in 3Dpol binding, with region II presenting the highest affinity. ELISA assays show that 3Dpol has higher binding affinity for 3B1 than for 3B2 or 3B3. Membrane-based pull-down assays show that 3B1 region II, and to a lesser extent also region I play essential roles in mediating the interaction of 3AB with the polymerase and its recruitment to intracellular membranes. |
| first_indexed | 2024-03-13T10:01:45Z |
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| id | doaj.art-f6c6fcc03dd34f1daf522c0fc8d26764 |
| institution | Directory Open Access Journal |
| issn | 1553-7366 1553-7374 |
| language | English |
| last_indexed | 2024-03-13T10:01:45Z |
| publishDate | 2023-05-01 |
| publisher | Public Library of Science (PLoS) |
| record_format | Article |
| series | PLoS Pathogens |
| spelling | doaj.art-f6c6fcc03dd34f1daf522c0fc8d267642023-05-23T05:31:34ZengPublic Library of Science (PLoS)PLoS Pathogens1553-73661553-73742023-05-01195e101137310.1371/journal.ppat.1011373Dual role of the foot-and-mouth disease virus 3B1 protein in the replication complex: As protein primer and as an essential component to recruit 3Dpol to membranes.Cristina Ferrer-OrtaDiego S FerreroNuria VerdaguerPicornavirus genome replication takes place in specialized intracellular membrane compartments that concentrate viral RNA and proteins as well as a number of host factors that also participate in the process. The core enzyme in the replication machinery is the viral RNA-dependent RNA polymerase (RdRP) 3Dpol. Replication requires the primer protein 3B (or VPg) attached to two uridine molecules. 3B uridylylation is also catalysed by 3Dpol. Another critical interaction in picornavirus replication is that between 3Dpol and the precursor 3AB, a membrane-binding protein responsible for the localization of 3Dpol to the membranous compartments at which replication occurs. Unlike other picornaviruses, the animal pathogen foot-and-mouth disease virus (FMDV), encodes three non-identical copies of the 3B (3B1, 3B2, and 3B3) that could be specialized in different functions within the replication complex. Here, we have used a combination of biophysics, molecular and structural biology approaches to characterize the functional binding of FMDV 3B1 to the base of the palm of 3Dpol. The 1.7 Å resolution crystal structure of the FMDV 3Dpol -3B1 complex shows that 3B1 simultaneously links two 3Dpol molecules by binding at the bottom of their palm subdomains in an almost symmetric way. The two 3B1 contact surfaces involve a combination of hydrophobic and basic residues at the N- (G5-P6, R9; Region I) and C-terminus (R16, L19-P20; Region II) of this small protein. Enzyme-Linked Immunosorbent Assays (ELISA) show that the two 3B1 binding sites play a role in 3Dpol binding, with region II presenting the highest affinity. ELISA assays show that 3Dpol has higher binding affinity for 3B1 than for 3B2 or 3B3. Membrane-based pull-down assays show that 3B1 region II, and to a lesser extent also region I play essential roles in mediating the interaction of 3AB with the polymerase and its recruitment to intracellular membranes.https://doi.org/10.1371/journal.ppat.1011373 |
| spellingShingle | Cristina Ferrer-Orta Diego S Ferrero Nuria Verdaguer Dual role of the foot-and-mouth disease virus 3B1 protein in the replication complex: As protein primer and as an essential component to recruit 3Dpol to membranes. PLoS Pathogens |
| title | Dual role of the foot-and-mouth disease virus 3B1 protein in the replication complex: As protein primer and as an essential component to recruit 3Dpol to membranes. |
| title_full | Dual role of the foot-and-mouth disease virus 3B1 protein in the replication complex: As protein primer and as an essential component to recruit 3Dpol to membranes. |
| title_fullStr | Dual role of the foot-and-mouth disease virus 3B1 protein in the replication complex: As protein primer and as an essential component to recruit 3Dpol to membranes. |
| title_full_unstemmed | Dual role of the foot-and-mouth disease virus 3B1 protein in the replication complex: As protein primer and as an essential component to recruit 3Dpol to membranes. |
| title_short | Dual role of the foot-and-mouth disease virus 3B1 protein in the replication complex: As protein primer and as an essential component to recruit 3Dpol to membranes. |
| title_sort | dual role of the foot and mouth disease virus 3b1 protein in the replication complex as protein primer and as an essential component to recruit 3dpol to membranes |
| url | https://doi.org/10.1371/journal.ppat.1011373 |
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