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Foot-and-mouth disease virus nonstructural protein 2C interacts with Beclin1, modulating virus replication.

ABSTRACT: Foot-and-mouth disease virus (FMDV), the causative agent of foot-and-mouth disease, is an Apthovirus within the Picornaviridae family. Replication of the virus occurs in association with replication complexes that are formed by host cell membrane rearrangements. The largest viral protein in the replication complex, 2C, is thought to have multiple roles during virus replication. However, studies examining the function of FMDV 2C have been rather limited. To better understand the role of 2C in the process of virus replication, we used a yeast two-hybrid approach to identify host proteins that interact with 2C. We report here that cellular Beclin1 is a specific host binding partner for 2C. Beclin1 is a regulator of the autophagy pathway, a metabolic pathway required for efficient FMDV replication. The 2C-Beclin1 interaction was further confirmed by coimmunoprecipitation and confocal microscopy to actually occur in FMDV-infected cells. Overexpression of either Beclin1 or Bcl-2, another important autophagy factor, strongly affects virus yield in cell culture. The fusion of lysosomes to autophagosomes containing viral proteins is not seen during FMDV infection, a process that is stimulated by Beclin1; however, in FMDV-infected cells overexpressing Beclin1 this fusion occurs, suggesting that 2C would bind to Beclin1 to prevent the fusion of lysosomes to autophagosomes, allowing for virus survival. Using reverse genetics, we demonstrate here that modifications to the amino acids in 2C that are critical for interaction with Beclin1 are also critical for virus growth. These results suggest that interaction between FMDV 2C and host protein Beclin1 could be essential for virus replication.

SUBMITTER: Gladue DP

PROVIDER: S-EPMC3486479 | biostudies-literature | 2012 Nov

REPOSITORIES: biostudies-literature

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Foot-and-mouth disease virus nonstructural protein 2C interacts with Beclin1, modulating virus replication.

Gladue D P DP O'Donnell V V Baker-Branstetter R R Holinka L G LG Pacheco J M JM Fernandez-Sainz I I Lu Z Z Brocchi E E Baxt B B Piccone M E ME Rodriguez L L Borca M V MV

Journal of virology 20120829 22

Foot-and-mouth disease virus (FMDV), the causative agent of foot-and-mouth disease, is an Apthovirus within the Picornaviridae family. Replication of the virus occurs in association with replication complexes that are formed by host cell membrane rearrangements. The largest viral protein in the replication complex, 2C, is thought to have multiple roles during virus replication. However, studies examining the function of FMDV 2C have been rather limited. To better understand the role of 2C in the ...[more]

PMID: 22933281

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Project description:Nonstructural protein 3A of foot-and-mouth disease virus (FMDV) is a partially conserved protein of 153 amino acids that is in most FMDVs examined to date, and it plays important roles in virus replication, virulence, and host range. To better understand the role of 3A during FMDV infection, we used coimmunoprecipitation followed by mass spectrometry to identify host proteins that interact with 3A in FMDV-infected cells. Here, we report that cellular vimentin is a host binding partner for 3A. The 3A-vimentin interaction was further confirmed by coimmunoprecipitation, glutathione S-transferase (GST) pull down, and immunofluorescence assays. Alanine-scanning mutagenesis indicated that amino acid residues 15 to 21 at the N-terminal region of the FMDV 3A are responsible for the interaction between 3A and vimentin. Using reverse genetics, we demonstrate that mutations in 3A that disrupt the interaction between 3A and vimentin are also critical for virus growth. Overexpression of vimentin significantly suppressed the replication of FMDV, whereas knockdown of vimentin significantly enhanced FMDV replication. However, chemical disruption of the vimentin network by acrylamide resulted in a significant decrease in viral yield, suggesting that an intact vimentin network is needed for FMDV replication. These results indicate that vimentin interacts with FMDV 3A and negatively regulates FMDV replication and that the vimentin-3A interaction is essential for FMDV replication. This study provides information that should be helpful for understanding the molecular mechanism of FMDV replication.IMPORTANCE Foot-and-mouth disease virus (FMDV) nonstructural protein 3A plays important roles in virus replication, host range, and virulence. To further understand the role of 3A during FMDV infection, identification of host cell factors that interact with FMDV 3A is needed. Here, we found that vimentin is a direct binding partner of FMDV 3A, and manipulation of vimentin has a negative effect on virus replication. We also demonstrated that amino acid residues 15 to 21 at the N-terminal region of the FMDV 3A are responsible for the interaction between 3A and vimentin and that the 3A-vimentin interaction is critical for viral replication since the full-length cDNA clone harboring mutations in 3A, which were disrupt 3A-vimentin reactivity, could not produce viable virus progeny. This study provides information that not only provides us a better understanding of the mechanism of FMDV replication but also helps in the development of novel antiviral strategies in the future.

Project description:The maintenance of viral protein homeostasis depends on the machinery of the infected host cells, giving us an insight into the interplay between host and virus. Accumulating evidence suggests that heat shock protein 60 (HSP60), as one molecular chaperone, is involved in regulating virus infection. However, the role of HSP60 during foot-and-mouth disease virus (FMDV) replication and its specific mechanisms have not been reported. We demonstrate that HSP60 modulates the FMDV life cycle. HSP60 plays a role at the postentry stage of the viral life cycle, including RNA replication and mRNA translation; however, HSP60 does not affect viral replication of Seneca Valley virus (SVA) or encephalomyocarditis virus (EMCV). We found that HSP60 is involved in FMDV replication complex (RC) formation. Furthermore, our results indicate that HSP60 interacts with FMDV nonstructural proteins 3A and 2C, key elements of the viral replication complex. We also show that HSP60 regulates the stability of 3A and 2C via caspase-dependent and autophagy-lysosome-dependent degradation, thereby promoting FMDV RNA synthesis and mRNA translation mediated by the RC. Additionally, we determined that the apical domain of HSP60 is responsible for interacting with 3A and 2C. The N terminus of 3A and ATPase domain of 2C are involved in binding to HSP60. Importantly, HSP60 depletion potently reduced FMDV pathogenicity in infected mice. Altogether, this study demonstrates a specific role of HSP60 in promoting FMDV replication. Furthermore, targeting host HSP60 will help us design the FMDV-specific antiviral drugs. IMPORTANCE FMDV is the leading cause of the foot-and-mouth disease (FMD), affecting cloven-hoofed animals with high morbidity and mortality. We determined that HSP60 is required for efficient viral RNA replication and mRNA translation during FMDV infection. Furthermore, we demonstrate that HSP60 interacts with FMDV nonstructural proteins 3A and 2C, the elements of the RC; HSP60 contributes to the stability of 3A and 2C to affect the formation and function of the RC. We also validated the potential role of HSP60 as the antiviral target in vivo using small interfering RNA. These findings deepen the understanding of the host-virus interaction and provide information supporting the design of novel therapeutics for FMDV infection.

Project description:Foot-and-mouth disease virus (FMDV) is the causative agent of foot-and-mouth disease, a highly contagious, economically important viral disease. The structural protein VP1 plays significant roles during FMDV infection. Here, we identified that VP1 interacted with host ribosomal protein SA (RPSA). RPSA is a viral receptor for dengue virus and classical swine fever virus infections. However, the incubation of susceptible cells using the anti-RPSA antibodies did not block the infection of FMDV. Overexpression of porcine RPSA in the insusceptible cells could not trigger FMDV infection, suggesting that RPSA was not responsible for FMDV entry and infection. On the contrary, we found that overexpression of RPSA suppressed FMDV replication, and knockdown of RPSA enhanced FMDV replication. We further determined that FMDV infection activated the mitogen-activated protein kinase (MAPK) pathway and demonstrated that MAPK pathway activation was critically important for FMDV replication. RPSA negatively regulated MAPK pathway activation during FMDV infection and displayed an antiviral function. FMDV VP1 interacted with RPSA to abrogate the RPSA-mediated suppressive role in MAPK pathway activation. Together, our study indicated that MAPK pathway activation was required for FMDV replication and that host RPSA played a negatively regulatory role on MAPK pathway activation to suppress FMDV replication. FMDV VP1 bound to RPSA to promote viral replication by repressing RPSA-mediated function and maintaining the activation of MAPK signal pathway.IMPORTANCE Identification of virus-cell interactions is essential for making strategies to limit virus replication and refine the models of virus replication. This study demonstrated that FMDV utilized the MAPK pathway for viral replication. The host RPSA protein inhibited FMDV replication by suppressing the activation of the MAPK pathway during FMDV infection. FMDV VP1 bound to RPSA to repress the RPSA-mediated regulatory effect on MAPK pathway activation. This study revealed an important implication of the MAPK pathway for FMDV infection and identified a novel mechanism by which FMDV VP1 has evolved to interact with RPSA and maintain the activation of the MAPK pathway, elucidating new information regarding the signal reprogramming of host cells by FMDV.

Project description:Vacuolar protein sorting 28 (Vps28), a component of the ESCRT-I (endosomal sorting complex required for transport I), plays an important role in the pathogen life cycle. Here, we investigated the reciprocal regulation between Vps28 and the foot-and-mouth disease virus (FMDV). Overexpression of Vps28 decreased FMDV replication. On the contrary, the knockdown of Vps28 increased viral replication. Subsequently, the mechanistic study showed that Vps28 destabilized the replication complex (RC) by associating with 3A rather than 2C protein. In addition, Vps28 targeted FMDV VP0, VP1, and VP3 for degradation to inhibit viral replication. To counteract this, FMDV utilized tactics to restrict Vps28 to promote viral replication. FMDV degraded Vps28 mainly through the ubiquitin-proteasome pathway. Additional data demonstrated that 2B and 3A proteins recruited E3 ubiquitin ligase tripartite motif-containing protein 21 to degrade Vps28 at Lys58 and Lys25, respectively, and FMDV 3Cpro degraded Vps28 through autophagy and its protease activity. Meantime, the 3Cpro-mediated Vps28 degradation principally alleviated the ability to inhibit viral propagation. Intriguingly, we also demonstrated that the N-terminal and C-terminal domains of Vps28 were responsible for the suppression of FMDV replication, which suggested the elaborated counteraction between FMDV and Vps28. Collectively, our results first investigate the role of ESCRTs in host defense against picornavirus and unveil underlying strategies utilized by FMDV to evade degradation machinery for triumphant propagation. IMPORTANCE ESCRT machinery plays positive roles in virus entry, replication, and budding. However, little has been reported on its negative regulation effects during viral infection. Here, we uncovered the novel roles of ESCRT-I subunit Vps28 on FMDV replication. The data indicated that Vps28 destabilized the RC and impaired viral structural proteins VP0, VP1, and VP3 to inhibit viral replication. To counteract this, FMDV hijacked intracellular protein degradation pathways to downregulate Vps28 expression and thus promoted viral replication. Our findings provide insights into how ESCRT regulates pathogen life cycles and elucidate additional information regarding FMDV counteraction of host antiviral activity.

Project description:UnlabelledNonstructural protein 3A of foot-and-mouth disease virus (FMDV) is a partially conserved protein of 153 amino acids in most FMDVs examined to date. The role of 3A in virus growth and virulence within the natural host is not well understood. Using a yeast two-hybrid approach, we identified cellular protein DCTN3 as a specific host binding partner for 3A. DCTN3 is a subunit of the dynactin complex, a cofactor for dynein, a motor protein. The dynactin-dynein duplex has been implicated in several subcellular functions involving intracellular organelle transport. The 3A-DCTN3 interaction identified by the yeast two-hybrid approach was further confirmed in mammalian cells. Overexpression of DCTN3 or proteins known to disrupt dynein, p150/Glued and 50/dynamitin, resulted in decreased FMDV replication in infected cells. We mapped the critical amino acid residues in the 3A protein that mediate the protein interaction with DCTN3 by mutational analysis and, based on that information, we developed a mutant harboring the same mutations in O1 Campos FMDV (O1C3A-PLDGv). Although O1C3A-PLDGv FMDV and its parental virus (O1Cv) grew equally well in LFBK-αvβ6, O1C3A-PLDGv virus exhibited a decreased ability to replicate in primary bovine cell cultures. Importantly, O1C3A-PLDGv virus exhibited a delayed disease in cattle compared to the virulent parental O1Campus (O1Cv). Virus isolated from lesions of animals inoculated with O1C3A-PLDGv virus contained amino acid substitutions in the area of 3A mediating binding to DCTN3. Importantly, 3A protein harboring similar amino acid substitutions regained interaction with DCTN3, supporting the hypothesis that DCTN3 interaction likely contributes to virulence in cattle.ImportanceThe objective of this study was to understand the possible role of a FMD virus protein 3A, in causing disease in cattle. We have found that the cellular protein, DCTN3, is a specific binding partner for 3A. It was shown that manipulation of DCTN3 has a profound effect in virus replication. We developed a FMDV mutant virus that could not bind DCTN3. This mutant virus exhibited a delayed disease in cattle compared to the parental strain highlighting the role of the 3A-DCTN3 interaction in virulence in cattle. Interestingly, virus isolated from lesions of animals inoculated with mutant virus contained mutations in the area of 3A that allowed binding to DCTN3. This highlights the importance of the 3A-DCTN3 interaction in FMD virus virulence and provides possible mechanisms of virus attenuation for the development of improved FMD vaccines.

Dataset Information

Foot-and-mouth disease virus nonstructural protein 2C interacts with Beclin1, modulating virus replication.

Publications

Foot-and-mouth disease virus nonstructural protein 2C interacts with Beclin1, modulating virus replication.

Similar Datasets

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