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Analysis of poliovirus protein 3A interactions with viral and cellular proteins in infected cells.

ABSTRACT: Poliovirus proteins 3A and 3AB are small, membrane-binding proteins that play multiple roles in viral RNA replication complex formation and function. In the infected cell, these proteins associate with other viral and cellular proteins as part of a supramolecular complex whose structure and composition are unknown. We isolated viable viruses with three different epitope tags (FLAG, hemagglutinin [HA], and c-myc) inserted into the N-terminal region of protein 3A. These viruses exhibited growth properties and characteristics very similar to those of the wild-type, untagged virus. Extracts prepared from the infected cells were subjected to immunoaffinity purification of the tagged proteins by adsorption to commercial antibody-linked beads and examined after elution for cellular and other viral proteins that remained bound to 3A sequences during purification. Viral proteins 2C, 2BC, 3D, and 3CD were detected in all three immunopurified 3A samples. Among the cellular proteins previously reported to interact with 3A either directly or indirectly, neither LIS1 nor phosphoinositol-4 kinase (PI4K) were detected in any of the purified tagged 3A samples. However, the guanine nucleotide exchange factor GBF1, which is a key regulator of membrane trafficking in the cellular protein secretory pathway and which has been shown previously to bind enteroviral protein 3A and to be required for viral RNA replication, was readily recovered along with immunoaffinity-purified 3A-FLAG. Surprisingly, we failed to cocapture GBF1 with 3A-HA or 3A-myc proteins. A model for variable binding of these 3A mutant proteins to GBF1 based on amino acid sequence motifs and the resulting practical and functional consequences thereof are discussed.

SUBMITTER: Teterina NL

PROVIDER: S-EPMC3126254 | biostudies-literature | 2011 May

REPOSITORIES: biostudies-literature

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Analysis of poliovirus protein 3A interactions with viral and cellular proteins in infected cells.

Teterina Natalya L NL Pinto Yuval Y Weaver Joseph D JD Jensen Kenneth S KS Ehrenfeld Ellie E

Journal of virology 20110223 9

Poliovirus proteins 3A and 3AB are small, membrane-binding proteins that play multiple roles in viral RNA replication complex formation and function. In the infected cell, these proteins associate with other viral and cellular proteins as part of a supramolecular complex whose structure and composition are unknown. We isolated viable viruses with three different epitope tags (FLAG, hemagglutinin [HA], and c-myc) inserted into the N-terminal region of protein 3A. These viruses exhibited growth pr ...[more]

PMID: 21345960

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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.

Dataset Information

Analysis of poliovirus protein 3A interactions with viral and cellular proteins in infected cells.

Publications

Analysis of poliovirus protein 3A interactions with viral and cellular proteins in infected cells.

Similar Datasets

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