Mammalian Two-Hybrid System: A Complementary Approach to the Yeast Two-Hybrid System

Luo, Ying; Batalao, Anna; Zhou, Helen; Zhu, Li

doi:10.2144/97222pf02

Cited by 85 publications

(57 citation statements)

References 10 publications

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“…1B), supporting the hypothesis that interaction between these two proteins occurs during viral infection. In addition, the confirmation of the 3A-DCTN3 interaction was performed (26). MCF10A cells were transfected with the indicated plasmids.…”

Section: Resultsmentioning

confidence: 99%

Interaction of Foot-and-Mouth Disease Virus Nonstructural Protein 3A with Host Protein DCTN3 Is Important for Viral Virulence in Cattle

Gladue

O’Donnell

Baker-Bransetter

et al. 2014

J Virol

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Nonstructural 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-␣v␤6, 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.

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Section: Resultsmentioning

confidence: 99%

Interaction of Foot-and-Mouth Disease Virus Nonstructural Protein 3A with Host Protein DCTN3 Is Important for Viral Virulence in Cattle

Gladue

O’Donnell

Baker-Bransetter

et al. 2014

J Virol

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“…Mammalian-two hybrid (M2H) was also used to complement the Y2H study, since mammalian cells used in the M2H system undergo more comprehensive post-translational modifications of proteins, hence may better mimic in vivo interactions [33]. M2H assays using Vero cells were conducted to validate the three selected A. aegypti putative protein candidates found interacting with their respective DENV-2 proteins.…”

Section: Identified Interactomes Validated In Mammalian Systemsmentioning

confidence: 99%

Protein-protein interactions between A. aegypti midgut and dengue virus 2: two-hybrid screens using the midgut cDNA library

Tham

Balasubramaniam

Chew

et al. 2015

J Infect Dev Ctries

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Introduction: Dengue virus (DENV) is principally transmitted by the Aedes aegypti mosquito. To date, mosquito population control remains the key strategy for reducing the continuing spread of DENV. The focus on the development of new vector control strategies through an understanding of the mosquito-virus relationship is essential, especially targeting the midgut, which is the first mosquito organ exposed to DENV infection. Methodology: A cDNA library derived from female adult A. aegypti mosquito midgut cells was established using the switching mechanism at the 5' end of the RNA transcript (SMART), in combination with a highly potent recombination machinery of Saccharomyces cerevisiae. Gal4-based yeast two-hybrid (Y2H) assays were performed against DENV-2 proteins (E, prM, M, and NS1). Mammalian two-hybrid (M2H) and double immunofluorescence assays (IFA) were conducted to validate the authenticity of the three selected interactions.Results: The cDNA library was of good quality based on its transformation efficiency, cell density, titer, and the percentage of insert size. A total of 36 midgut proteins interacting with DENV-2 proteins were identified, some involved in nucleic acid transcription, oxidoreductase activity, peptidase activity, and ion binding. Positive outcomes were obtained from the three selected interactions validated using M2H and double IFA assays. Conclusions: The identified proteins have different biological activities that may aid in the virus replication pathway. Therefore, the midgut cDNA library is a valuable tool for identifying DENV-2 interacting proteins. The positive outcomes of the three selected proteins validated supported the quality of the cDNA library and the robustness of the Y2H mechanisms.

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“…Indeed, modified two hybrid assays were developed to detect proteinprotein interaction directly in mammalian cells. Thus, this assay better mimics the situation which occurs in vivo (17)(18)(19). In addition, the use of a mammalian system for proteins of mammalian origin is preferable for a reverse two hybrid approach (20)(21)(22).…”

Section: Introductionmentioning

confidence: 99%

A novel in vivo assay for the analysis of protein-protein interaction

Maroun

Aronheim

1999

Nucleic Acids Research

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The Ras Recruitment System (RRS) is a method for identification and isolation of protein-protein interaction. The method is based on translocation of cytoplasmic mammalian Ras protein to the inner leaflet of the plasma membrane through protein-protein interaction. The system is studied in a temperaturesensitive yeast strain where the yeast Ras guanyl nucleotide exchange factor is inactive at 36°°°°C. Protein-protein interaction results in cell growth at the restrictive temperature. We developed a gene reporter assay for the analysis of protein-protein interaction in mammalian cells. Ras activation in mammalian cells induces the mitogen-activated kinase cascade (MAPK), which can be monitored using Ras-dependent reporter genes. This greatly extends the usefulness of the system and provides a novel assay for protein-protein interaction in mammalian cells.

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Mammalian Two-Hybrid System: A Complementary Approach to the Yeast Two-Hybrid System

Abstract: Here we demonstrate the use of a mammalian two-hybrid system to study protein-protein

Cited by 85 publications

References 10 publications

Interaction of Foot-and-Mouth Disease Virus Nonstructural Protein 3A with Host Protein DCTN3 Is Important for Viral Virulence in Cattle

Interaction of Foot-and-Mouth Disease Virus Nonstructural Protein 3A with Host Protein DCTN3 Is Important for Viral Virulence in Cattle

Protein-protein interactions between A. aegypti midgut and dengue virus 2: two-hybrid screens using the midgut cDNA library

A novel in vivo assay for the analysis of protein-protein interaction

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