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Title: Analysis of the interaction between host factor Sam68 and viral elements during foot-and-mouth disease virus infections

Journal Article · · Virology Journal
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  1. US Dept. Agriculture, Greenport, NY (United States). Plum Island Animal Disease Center. Foreign Animal Disease Research Unit
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Background: The nuclear protein Src-associated protein of 68 kDa in mitosis (Sam68) is known to bind RNA and be involved in cellular processes triggered in response to environmental stresses, including virus infection. Interestingly, Sam68 is a multi-functional protein implicated in the life cycle of retroviruses and picornaviruses and is also considered a marker of virus-induced stress granules (SGs). Recently, we demonstrated the partial redistribution of Sam68 to the cytoplasm in FMDV infected cells, its interaction with viral protease 3Cpro, and found a significant reduction in viral titers as consequence of Sam68-specific siRNA knockdowns. Despite of that, details of how it benefits FMDV remains to be elucidated. Methods: Sam68 cytoplasmic localization was examined by immunofluorescent microscopy, counterstaining with antibodies against Sam68, a viral capsid protein and markers of SGs. The relevance of RAAA motifs in the IRES was investigated using electromobility shift assays with Sam68 protein and parental and mutant FMDV RNAs. In addition, full genome WT and mutant or G-luc replicon RNAs were tested following transfection in mammalian cells. The impact of Sam68 depletion to virus protein and RNA synthesis was investigated in a cell-free system. Lastly, through co-immunoprecipitation, structural modeling, and subcellular fractionation, viral protein interactions with Sam68 were explored. Results: FMDV-induced cytoplasmic redistribution of Sam68 resulted in it temporarily co-localizing with SG marker: TIA-1. Mutations that disrupted FMDV IRES RAAA motifs, with putative affinity to Sam68 in domain 3 and 4 cause a reduction on the formation of ribonucleoprotein complexes with this protein and resulted in non-viable progeny viruses and replication-impaired replicons. Furthermore, depletion of Sam68 in cell-free extracts greatly diminished FMDV RNA replication, which was restored by addition of recombinant Sam68. The results here demonstrated that Sam68 specifically co-precipitates with both FMDV 3Dpol and 3Cpro consistent with early observations of FMDV 3Cpro-induced cleavage of Sam68. Conclusion: We have found that Sam68 is a specific binding partner for FMDV non-structural proteins 3Cpro and 3Dpol and showed that mutations at RAAA motifs in IRES domains 3 and 4 cause a decrease in Sam68 affinity to these RNA elements and rendered the mutant RNA non-viable. Interestingly, in FMDV infected cells re-localized Sam68 was transiently detected along with SG markers in the cytoplasm. These results support the importance of Sam68 as a host factor co-opted by FMDV during infection and demonstrate that Sam68 interact with both, FMDV RNA motifs in the IRES and viral non-structural proteins 3Cpro and 3Dpol.

Research Organization:
Oak Ridge Institute for Science and Education (ORISE), Oak Ridge, TN (United States)
Sponsoring Organization:
USDOE Office of Science (SC)
Grant/Contract Number:
SC0014664
OSTI ID:
1626911
Journal Information:
Virology Journal, Vol. 12, Issue 1; ISSN 1743-422X
Publisher:
BioMed CentralCopyright Statement
Country of Publication:
United States
Language:
English

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The DEAD-Box RNA Helicase DDX1 Interacts with the Viral Protein 3D and Inhibits Foot-and-Mouth Disease Virus Replication journal July 2019
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Strategies for Success. Viral Infections and Membraneless Organelles journal October 2019
The Roles of Picornavirus Untranslated Regions in Infection and Innate Immunity journal March 2018
Insights into Structural and Mechanistic Features of Viral IRES Elements journal January 2018
Picornaviruses and Apoptosis: Subversion of Cell Death journal November 2017

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59 BASIC BIOLOGICAL SCIENCES
Virology
Sam68
FMDV
3C protease
3D polymerase
IRES
Stress granules
RNA replication