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

Abstract

BackgroundThe 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.MethodsSam68 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.ResultsFMDV-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.ConclusionWe 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.Electronic supplementary materialThe online version of this article (doi:10.1186/s12985-015-0452-8) contains supplementary material, which is available to authorized users.

Foot-and-Mouth Disease Virus Leader Protease Cleaves G3BP1 and G3BP2 and Inhibits Stress Granule Formation

“…We found a strongly reduced amount of G3BP1-containing SGs in cells infected with wild-type FMDV. This observation is in agreement with previous observations that some small SGs that contain TIA-1 and Sam68, but lack G3BP1, are formed in FMDV-infected cells (51,52). These small SGs resemble the small "atypical" SGs observed in cells expressing enterovirus 2A pro (25,48).…”

Foot‐and‐mouth disease virus nonstructural protein 2B interacts with cyclophilin A, modulating virus replication

“…The potential mechanisms of interaction of FMDV proteins with host cell proteins are not fully understood. Some host cell proteins that interact with FMDV VP1, 2C, and 3A proteins have been identified with the yeast 2-hybrid system (12)(13)(14)(15). In the current study, for the first time, using the yeast 2-hybrid system, we showed that the FMDV Figure 7.…”

The DEAD-Box RNA Helicase DDX1 Interacts with the Viral Protein 3D and Inhibits Foot-and-Mouth Disease Virus Replication

“…3D is an important viral protein that has multiple roles in FMDV replication and pathogenesis (Rai et al 2013;Herod et al 2016;Rai et al 2017). An interaction between the host factor Sam68 and 3D has been reported (Rai et al 2015). To better understand the role of FMDV 3D in viral replication and virulence, we sought to identify new host cell proteins that interact with 3D using the yeast two-hybrid system.…”