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

item RAI, DEVENDRA - Oak Ridge Institute For Science And Education (ORISE)
item Lawrence, Paul
item Schafer, Elizabeth
item KLOC, ANNA - Oak Ridge Institute For Science And Education (ORISE)
item Rieder, Aida - Elizabeth

Submitted to: Virology Journal
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 12/8/2015
Publication Date: 12/23/2015
Citation: Rai, D., Lawrence, P.J., Schafer, E.A., Kloc, A., Rieder, A.E. 2015. Analysis of the interactions between host factor Sam68 and viral elements during foot-and-mouth disease virus infection. Virology Journal. 12:224.

Interpretive Summary: Foot-and-mouth disease virus (FMDV) is a devastating disease of cattle and other cloven-hoofed animals that is responsible for millions of dollars in losses around the globe each year, prompting researchers in both the U.S. and other countries to develop more effective vaccines. In previous work, we had found that a cellular protein called Sam68 interacts with and is modified by a FMDV protein called 3C and this process is necessary for the virus to replicate in cells. In this study we provide evidence that Sam68 also binds FMDV genetic material and a viral protein called 3D^pol, which is responsible for copying the viral genetic information during replication. This work adds to the knowledge of virus-unique processes that have potential for the development of disease control strategies such as better vaccines.

Technical Abstract: 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 3C^pro, and found a significant reduction in viral titers as consequence of Sam68-specific siRNA knockdowns. Despite of that, details of how it benefits FMDV remain unclear. FMDV-induced partial cytoplasmic redistribution of Sam68 resulted in it temporarily co-localizing with SG markers: TIA-1 and G3BP. It also localized with nuclear export factor NXF1. Mutations that disrupted FMDV IRES UAAA motifs, with putative affinity to Sam68 in domain 4 cause a reduction on the formation of ribocnucleoprotein complexes with this protein and resulted in non-viable progeny viruses. Furthermore, depletion of Sam68 in cell-free extracts greatly diminished FMDV RNA replication, which was restored by addition of recombinant Sam68. The results also showed that Sam68 specifically co-precipitated with both FMDV 3D^pol and 3C^pro consistent with early observations of FMDV 3C^pro-induced cleavage of Sam68. We have found that Sam68 is a specific binding partner for FMDV non-structural proteins 3C^pro and 3D^pol and showed that mutations at UAAA motifs in IRES domains 4 cause a decrease in Sam68 affinity to this loop and rendered the mutant RNA non-viable. Interestingly, in FMDV infected cells re-localized Sam68 was detected along with SG markers and NXF1 in the cytoplasm. These results add to the evidence that Sam68 is an important host factor co-opted by FMDV during infection.