Interaction of TIA-1/TIAR with West Nile and dengue virus products in infected cells interferes with stress granule formation and processing body assembly Mohamed M. Emara and Margo A. Brinton* Department of Biology, Georgia State University, Atlanta, GA 30302 Communicated by Hilary Koprowski, Thomas Jefferson University, Philadelphia, PA, April 11, 2007 (received for review February 23, 2007) -The West Nile virus minus-strand 3؅ terminal stem loop (SL) RNA cells and tissues (8, 9). TIA-1 and TIAR contain three N was previously shown to bind specifically to cellular stress granule terminal RNA recognition motifs (RRM) (10) and a glutamine- (SG) components, T cell intracellular antigen-1 (TIA-1) and the rich C-terminal domain, called prion-related domain (PRD), related protein TIAR. In vitro TIAR binding was 10 times more that shares structural and functional characteristics with the efficient than TIA-1. The 3؅(؊)SL functions as the promoter for aggregation domains of mammalian and yeast prion proteins genomic RNA synthesis. Colocalization of TIAR and TIA-1 with the (11). A recombinant TIA-1 protein lacking the three RNA- viral replication complex components dsRNA and NS3 was ob- binding domains was unable to bind poly(A)ϩ RNA and recruit served in the perinuclear regions of West Nile virus- and dengue it to SGs (2). Deletion of the TIA-1 PRD inhibited protein virus-infected cells. The kinetics of accumulation of TIAR in the aggregation and SG assembly (11). PRD aggregation is regulated perinuclear region was similar to those of genomic RNA synthesis. by the molecular chaperone heat shock protein 70 (HSP70), and In contrast, relocation of TIA-1 to the perinuclear region began only overexpression of HSP70 prevented cytosolic aggregation of after maximal levels of RNA synthesis had been achieved, except PRD (11). TIA-1 and TIAR also regulate translational silencing when TIAR was absent. Virus infection did not induce SGs and of a subset of cellular mRNAs by binding to AU-rich sequences progressive resistance to SG induction by arsenite developed in the 3Ј noncoding regions (NCRs) of these mRNAs (12). In coincident with TIAR relocation. A progressive decrease in the addition, TIA-1 and TIAR proteins interact specifically with the number of processing bodies was secondarily observed in infected 3Ј-terminal stem loop (SL) structure of the West Nile virus cells. These data suggest that the interaction of TIAR with viral minus-strand RNA [WNV3Ј(Ϫ)SL] (13). components facilitates flavivirus genome RNA synthesis and in- WNV is classified in the family Flaviviridae, genus Flavivirus, hibits SG formation, which prevents the shutoff of host translation. along with a number of other human pathogens, including dengue virus (DV), Japanese encephalitis virus, yellow fever T cell intracellular antigen-related protein ͉ T cell intracellular antigen-1 ͉ virus, and tick-borne encephalitis virus. The flavivirus genome viral replication complexes is a single-stranded, positive sense RNA of Ϸ11 kb containing a single ORF and is the only viral mRNA produced during the ukaryotic cells respond to environmental stresses, such as virus replication cycle. Flavivirus replication takes place in the Eoxidative stress, heat shock, UV irradiation, endoplasmic perinuclear region of the cytoplasm of infected cells. Three reticulum (ER) stress, and some viral infections, by altering the structural (capsid, membrane, and envelope) and seven non- protein expression machinery (1). The expression of proteins structural (NS1, NS2a, NS2b, NS3, NS4a, NS4b, and NS5) viral responsible for damage repair is increased, whereas translation proteins are produced by proteolytic processing of the single of constitutively expressed proteins is aborted by redirection of polyprotein by viral and cellular proteases (14, 15). The C- these mRNAs from polysomes to discrete cytoplasmic foci terminal portion of NS5 is the RNA-dependent RNA polymer- known as stress granules (SGs) for transient storage (2). Several ase (RdRp), whereas the N-terminal part is a methyltransferase cell proteins, including T cell intracellular antigen-1 (TIA-1), involved in RNA capping (16). The N terminus of NS3 has serine TIA-1-related protein (TIAR) (2), and Ras-Gap-SH3 domain- protease activity, whereas the C terminus contains helicase, binding protein (G3BP) are involved in SG assembly (3). Phos- NTPase, and triphosphatase activities (14, 15). The NS3, NS5, phorylation of eukaryotic translation initiation factor 2␣ (eIF2␣) and NS1 proteins, as well as the hydrophobic NS2a, NS2b, NS4a, by protein kinase R (PKR) and other kinases prevents the assembly of the active ternary preinitiation complex eIF2-GTP- and NS4b proteins, were previously shown to be components of tRNAMet and inhibits translation initiation and polysome assem- membrane bound viral RNA replication complexes located in bly (4). TIA-1 and TIAR bind to these inactive translation the perinuclear region of infected cells (17–19). initiation complexes as well as to the mRNA poly(A)ϩ and In this study, both TIA-1 and TIAR were shown to colocalize self-aggregate, promoting the assembly of SGs (2). Processing with the WNV and DV NS3 protein as well as with viral dsRNA, bodies (PBs), another type of foci identified in the cytoplasm of a marker for viral replication complexes, in infected mammalian eukaryotic cells (5), contain components of the 5Ј–3Ј mRNA cells. The data obtained suggest that these interactions facilitate degradation pathway as well as the miRNA-dependent silencing protein GW182 (6). PBs do not require eIF2␣ phosphorylation Author contributions: M.M.E. and M.A.B. designed research; M.M.E. performed research; for their assembly (7). Although SGs and PBs differ in their size M.M.E. and M.A.B. analyzed data; and M.M.E. and M.A.B. wrote the paper. MICROBIOLOGY and shape as well as in their mechanism of assembly, the two The authors declare no conflict of interest. types of foci are found side by side in mammalian cells and a Abbreviations: BHK, baby hamster kidney; WNV, West Nile virus; TIA-1, T cell intracellular physical association between them was demonstrated by real- antigen-1; TIAR, T cell intracellular antigen-related protein; SG, stress granule; PB, process- time fluorescence imaging (7). This study also showed that PBs ing body. are highly motile, whereas the positions of SGs are relatively *To whom correspondence should be addressed at: Department of Biology, Georgia State fixed, and that SGs deliver mRNAs to associated PBs for University, P.O. Box 4010, Atlanta, GA, 30302-4010. E-mail: [email protected]. degradation. This article contains supporting information online at www.pnas.org/cgi/content/full/ TIA-1 and TIAR are essential, multifunctional, nucleocyto- 0703348104/DC1. plasmic shuttling proteins that are expressed in most types of © 2007 by The National Academy of Sciences of the USA www.pnas.org͞cgi͞doi͞10.1073͞pnas.0703348104 PNAS ͉ May 22, 2007 ͉ vol. 104 ͉ no. 21 ͉ 9041–9046 Downloaded by guest on September 30, 2021 contrast, only a few bright cytoplasmic foci of TIA-1 colocalized A E with the WNV proteins by 24 hpi (Fig. 1F, merge; a pattern similar to that seen at 12 hpi with TIAR). Strong colocalization of TIA-1 with the WNV proteins was observed by 36 hpi (Fig. 1G, merge). Quantification of the amount of TIAR or TIA-1 in B F the nucleus confirmed that the distribution of both TIA-1 and TIAR was altered with time after WNV infection but the redistribution of TIAR occurred more rapidly (Fig. 1H). It was previously reported that the expression of TIA-1 was C G three times higher in TIARϪ/Ϫ than in wild-type MEFs (13). To determine whether the time course of TIA-1 redistribution was altered when TIAR was not present, TIARϪ/Ϫ MEFs were D infected with WNV (MOI of 5), and the locations of TIA-1 and H WNV proteins were analyzed at 12 and 24 hpi. TIA-1 was equally distributed in both the cytoplasm and nucleus of mock-infected TIARϪ/Ϫ MEFs (SI Fig. 5A, green), but these cells were significantly larger than BHK cells. The time course of appear- ance, staining intensity, and distribution of the WNV proteins in these cells (SI Fig. 5 B and C, red) were similar to those observed Fig. 1. Colocalization of TIA-1 and TIAR with WNV proteins in infected BHK with infected BHK cells. At 12 hpi, a few bright foci of TIA-1 that cells. (A–D) Laser scanning confocal microscopy of mock-infected (A) or WNV- colocalized with WNV proteins were observed (SI Fig. 5B, infected (MOI of 5) (B–D) BHK cells stained with anti-WNV (red) and anti-TIAR merge), similar to what was observed by 12 hpi for TIAR in BHK antibody (green) at the indicated times after infection. (E–G) BHK stained with cells. By 24 hpi, a significant increase in TIA-1 in the perinuclear anti-WNV (red) and anti-TIA-1 antibody (green) at the indicated times after region and strong colocalization with viral proteins was observed WNV (F and G) or mock (E) infection. Nuclear DNA (blue) was stained with (SI Fig. 5C). At all times, a significant portion of TIA-1 remained Hoechst 33258 dye. (H) Quantification of the amount of TIAR (Left) or TIA-1 in the nucleus. These results indicate that, in the absence of (Right) in the nucleus of mock-infected cells (M) and WNV-infected cells at the TIAR, colocalization of TIA-1 with the viral proteins occurred indicated times after infection. The relative pixel intensity in the nuclei of 20 more quickly, with a time course similar to that of TIAR cells at each time after infection was measured, and the mean values were plotted. Error bars indicate the standard deviation of the mean.