Dengue virus (DENV) is a rapidly re-emerging flavivirus that causes dengue fever (DF), dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS), diseases for which there are no available therapies or vaccines. The DENV-2 positive-strand RNA genome contains 5' and 3' untranslated regions (UTRs) that have been shown to form secondary structures required for virus replication and interaction with host cell proteins. In order to comprehensively identify host cell factors that bind the DENV-2 UTRs, we performed RNA chromatography, using the DENV-2 5' and 3' UTRs as "bait", combined with quantitative mass spectrometry. We identified several proteins, including DDX6, G3BP1, G3BP2, Caprin1, and USP10, implicated in P body (PB) and stress granule (SG) function, and not previously known to bind DENV RNAs. Indirect immunofluorescence microscopy showed these proteins to colocalize with the DENV replication complex. Moreover, DDX6 knockdown resulted in reduced amounts of infectious particles and viral RNA in tissue culture supernatants following DENV infection. DDX6 interacted with DENV RNA in vivo during infection and in vitro this interaction was mediated by the DB1 and DB2 structures in the 3' UTR, possibly by formation of a pseudoknot structure. Additional experiments demonstrate that, in contrast to DDX6, the SG proteins G3BP1, G3BP2, Caprin1 and USP10 bind to the variable region (VR) in the 3' UTR. These results suggest that the DENV-2 3' UTR is a site for assembly of PB and SG proteins and, for DDX6, assembly on the 3' UTR is required for DENV replication.

The microRNA miR-122 and DDX6/Rck/p54, a microRNA effector, have been implicated in hepatitis C virus (HCV) replication. In this study, we demonstrated for the first time that HCV-JFH1 infection disrupted processing (P)-body formation of the microRNA effectors DDX6, Lsm1, Xrn1, PATL1, and Ago2, but not the decapping enzyme DCP2, and dynamically redistributed these microRNA effectors to the HCV production factory around lipid droplets in HuH-7-derived RSc cells. Notably, HCV-JFH1 infection also redistributed the stress granule components GTPase-activating protein (SH3 domain)-binding protein 1 (G3BP1), ataxin-2 (ATX2), and poly(A)-binding protein 1 (PABP1) to the HCV production factory. In this regard, we found that the P-body formation of DDX6 began to be disrupted at 36 h postinfection. Consistently, G3BP1 transiently formed stress granules at 36 h postinfection. We then observed the ringlike formation of DDX6 or G3BP1 and colocalization with HCV core after 48 h postinfection, suggesting that the disruption of P-body formation and the hijacking of P-body and stress granule components occur at a late step of HCV infection. Furthermore, HCV infection could suppress stress granule formation in response to heat shock or treatment with arsenite. Importantly, we demonstrate that the accumulation of HCV RNA was significantly suppressed in DDX6, Lsm1, ATX2, and PABP1 knockdown cells after the inoculation of HCV-JFH1, suggesting that the P-body and the stress granule components are required for the HCV life cycle. Altogether, HCV seems to hijack the P-body and the stress granule components for HCV replication.

Alphaviruses represent a serious public health threat and cause a wide variety of diseases, ranging from severe encephalitis, which can result in death or neurological sequelae, to mild infection, characterized by fever, skin rashes, and arthritis. In the infected cells, alphaviruses express only four nonstructural proteins, which function in the synthesis of virus-specific RNAs and in modification of the intracellular environment. The results of our study suggest that Sindbis virus (SINV) infection in BHK-21 cells leads to the formation of at least two types of nsP3-containing complexes, one of which was found in association with the plasma membrane and endosome-like vesicles, while the second was coisolated with cell nuclei. The latter complexes could be solubilized only with the cytoskeleton-destabilizing detergent. Besides viral nsPs, in the mammalian cells, both complexes contained G3BP1 and G3BP2 (which were found in different ratios), YBX1, and HSC70. Rasputin, an insect cell-specific homolog of G3BP1, was found in the nsP3-containing complexes isolated from mosquito cells, which was suggestive of a high conservation of the complexes in the cells of both vertebrate and invertebrate origin. The endosome- and plasma membrane-associated complexes contained a high concentration of double-stranded RNAs (dsRNAs), which is indicative of their function in viral-RNA synthesis. The dsRNA synthesis is likely to efficiently proceed on the plasma membrane, and at least some of the protein-RNA complexes would then be transported into the cytosol in association with the endosome-like vesicular organelles. These findings provide new insight into the mechanism of SINV replication and virus-host cell interactions.

Viral RNA-host protein interactions are critical for replication of flaviviruses, a genus of positive-strand RNA viruses comprising major vector-borne human pathogens including dengue viruses (DENV). We examined three conserved host RNA-binding proteins (RBPs) G3BP1, G3BP2 and CAPRIN1 in dengue virus (DENV-2) infection and found them to be novel regulators of the interferon (IFN) response against DENV-2. The three RBPs were required for the accumulation of the protein products of several interferon stimulated genes (ISGs), and for efficient translation of PKR and IFITM2 mRNAs. This identifies G3BP1, G3BP2 and CAPRIN1 as novel regulators of the antiviral state. Their antiviral activity was antagonized by the abundant DENV-2 non-coding subgenomic flaviviral RNA (sfRNA), which bound to G3BP1, G3BP2 and CAPRIN1, inhibited their activity and lead to profound inhibition of ISG mRNA translation. This work describes a new and unexpected level of regulation for interferon stimulated gene expression and presents the first mechanism of action for an sfRNA as a molecular sponge of anti-viral effectors in human cells.

Upon exposure to various environmental stresses such as arsenite, hypoxia, and heat shock, cells inhibit their translation and apoptosis and then repair stress-induced alterations, such as DNA damage and the accumulation of misfolded proteins. These types of stresses induce the formation of cytoplasmic RNA granules called stress granules (SGs). SGs are storage sites for the many mRNAs released from disassembled polysomes under these stress conditions and are essential for the selective translation of stress-inducible genes. Ras-GTPase-activating protein SH3 domain-binding protein 1 (G3BP1) is a component of SGs that initiates the assembly of SGs by forming a multimer. In this study, we examined the role of G3BP2, a close relative of G3BP1, in SG formation. Although single knockdown of either G3BP1 or G3BP2 in 293T cells partially reduced the number of SG-positive cells induced by arsenite, the knockdowns of both genes significantly reduced the number. G3BP2 formed a homo-multimer and a hetero-multimer with G3BP1. Moreover, like G3BP1, the overexpression of G3BP2 induced SGs even without stress stimuli. Collectively, these results suggest that both G3BP1 and G3BP2 play a role in the formation of SGs in various human cells and thereby recovery from these cellular stresses.

Long noncoding RNAs (lncRNA) have been associated with various types of cancer; however, the precise role of many lncRNAs in tumorigenesis remains elusive. Here we demonstrate that the cytosolic lncRNA P53RRA is downregulated in cancers and functions as a tumor suppressor by inhibiting cancer progression. Chromatin remodeling proteins LSH and Cfp1 silenced or increased P53RRA expression, respectively. P53RRA bound Ras GTPase-activating protein-binding protein 1 (G3BP1) using nucleotides 1 and 871 of P53RRA and the RRM interaction domain of G3BP1 (aa 177-466). The cytosolic P53RRA-G3BP1 interaction displaced p53 from a G3BP1 complex, resulting in greater p53 retention in the nucleus, which led to cell-cycle arrest, apoptosis, and ferroptosis. P53RRA promoted ferroptosis and apoptosis by affecting transcription of several metabolic genes. Low P53RRA expression significantly correlated with poor survival in patients with breast and lung cancers harboring wild-type p53. These data show that lncRNAs can directly interact with the functional domain of signaling proteins in the cytoplasm, thus regulating p53 modulators to suppress cancer progression.Significance: A cytosolic lncRNA functions as a tumor suppressor by activating the p53 pathway. Cancer Res; 78(13); 3484-96. ©2018 AACR.