G3BP1, G3BP2 and CAPRIN1 interact with DENV-2 gRNA and sfRNA in infected cells.

(A) The DENV-2 NGC 3′UTR variable region (VR) is predicted to contain five stem-loops (SL-I to SL-V) and two highly conserved pseudoknots PKSL-II and PKSL-IV. Sequences shared by DENV-2 gRNA and sfRNA are highlighted in grey. To relatively quantify these two RNAs species, a differential real-time RT-PCR strategy was designed in which one primer pair, QG, detects DENV-2 gRNA only, while the other primer pair, QGSF, amplifies sequences shared by the gRNA and sfRNA. sfRNA levels are obtained by subtraction of absolute levels of amplicons obtained from both primer pairs calculated against a standard curve (for more details refer to Figure S7). (B–C) HuH-7 cells were infected with DENV-2 at MOI = 1 for 24 h and binding of host RBPs to viral and cellular RNAs lysates was analyzed by RNA immunoprecipitation (IP). (B) A representative western blot shows robust enrichment of G3BP1 and KSRP in the specific IP. (C) Pellet fractions from IP with anti-G3BP1 or anti-KSRP antibodies were analyzed for DENV-2 gRNA and sfRNA as described above. Results are presented as mean ± SEM of the ratio of aforementioned RNAs over GAPDH mRNA in the pellet fraction, normalized to same value for control α-IgG IP. (D–E) RNAs containing a 5′ terminal binding aptamer were used to identify sequences required for G3BP1, G3BP2 and CAPRIN1 binding. RNA matrices DENV-2 3′UTR, the same deleted of SL-II (dSLII), or containing two point mutations in the terminal loop of SLII, which are predicted to disrupt PKSLII (D) were incubated with uninfected cell lysates and bound host RBPs eluted as previously described. The DENV-2 NS2A ORF was used as a negative control [17]. The binding of TIAR (TIAL1), DDX6, G3BP1, G3BP2, or CAPRIN1 was interrogated using specific antibodies. DDX6, which was shown to bind DENV-2 DB region, was used as a positive control [17]. TIAR, which was shown not to interact with DENV-2 positive strand RNA, was used as a negative control [64]. One representative western blot (E) of three done is shown.