Figure 4.

Inducible expression of a constitutively active 4E-BP1 repressor promotes reactivation. (A) The 4E-BP1 (AA) mutant is unresponsive to mTORC1-mediated negative regulation and constitutively sequesters eIF4E (4E), suppressing cap-dependent mRNA translation. (m⁷) m⁷GTP cap at the mRNA 5′ end. Latently infected neurons were transduced with a dox-inducible lentivirus encoding HA-tagged 4E-BP1 wild-type (WT), AA, or empty vector. After dox induction, total protein was analyzed by immunoblotting as described in Figure 2B. The black arrowhead at the left of the panel denotes slower-migrating (HA)-tagged 4E-BP1; endogenous 4E-BP1 migrates faster. (B) Total protein was isolated from cultures metabolically labeled with ³⁵S amino acids for 3 h, and the relative levels of radioactivity incorporated compared with the empty vector control were determined. (C) RNA was collected 72 h after dox induction; ICP27 mRNA levels were quantified by qRT–PCR and normalized to cultures transduced with empty vector control. Rapamycin (100 nM) and DMSO-treated cultures were not transduced with lentivirus and, respectively, serve as ±reactivation controls. Error bars indicate SEM. (D) Cultures latently infected with EGFP-expressing HSV1 and transduced with one of two lentiviruses (22904 and 3159) expressing shRNA that depleted p70 S6K1 or a NS shRNA control were established in the presence of ACV as described (Camarena et al. 2010; Kobayashi et al. 2012). After 6 d, ACV was removed, and 20 h later, total RNA was isolated. ICP27 mRNA levels were quantified by qRT–PCR as in C. (Inset) Immunoblot of neuronal lysates validating S6K1 silencing. Tubulin is a loading control. Rapamycin was included as a positive control.