Chemical inhibitor of Vps34 inhibits TBSV replication and decreases the stability of the viral replication protein.

(A) Inhibition of TBSV replication by the PI3K inhibitor AS604850 in yeast. See further details in Fig 1A. (B) Western blot analysis of TBSV p33 replication protein level in the presence or absence of AS604850 inhibitor. Yeast spheroplasts expressing p33 were treated with MG132 proteasome inhibitor or DMSO as shown. As a loading control, the cellular PGK1 was detected with anti-PGK1 antibody (bottom panel). The samples were taken after 2 h of treatments of spheroplasts. (C) Reduced TBSV RNA production by the tombusvirus replicase assembled in vitro in cell-free extract (CFE) prepared from vps34Δ yeast in comparison with a similar CFE preparation obtained from wt yeast. Purified recombinant p33 and p92^pol replication proteins of TBSV and in vitro transcribed TBSV DI-72 (+)repRNA were added to the CFEs. Nondenaturing PAGE analysis shows the ³²P-labeled TBSV repRNA products, including the (+)repRNA progeny and the dsRNA replication intermediate, made by the reconstituted TBSV replicase. (D) Reduced TBSV RNA production by the tombusvirus replicase assembled in vitro in CFEs prepared from Wortmannin or DMSO-treated WT yeast. See further details in panel C above. Each experiment was repeated.