Figure 5. A subset of human DNAJ cochaperones is required for the DENV life cycle.

(A) Screen for human DNAJ involvement in DENV replication. Forty-five human DNAJs were tested for effect on DENV by KD using three different shRNAs. Indicated DNAJ-KD cells were established as for Hsp70s in Fig. 1, and infected with DENV2 for 36 h. Extracellular virus production was measured by FFA. Ctrl: shRNA against luciferase.

(B-C) Validation of KD of different DNAJ isoforms by assessing mRNA levels (B) and DNAJ protein (C).

(D-F) Identification of DNAJs involved in post-entry stages of the DENV life cycle. DENV vRNA was electroporated into DNAJ-KD cells. Extracellular viral infectivity (D); intracellular viral RNA (E) and viral proteins (F) were measured after 72 h, by FFA, qRT-PCR and immunoblotting respectively.

(G) Isoform DnaJB6b is required for DENV propagation. (i) Splicing creates DnaJB6 variants. KD cells were established for each variant, and infected with DENV2 for 36 h. The levels of each variant transcript (ii) and extracellular viral production (iii) were measured by qRT-PCR with variant-specific primers and FFA respectively. Data are representative of three independent experiments and expressed as mean ± SD of triplicates. * P < 0.01.