Figure 2.

TRAF6 inhibits CSFV replication. PAMs stably overexpressing TRAF6 and knockdown of TRAF6 were constructed via lentivirus. (a) Confirmation of CMV-TRAF6 recombinant lentivirus infection by fluorescence detection of the GFP reporter expressed in PAMs. (i) Mock-infected PAMs. (ii) PAMs infected with lentiviruses expressing CMV. (iii) PAMs infected with lentiviruses expressing CMV-TRAF6. (b) Western blot for TRAF6 expression in stable CMV-TRAF6 cells. β-actin was used as an internal control. (c) CSFV genome RNA in TRAF6-overexpressing cells. Cells stably expressing CMV or CMV-TRAF6 were infected with CSFV. RT-PCR determined CSFV genome RNA levels at 24 and 48 hpi. (d) Infectious progeny viral titres in supernatants from TRAF6-overexpressing cells. Viral titres from the supernatant collected at 24 and 48 hpi were determined and expressed as TCID₅₀/ml. (e) Confirmation of TRAF6-knockdown lentivirus infection by fluorescence detection of the GFP reporter expressed in PAMs. (i) Mock-infected PAMs. (ii) PAMs infected with lentiviruses expressing shN. (iii) PAMs infected with TRAF6-knockdown lentivirus. (f) Western blot for TRAF6 expression in TRAF6 knockdown cells. Cells stably expressing TRAF6-sh1, TRAF6-sh2, TRAF6-sh3, or shN were constructed, and Western blot determined endogenous TRAF6 expression. (g) CSFV genome RNA in TRAF6-sh2 cells. Stable shN and TRAF6-sh2 cells were infected with CSFV, and RT-PCR determined CSFV genome RNA levels at 24 and 48 hpi. (h) Viral titres of infectious progeny in supernatant from TRAF6-sh2 cells. Viral titres from the supernatant collected at 24 and 48 hpi were determined and expressed as TCID₅₀/ml. Error bars represent the mean ± SD of three independent experiments. **P < 0.01 and ***P < 0.001 compared with the controls calculated using Student’s t-test.