Fig. 8

Immunofluorescence and WB analysis after superinfection. A Immunofluorescence of PERV proteins after superinfection of PERV-C(5683) positive ST-IOWA cells using antibodies directed against PERV-C Env and HA-tag. Protein expression was monitored at day 56 p.i.. A1-4: non-infected ST-IOWA (ctr−), B1-4: PERV-C(5683) positive ST-IOWA cells (ctr+), C1-4: ST-IOWA cells infected with SP-HA (ctr+) or D1-4: RPep-HA (ctr.+), E1-E4: ST-IOWA cells superinfected with SP-HA or F1-4: RPep-HA. PERV-C Env proteins were observed for all cells except for the (ctr−), while HA proteins only were present in primary infected control cells. B WB analysis of PERV-C proteins using antibodies directed against (a) the HA-tag or (b) PERV-C Env. WB was performed 56 days after superinfection of ST-IOWA-PERV-C(5683) cells with PERV-C(5683)-HA viruses. Native ST-IOWA cells were used as negative control (ctr−). ST-IOWA cells infected with virus present in SN that was also used for superinfection served as positive controls (ctr+). Lane 1: ST-IOWA (ctr−, lane 2: PERV-C(5683) positive ST-IOWA cells, lane 3–4: ST-IOWA cells infected with (3) SP-HA (ctr+) or (4) RPep-HA (ctr+), lane 5–6: PERV-C(5683) positive ST-IOWA cells superinfected with (5) SP-HA or (6) RPep-HA. A The EnvC-HA precursor (72.1 kDa) was detected for both SP-HA and RPep-HA. The corresponding cleaved product of SP-HA-SU is slightly below the precursor, thus indicated by a shadow. Since the HA-tag is located C-terminal in RPep-HA only the cleaved product TM-HA was detected. B For differentiation of EnvC and EnvC-HA, an anti-PERV-C Env(498) antiserum was used. PERV-C Env proteins were detected for all cells except for the negative control. C An anti‐beta‐actin WB was used as loading control