Figure 3

Intracellular expression and secretion of GP1 in cells transfected with intron-A containing and intronless LASV GPC constructs. (A) Intracellular expression of glycoprotein constructs [C]. The 42 KDa GP1 protein was readily detected in intron-containing GPC (lane 2), GP1-TM (lane 4), and sGP1-RRAA-FLAG (lane 5) cell extracts. Expression of GPC from an intronless construct resulted in significantly lower levels of detectable GP1 (lane 3). In vector control (lane 1) only endogenous proteins ca. 47 and 55 KDa were detected (end.). Relative average expression levels of each glycoprotein compared to intron-A containing GPC are indicated below the respective lanes, as estimated by densitometry. (B) Secreted GP1 was detected only in intron-A containing GPC and sGP1-RRAA-FLAG supernatants [S]. Relative level of secreted GP1 generated by sGP1-RRAA-FLAG compared to GPC, as estimated by densitometry, is indicated below lane 5B. (C) Immunoprecipitation of GP1 from GPC, GP1-TM, and sGP1-RRAA-FLAG transfected cell supernatants [IP]. Immunoprecipitated GP1 was detected in intron-A containing GPC (lane 2C) and sGP1-RRAA-FLAG (lane 5C), and at significantly lower levels in intronless GPC (lane 3C) expressing cell supernatants. A very faint GP1 band was detected in the immunoprecipitated GP1-TM sample (lane 4C). Vector control reactions did not show reactivity (lane 1C). Relative volume of each IP reaction loaded per lane is indicated above panel C. (D) Secreted GP1 profile in VERO cells transfected with LASV glycoprotein expression constructs. Average levels of GP1 from intronless GPC, GP1-TM, and sGP1-RRAA-FLAG, compared to those from intron-A containing GPC expression are shown below each lane in panels C and D (N = 4).