Expression and trafficking of fluorescent viral membrane proteins in baculovirus-transduced BHK cells.
Baculovirus vectors show promise as a novel tool for gene delivery into mammalian cells and gene transfer with wild-type baculovirus has been demonstrated both in vitro and in vivo. To study expression and intracellular trafficking of foreign viral membrane proteins in baculovirus-transduced mammalian cells, the envelope proteins, E1 and E2, of rubella virus (RV) were chosen as a model. The enhanced green fluorescent protein (EGFP) and a red fluorescent protein (RFP) were fused to the C-terminus of E1 and E2, respectively. The proteins were cloned under a cytomegalovirus (CMV) promoter and expressed as fluorescent fusion proteins in baculovirus-transduced baby hamster kidney (BHK) cells. Expression of the chimeric proteins in these cells showed that E1 was retained within the ER and cis-Golgi when expressed alone. In contrast, E2 was efficiently transported to the trans-Golgi network (TGN). However, when expressed together, E1 co-localized with E2 in TGN and to some extent in the lysosomes. The recombinant baculovirus vectors were able to transduce the BHK cells efficiently and the fluorescent fusion constructs allowed easy detection of the trafficking events in the transduced mammalian cells. Consequently, this technique should have wide applications when intracellular analysis of protein synthesis and maturation is under study.
Ojala
Kirsi
K
Department of Biological and Environmental Science, University of Jyväskylä, P.O. Box 35, FIN-40014 Jyväskylä, Finland.
Tikka
Päivi J
PJ
Kautto
Liisa
L
Käpylä
Pirjo
P
Marjomäki
Varpu
V
Oker-Blom
Christian
C
D000818:Animals; D016367:Baculoviridae / Q000235:genetics* / Q000378:metabolism; D002460:Cell Line; D006224:Cricetinae; D020869:Gene Expression Profiling / Q000379:methods*; D005786:Gene Expression Regulation / Q000502:physiology; D005822:Genetic Vectors; D007668:Kidney / Q000166:cytology* / Q000378:metabolism*; D008856:Microscopy, Fluorescence / Q000379:methods*; D021381:Protein Transport / Q000502:physiology*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D014162:Transfection / Q000379:methods*; D014759:Viral Envelope Proteins / Q000235:genetics / Q000378:metabolism*
D011993:Recombinant Fusion Proteins; D014759:Viral Envelope Proteins; C061826:E1 envelope protein, Rubella virus; C061827:E2 envelope protein, Rubella virus