Fluorescent tagging the NS1 protein in yellow fever virus: Replication-capable viruses which produce the secretory GFP-NS1 fusion protein.

Yellow fever virus, the prototype in the genus Flavivirus, was used to develop viruses in which the nonstructural protein NS1 is genetically fused to GFP in the context of viruses capable of autonomous replication. The GFP-tagging of NS1 at the amino-terminus appeared possible despite the presence of a small and functionally important domain at the NS1's amino-terminus which can be distorted by such fusing. GFP-tagged NS1 viruses were rescued from DNA-launched molecular clones. The initially produced GFP-tagged NS1 virus was capable of only poor replication. Sequential passages of the virus in cell cultures resulted in the appearance of mutations in GFP, NS4A, NS4B and NS5. The mutations which change amino acid sequences of GFP, NS4A and NS5 have the adaptive effect on the replication of GFP-tagged NS1 viruses. The pattern of GFP-fluorescence indicates that the GFP-NS1 fusion protein is produced into the endoplasmic reticulum. The intracellular GFP-NS1 fusion protein colocalizes with dsRNA. The discovered forms of extracellular GFP-NS1 possibly include tetramers and hexamers.

Towards development of plasmacytoma cells-based expression systems utilizing alphavirus vectors: An NS0-VEE model.

Plasmacytoma (myeloma) cells have a large protein expression capacity, although their industrial use is confined to stable expression systems. Vectors derived from genomes of viruses from the genus Alphavirus allow obtaining of high yields of target proteins but their use is limited to transient expression. Little information has been published to date on attempts to combine the myeloma cells as hosts with alphaviruses as expression vectors. A plasmid construct which allows rescue of a model alphavirus Venezuelan equine encephalitis virus (VEE) upon transfection of a cell culture was created. Mutations in the capsid and nsP2 genes allow for less cytopathogenic propagation of the virus. A cDNA-copy of the genome was placed in a plasmid under the control of the CMV promoter for virus rescue following DNA transfection. Parameters for the virus rescue by electroporating of the infectious clone in murine myeloma cells (NS0) were optimized. The highest FFU counts (1.2 × 105 FFU per 10 ug DNA) were produced with 2 pulses (voltage 250 V, capacitance 960 u F) and the best electroporation buffer was selected from eight buffers. Self-sustained VEE infection was established in NS0 cultures with high titers (8 × 108 FFU/ml) of the virus, despite a fraction of infected cells dying during 5-days observation. Further development of the NS0-VEE expression system may require addressing of apoptosis induced by VEE.