Glycoprotein is enough for sindbis virus-derived DNA vector to express heterogenous genes

Abstract

To investigate the necessity and potential application of structural genes for expressing heterogenous genes from Sindbis virus-derived vector, the DNA-based expression vector pVaXJ was constructed by placing the recombinant genome of sindbis-like virus XJ-160 under the control of the human cytomegalovirus (CMV) promoter of the plasmid pVAX1, in which viral structural genes were replaced by a polylinker cassette to allow for insertion of heterologous genes. The defect helper plasmids pVaE or pVaC were developed by cloning the gene of glycoprotein E3E26KE1 or capsid protein of XJ-160 virus into pVAX1, respectively. The report gene cassette pVaXJ-EGFP or pV-Gluc expressing enhanced green fluorescence protein (EGFP) or Gaussia luciferase (G.luc) were constructed by cloning EGFP or G.luc gene into pVaXJ. EGFP or G.luc was expressed in the BHK-21 cells co-transfected with report gene cassettes and pVaE at levels that were comparable to those produced by report gene cassettes, pVaC and pVaE and were much higher than the levels produced by report gene cassette and pVaC, suggesting that glycoprotein is enough for Sindbis virus-derived DNA vector to express heterogenous genes in host cells. The method of gene expression from Sindbis virus-based DNA vector only co-transfected with envelop E gene increase the conveniency and the utility of alphavirus-based vector systems in general.

Research on basis of reverse genetics system of a Sindbis-like virus XJ-160

“…Compared with this method, there is no need for in vitro transcription and mRNA capping for DNA-based vectors. We have constructed XJ-160 virus-based DNA vector by placing its recombinant genome under the control of the human cytomegalovirus (CMV) promoter of the plasmid pVAX1 (Invitrogen, USA), in which viral structure genes were replaced by the sequence of multiple cloning site (MCS) to allow for insertion of heterologous genes [ 33 ]. And a packaging cell lines (PCLs) BHK-21 E+Capsid for XJ-160 virus was constructed by two times of selections with G418 and Hygromycin., which not only highly increased packaging efficiency of the replicon vector from XJ-160 virus, but also provided packaging function for the replicon vector from Semliki Forest virus [ 34 ].…”

Specific Nucleotide Changes in the Subgenomic Promoter Region Influence Infectivity of the Sindbis Virus

“…To further examine the relationship between the SP sequence and its function, reporter gene assays were performed as described previously [10]. Specific green fluorescence was observed in BHK-21 cells transfected with XJ-12 to XJ-15 virus, while no EGFP expression was observed following transfection with pBR-XJ11 (fig.…”