Glycoprotein is enough for sindbis virus-derived DNA vector to express heterogenous genes
© Zhu et al; licensee BioMed Central Ltd. 2011
Received: 14 May 2011
Accepted: 10 July 2011
Published: 10 July 2011
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.
The concept that alphaviruses can be developed as expression vectors was first established by Xiong et al. . Since then, Sindbis virus (SINV), a member of alphavirus, has been developed as vectors for the expression of heterologous gene [2–4], gene therapy and vaccine application [5–8]. Sindbis virus genome is a single strand of positive-sense RNA of approximately 12 kb which is capped at the 5' terminus and polyadenylated at the 3' terminus . The 5' two-thirds of this RNA encode the nonstructural proteins (nsP1 through 4). The 3' one-third is initially translated as a polyprotein (NH2-C-E3-E2-6K-E1-COOH) that is processed co- and posttranslationally to produce the structural proteins (SPs) (capsid, El and E2). In infected cells, the virion structural proteins are translated from a subgenomic mRNA (26S RNA) and produced by transcription of genome-length complementary (minus) strand from a highly active subgenomic promoter. Since the nonstructural protein genes and the structural protein genes are expressed from two different mRNAs, they may be expressed independently of one another . Thus, the high levels of expression of heterologous products are achieved when the viral structural genes are replaced by the heterologous coding sequences. Such recombinant vectors are self-replicating (replicons) and can be introduced into cells as naked RNA or plasmid DNA.
As a Sindbis-like virus, XJ-160 virus (GenBank No. AF103728) was isolated from a pooled sample of Anopheles mosquitoes collected in Xinjiang, China, in 1990 . Recombinant plasmid pBR-XJ160 is an infectious full-length cDNA clone of XJ-160 virus, from which rescued virus BR-XJ160 can be obtained by transcription in vitro and transfection. The BR-XJ160 virus raised in BHK-21 cells was indistinguishable from the XJ-160 virus in its biological properties, including its plaque morphology, growth kinetics and suckling mouse neurovirulence . On basis of pBR-XJ160, the effects of the substitutions of 169 Lys and 173Thr in nonstructural protein 1 (nsP1) as well as nsP2-726 Pro on the infectivity and pathogenesis of Sindbis virus have been investigated [13, 14]. We have also confirmed the essential role of E2 glycoprotein, especially the domain of 145-150 aa, in Sindbis virus infection through the interaction with cellular heparan sulfate [15, 16]. In addition, we have developed XJ-160 virus-based RNA vector system, including replicon vector pBRepXJ, a defective helper (DH) plasmid (pBR-H) and the packaging cell lines (PCLs) [17, 18].
DNA-based vector derived from alphavirus can be initiated by transfection of plasmid DNA [4, 19]. In this strategy, full-length 5'-capped RNAs are transcribed in the nucleus using a polymerase II promoter, and transported to the cytoplasm. Then autocatalytic amplification of the vector, proceeds according to the Sindbis virus replication cycle and results in expression of the heterologous gene. Thus, as a double-stranded DNA, DNA-based vector can be directly transfected into the host cells dispensing with the process of in vitro transcription and mRNA capping, which is required for the transfection of previously described Sindbis virus-derived RNA vectors. In this study, we have constructed DNA-based vector pVaXJ from XJ-160 virus, which could efficiently express reporter genes in the transfected cells. The XJ-160 virus-derived DNA vectors described here increase the utility of alphavirus-based vector systems in general and also provide a vector with broad potential applications for genetic immunization.
The SINV-derived replicons lacking viral structural genes are suicide vectors, incapable of packaging into infectious particles and causing productive infection. To obtain viable particles, vector replicon can be packaged into infectious particles by cotransfection of cultured cells with a defective helper vector, which provides the virion structural proteins in trans . Two principal strategies are being performed for construction of DH vector: (i) engineering recombinant plasmid that expresses viral subgenomic RNAs (NH2-C-E3-E2-6K-E1-COOH) and (ii) developing two DH vectors, one that expresses the capsid protein and a second that expresses the viral glycoproteins. High levels of expression of heterologous genes were achieved by using both of two methods. However, it is not clear which of the two SPs or both is necessary for SINV-derived vector to express heterologous coding sequences. In this investigation, based on the XJ-160 virus-derived DNA vector, our findings clearly prove 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 increases the conveniency and the utility of alphavirus-based vector systems in general.
This work was supported by the grants from the National Natural Science Foundation of China (No. 30970160), the Development Grant of State Key Laboratory for Infectious Disease Prevention and Control (2011SKLID205), the Major Science and Technology Project for Infectious Disease (No. 2008ZX10004-001; 2009ZX10004-705).
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