Expression of Two Foreign Genes by a Newcastle Disease Virus Vector From the Optimal Insertion Sites through a Combination of the ITU and IRES-Dependent Expression Approaches.

Lei He,Qingzhong Yu,Zhenyu Zhang

doi:10.3389/fmicb.2020.00769

Abstract

Many Newcastle disease virus (NDV) strains have been developed as vectors to express a foreign gene (FG) for vaccine and cancer therapy purposes. The non-coding region between the phosphoprotein (P) and matrix protein (M) genes and the non-coding region behind the NP gene open reading frame (ORF) in the NDV genome have been identified as the optimal insertion sites for efficient FG expression through the independent transcription unit (ITU) and the internal ribosomal entry site (IRES) dependent expression approaches, respectively. To date, however, the majority of these NDV vectors express only a single or two FGs from suboptimal insertion sites in the NDV genome, obtaining various levels of FG expression. To improve the FG expression, we generated NDV LaSota vaccine strain-based recombinant viruses expressing two FGs, GFP, and RFP, from the identified optimal insertion sites through a combination of the ITU and IRES-dependent approaches. Biological assessments of the recombinant viruses indicated that the recombinants expressing two FGs were slightly attenuated with approximately one order of magnitude lower in virus titers when compared to the viruses containing a single FG. The FG expression efficiencies from the two-FG viruses were also lower than those from the single-FG viruses. However, the expression of two FGs from the optimal insertion sites was significantly (p < 0.05) higher than those from the suboptimal insertion sites. The expressions of FGs as monocistronic ITU were approximately 4-fold more efficient than those expressed by the bicistronic IRES-dependent approach. These results suggest that the NDV LaSota vector could efficiently express two FGs from the identified optimal insertions sites. The ITU strategy could be used for “vectoring” FGs in circumstances where high expression of gene products (e.g., antigens) is warranted, whereas, the IRES-dependent tactic might be useful when lower amounts of IRES-directed FG products are needed.

Highlights

Avian orthoavulavirus 1, commonly known as Newcastle disease virus (NDV, used hereafter), is a member of the genus orthoavulavirus within the subfamily Avulavirinae of the family Paramyxoviridae1 (Amarasinghe et al, 2019)
After co-transfection of the full-length cDNA clones and supporting plasmids into HEp-2 cells and subsequent propagation in SPF chicken embryonated eggs, the LaSota strainbased recombinant viruses containing either a single foreign gene (FG) (GFP or red fluorescent protein (RFP)) or two FGs (GFP and RFP) in the optimal insertion sites were successfully generated. These rescued recombinant viruses were designated as rLS-I-RFP-green fluorescent protein (GFP), rLS-I-GFP-RFP, rLSI-GFP, and rLS-RFP, respectively
The rLS-I-RFP-GFP virus expressed significantly higher levels of GFP (p = 0.027) and RFP (p = 0.007) than the rLS/internal ribosomal entry site (IRES)-RFP/GFP, in which the I-RFP was inserted in the F gene as a 2nd open reading frame (ORF), and the GFP gene was inserted between the F and HN genes in the NDV genome (Hu et al, 2018). These results demonstrated that the independent transcription unit (ITU) expression approach was more efficient than the IRES-dependent method for the FG expression

Summary

Introduction

Avian orthoavulavirus 1, commonly known as Newcastle disease virus (NDV, used hereafter), is a member of the genus orthoavulavirus within the subfamily Avulavirinae of the family Paramyxoviridae (Amarasinghe et al, 2019). NDV contains a single-stranded, non-segmented, negativesense RNA of approximately 15.2 kb in length that consists of six genes flanked by a 3 Leader and 5 Trailer in the order 3 -nucleocapsid protein (NP)-phosphoprotein (P)-matrix protein (M)-fusion protein (F)-hemagglutininneuraminidase (HN)-large polymerase (L)-5 (de Leeuw and Peeters, 1999; Peeters et al, 1999; Samal, 2011). Unlike positive-stranded RNA viruses, the naked genomic RNA of NDV is not infectious. It must be encapsidated with the NP protein and associated with the P and L proteins, forming a ribonucleocapsid, to act as a template for RNA transcription and replication (Peeters et al, 1999)

Methods

Results

Conclusion