The effect of 5' and 3' non-translated regions on the expression of a transgene from a Newcastle disease virus vector

Abstract

Newcastle disease virus (NDV) is an avian virus and a promising vector for the development of vaccines for veterinary and human use. The optimal vaccine vector performance requires a stable high-level expression of a transgene. The foreign genes are usually incorporated in the genome of NDV as individual transcription units, whose transcription and subsequent translation of the mRNA are regulated by the 5' and 3' untranslated regions (UTRs) flanking the open reading frame of the transgene. Here, we investigated if the UTRs derived from the cognate NDV genes would increase the expression of a model protective antigene from an NDV vector. Our results show that in chicken DF1 cells, none of the UTRs tested significantly outperformed generic short sequences flanking the transgene, while in human HeLa cells, UTRs derived from the M gene of NDV statistically significantly increased the expression of the transgene. The UTRs derived from the HN gene significantly downregulated the transgene expression in both cell cultures. Further experiments demonstrated that NDV UTRs differently affect the mRNA abundance and translation efficacy. While both M and HN UTRs decreased the level of the transgene mRNA in infected cells compared to the mRNA flanked by generic UTRs, M, and particularly, HN UTRs strongly increased the mRNA translation efficacy. The major determinants of translation enhancement are localized in the 5'UTR of HN. Thus, our data reveal a direct role of NDV UTRs in translational regulation, and inform future optimization of NDV vectors for vaccine and therapeutic use.

Keywords: Newcastle disease virus vector; Transgene expression; UTR translation control; Vectored vaccines.

Fig. 1

Construction of the NDV vectors expressing HA protein under the control of cognate UTRs. A. A scheme of the NDV genome with the insert position and a table showing the design of the HA inserts flanked by the control and NDV-derived UTRs (sequences are shown in positive mRNA polarity). The HA initiation codon is in red, the Kozak sequence is underlined, the stop codon is in blue. NDV gene start and gene end sequences in the inserts are highlighted in green and purple, respectively. The PmeI site in the 3′UTR is in underlined italics. B. The growth kinetics of the recombinant viruses in DF1 cells infected with an MOI of 0.01. The medium aliquots were collected at the indicated time points and the virus titer was determined by an immunofluorescence assay.

Fig. 2

M-derived UTRs increase the expression of the transgene in human cells. DF1 or HeLa cells were infected with recombinant NDVs (or NDV control without the insert) at an MOI of 10 and processed for western blot at 18 h p.i. The expression of HA was normalized to that of the NDV nuclear protein (NP), and the ratio in the control sample infected with the NDV expressing HA under generic UTRs was set as 100 % for relative expression calculation. The experiments in each cell culture were performed at least four times, representative images are shown. Actin represents a loading control.

Fig. 3

Cognate UTRs do not increase the amount of the transgene mRNA. DF1 or HeLa cells were infected with recombinant NDVs (or control NDV without the insert) at an MOI of 10 and the poly-A-containing RNA fraction was isolated at 18 hp.i. A. The analysis of amplicons used for qPCR quantitation of HA (recombinant insert) and NP (normalization control) mRNAs isolated from HeLa cells infected with the NDV expressing HA under generic UTRs. Actin amplicon was used to evaluate the quality of polyA RNA preparations. B. The amount of HA mRNAs expressed under the control of M and HN UTRs relative to that expressed under the control of generic UTRs.

Fig. 4

M and HN UTRs increase the mRNA translation. A. A scheme of the templates used for the generation of capped and oligo-adenylated mRNAs coding for the firefly luciferase flanked by the specific UTRs is shown on top. DF1 or HeLa cells were transfected with in vitro synthesized purified capped and oligo-adenylated mRNAs coding for Firefly luciferase flanked by the indicated UTRs, the luciferase expression was determined at 18 h post-transfection. The results are normalized to the luciferase signal of the control construct with the same UTRs as used in the HA control vector. B. The translational efficacies of the mRNAs with the luciferase ORF flanked by either 5′ or 3′ HN-derived UTRs were compared to those of the control mRNAs with both HN-specific or generic UTRs (shown on top) in the same experimental conditions as in A.

Fig. 5

The M UTRs do not significantly affect the transgene stability. A. The immunofluorescence assay was used for the quantitation of individual infectious NDV virions expressing or non-expressing the HA transgene. HeLa cell monolayers were infected with a low MOI of recombinant NDVs in the absence of exogenous proteases to limit the virus spread beyond the originally infected cells. The cells were fixed and processed for immunofluorescence with anti-NDV and anti-HA antibodies at 18 h p.i. The individual antigen staining is shown in the NDV and HA panels, and the composed image in the NDV+HA+Hoechst panel, scale bar is 20 µm. B. The analysis of HA expression by recombinant NDVs coding for the HA gene under the control of generic (control) or M UTRs from passages 1 and 10 in embryonated chicken eggs using the assay described in A.