Role of untranslated regions in regulation of gene expression, replication, and pathogenicity of Newcastle disease virus expressing green fluorescent protein

Abstract

To gain insight into the role of untranslated regions (UTRs) in regulation of foreign gene expression, replication, and pathogenicity of Newcastle disease virus (NDV), a green fluorescent protein (GFP) gene flanked by 5' and 3' UTRs of each NDV gene was individually expressed by recombinant NDVs. UTRs of each gene modulated GFP expression positively or negatively. In particular, UTRs of the M and F genes enhanced levels of GFP expression at the junction of the P and M genes without altering replication of NDV, suggesting that UTRs could be used for enhanced expression of a foreign gene by NDV.

FIG. 1.

Generation and in vitro replication of rBCs containing GFP genes flanked by UTRs of the six NDV genes. GFP gene was flanked by a conserved gene start (GS) and 5′ UTR of individual NDV gene upstream and 3′ UTR of corresponding NDV gene and a conserved gene end (GE) downstream. Each transcriptional unit was inserted between the P and M genes (A) or between the HN and L genes (B) in the NDV genome. In vitro replication of the recovered viruses was determined in virus-infected DF-1 cells at an MOI of 0.01. The viral titers were determined by plaque assay.

FIG. 1.

Generation and in vitro replication of rBCs containing GFP genes flanked by UTRs of the six NDV genes. GFP gene was flanked by a conserved gene start (GS) and 5′ UTR of individual NDV gene upstream and 3′ UTR of corresponding NDV gene and a conserved gene end (GE) downstream. Each transcriptional unit was inserted between the P and M genes (A) or between the HN and L genes (B) in the NDV genome. In vitro replication of the recovered viruses was determined in virus-infected DF-1 cells at an MOI of 0.01. The viral titers were determined by plaque assay.

FIG. 2.

Effect of the NDV UTRs on GFP transcription and translation in DF-1 cells infected with rBCs expressing GFP. (A) The levels of GFP mRNA transcription were determined by quantitative RT-PCR (qRT-PCR). DF-1 cells in 6-well plates were infected with rBCs at an MOI of 0.1. At 20 h postinfection, total RNAs were isolated using Trizol, treated with DNase to remove residual genomic DNA. The RNA (1.5 μg each) was reverse transcribed using SuperScript II. The prepared cDNA was subjected to qRT-PCR. Primers used for the GFP gene were the forward primer 5′-CGA CGG CAA CTA CAA GAC-3′ and the reverse primer 5′-TAG TTG TAC TCC AGC TTG TGC-3′. For internal normalization, the β-actin gene was amplified using the forward primer 5′-GAG AAA TTG TGC GTG ACA TCA-3′ and the reverse primer 5′-CCT GAA CCT CTC ATT GCC A-3′. (B) For Western blot analysis of GFP production, total proteins were collected from virus-infected DF-1 cells at 24 h postinfection, electrophoresed, transferred to a nitrocellulose membrane, and immunostained using a monoclonal antibody against GFP protein or the NDV M protein as a loading control. (C) GFP expression in virus-infected DF-1 cells at an MOI of 0.1 was quantitated by fluorescence-activated cell sorting analysis. At 24 h and 32 h postinfection, the cells were harvested, fixed with 4% paraformaldehyde, and analyzed by flow cytometry. Mean fluorescence intensity and percent GFP-positive cells were analyzed using Flowjo software. The analyses were independently performed three times.

FIG. 3.

Incorporation of structural proteins of rBCs expressing GFP into virions. (A) Viruses were harvested from allantoic fluids and purified through a 30% sucrose cushion. The viral proteins were separated on an SDS-PAGE gel (8%) and stained with Coomassie brilliant blue. (B and C) Ratios of the HN protein (B) or the L protein (C) to the M protein levels from rBC and rBCs expressing GFP were quantified. This assay was performed by determining the ratios of the P protein to M protein. Since similar levels of the P and M proteins were present among the different viruses, the levels of HN protein or L protein incorporation into the virus particles was measured by determining their ratios to the M protein. This analysis was independently performed three times.