Role of transcription regulatory sequence in regulation of gene expression and replication of porcine reproductive and respiratory syndrome virus
© The Author(s) 2017
Received: 9 May 2017
Accepted: 31 July 2017
Published: 10 August 2017
In order to gain insight into the role of the transcription regulatory sequences (TRSs) in the regulation of gene expression and replication of porcine reproductive and respiratory syndrome virus (PRRSV), the enhanced green fluorescent protein (EGFP) gene, under the control of the different structural gene TRSs, was inserted between the N gene and 3′-UTR of the PRRSV genome and EGFP expression was analyzed for each TRS. TRSs of all the studied structural genes of PRRSV positively modulated EGFP expression at different levels. Among the TRSs analyzed, those of GP2, GP5, M, and N genes highly enhanced EGFP expression without altering replication of PRRSV. These data indicated that structural gene TRSs could be an extremely useful tool for foreign gene expression using PRRSV as a vector.
Introduction, methods, and results
Porcine reproductive and respiratory syndrome virus (PRRSV) is the causative agent of porcine reproductive and respiratory syndrome (PRRS), which leads to a highly contagious respiratory disease in nursery pigs and reproductive failure in sows . PRRS was first reported in 1987 in North America and has become pandemic within a few years. PRRS is still one of the most economically important diseases for the swine industry worldwide [2, 3]. PRRSV is a member of the genus Arterivius of the family Arteriviridae within the order Nidovirales. This enveloped virus bears a single-stranded, positive-sense RNA genome containing at least seven genes, encoding the replicase (ORF 1a and ORF 1b) and the structural proteins E, GP2 or GP2a, GP3, GP4, 5a, GP5, M, and N in the order 5′-ORF1-E-GP2-GP3-GP4-5a-GP5-M-N-3′ [4–6]. The structural proteins are expressed by a nested series of subgenomic (sg) RNAs, which are produced during viral transcription. The structure of the arterivirus and coronavirus sg mRNAs derives from the discontinuous step of minus-strand RNA synthesis, which is guided by conserved AU-rich transcription-regulating sequences (TRS) [7–9]. There are two key elements of TRSs that are present both at the 3′ end of the leader sequence (leader TRS) and at the 5′ end of each gene in the 3′-proximal region of the genome (body TRSs). The body TRS motifs are found preceding almost all structural genes, while a leader TRS is present at the 5′ end of the genome. Minus-strand RNA synthesis is guided by base-pairing between the genomic leader TRS and the copy of the body TRS present in the 3′ end of the nascent minus strand. Next, the nascent strands are extended with the complement of the genomic leader sequence, generating a nested set of minus-strand templates that can be directly copied into the sg mRNAs [10, 11]. Leader TRS is highly conserved among Arteriviridae. For example, in equine arteritis virus (EAV) the TRS contains the conserved hexanucleotide sequence UCAACU , highly related with those found in lactate dehydrogenase-elevating virus (LDV) (UAUAACC)  and in simian hemorrhagic fever virus (SHFV) (UUAACC) . In case of PRRSV, the leader TRS is also highly conserved and appears to be UUAACC regardless of the PRRSV genotype (type 1 or type 2, also called European and North American genotypes, respectively) [15, 16]. In contrast, different body TRSs have been shown to be diverse: [U/A/G][U/A/G][A/C][A/G][C/U]C among North American genotype viruses, and U[A/U/C][A/G][A/C]CC among European genotype viruses. Furthermore, the number of body TRSs and sites upstream of the start codon of each ORF vary in length and sequence [16–18]. Others and we have previously shown that the infectious clone of PRRSV can be engineered as an expression vector, in which a foreign gene could be expressed under the control of a body TRS as a separate transcription unit. These findings have confirmed the potential use of PRRSV as a vaccine vector against swine pathogens [19–23].
Previous studies and our own findings have suggested that the body TRS2 and TRS6 of PRRSV can play important roles in the regulation of viral transcription and translation [19–23]. However, there have been no studies, as far as we know, that have addressed the roles of the other body TRSs in gene expression regulation, replication, and transcription of PRRSV because the overlapping genes in PRRSV genome made mutation analysis challenging. It is well-accepted that leader TRS and body TRSs are the two key elements of PRRSV transcription. However, the roles and efficiency of base-pairing interaction between the leader TRS and different body TRSs on the expression of different structural proteins are not discussed in this paper due to the space limitation. Nonetheless, whether the distance between TRS and downstream gene governs the efficiency of the transcription and (possibly) translation is the objective of our further research. Therefore, we report here, for the first time, only the data that allow comparison of the effects of body TRSs on the expression of a foreign gene. We generated a series of recombinant HP-PRRSVs expressing EGFP gene driven by the six individual body TRSs of each HP-PRRSV structural genes, respectively. Each transcriptional unit, including the individual body TRS and EGFP gene, was inserted between the N protein and 3′-UTR in a full-length cDNA infectious clone of HP-PRRSV/SD16 strain. Importantly, all six recombinant HP-PRRSVs showed similar patterns of growth rate and maximum titers in comparison with the parental virus. These data indicated that the site between N gene and 3′-UTR can tolerate the addition of a foreign gene without reduction of the level of the viral replication.
Insertion of an additional transcriptional unit into the virus genome might affect the efficient incorporation of structural proteins into virions [27–29]. In this study, six recombinant HP-PRRSVs were subjected to western blot by measuring the ratios of the N protein and EGFP protein, respectively. The present results and our earlier data showed that insertion of the EGFP transcriptional units between N gene and 3′-UTR did not affect the incorporation of viral protein into the virions by measuring the ratios of the N protein and EGFP protein [20–22]. Moreover, six recombinant HP-PRRSVs were subjected to Northern blot analysis by measuring the ratios of the EGFP mRNA. Overall, the body TRSs of GP2, GP5, M and N genes produced the higher level of EGFP expression when this reporter gene is cloned upstream of the N gene and 3′-UTR, suggesting that these body TRSs at this position would assure effective regulation of the gene of interest. It is possible that HP-PRRSV has evolved to have unique body TRSs for each structural gene, and they are most effective in regulating the expression of the corresponding structural genes at their original positions. In summary, we have evaluated the role of six PRRSV body TRSs in expression of a foreign gene by using HP-PRRSV reverse genetics system. We showed that HP-PRRSV body TRSs have the ability to regulate gene expression, replication, and transcription of the foreign gene at different levels. Moreover, our results and the previous findings all indicate that the PRRSV body TRSs could be a useful tool for controlling foreign gene expression. Compared with the expression levels of six different recombinant PRRSVs expressing EGFP gene, body TRSs of GP2, GP5, M and N genes have shown relatively higher levels of EGFP expression without altering the viral replication. Therefore, our results provide new clues useful for the rational design of next generation effective PRRSV vaccine vectors.
The authors declare that they have no competing interests.
CBW, LA, and YMZ carried out all the experiments (except for northern blot) and drafted the manuscript. HM carried out the plasmid construction, western blot analysis, and Additional file 1: Table S1. YJG assisted with the sequence of the HP-PRRSV genome and the analysis of virus growth kinetics. HG carried out the northern blot analysis. KKG assisted with the cell culture and quantification analysis of EGFP fluorescence. FA, IS, and LE engineered a BAC backbone to clone EGFP-tagged HP-PRRSV cDNA, provided comments, and interpreted the data. All authors read and approved the final manuscript.
The authors thank M.Sc. Mackenzie Waltke for proofreading the manuscript.
This study was funded by grants to Chengbao Wang from the Natural Science Basic Research Plan in Shaanxi Province of China (Program No. 2016JQ3010), project funded by China Postdoctoral Science Foundation (2017M610659), the State Key Laboratory of Veterinary Etiological Biology, Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences (SKLVEB2016KFKT014) and the National Natural Science Foundation of China (31302103), the European Community`s Seventh Frame-work Programme (PoRRSCon FP7-KBBE-2009-3-245141), and the Ministry of Science and Innovation of Spain (MCINN) (BIO2010-16075); by the start-up grants from the Université de Montréal to Levon Abrahamyan.
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