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Human rhinovirus internal ribosome entry site element enhances transgene expression in transfected CHO-S cells.

ABSTRACT: Chinese hamster ovary (CHO) cells are mainly used for recombinant protein production. However, the unstable transgene expression and lower transgene copy numbers are the major issues need to be resolved. Here, eleven internal ribosome entry site (IRES) elements from viral and cellular IRES were evaluated for foreign gene expression in CHO-S cells. We constructed eleven fusing plasmids containing different IRES sequences downstream of the enhanced green fluorescent protein (EGFP) gene. EGFP expression was detected by flow cytometry and the transgene copy number was evaluated by quantitative PCR. The erythropoietin (EPO) protein was also used to assess the stronger IRES. The results showed that IRES from human rhinovirus (HRV) exhibited the highest EGFP expression level under transient and stable transfections. The EGFP expression level of vector with IRES from HRV was related to the gene copy number in stably transfected CHO-S cells. Moreover, IRES from HRV induced higher expression level of EPO compared with one mutant IRES from EMCV in transfected cells. In conclusion, IRES from HRV can function as a strong IRES element for stable expression in CHO-S cells, which could potentially guide more effective foreign gene expression in CHO-S cells.

SUBMITTER: Chai YR

PROVIDER: S-EPMC5923211 | biostudies-literature | 2018 Apr

REPOSITORIES: biostudies-literature

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Human rhinovirus internal ribosome entry site element enhances transgene expression in transfected CHO-S cells.

Chai Yu-Rong YR Ge Meng-Meng MM Wei Ting-Ting TT Jia Yan-Long YL Guo Xiao X Wang Tian-Yun TY

Scientific reports 20180427 1

Chinese hamster ovary (CHO) cells are mainly used for recombinant protein production. However, the unstable transgene expression and lower transgene copy numbers are the major issues need to be resolved. Here, eleven internal ribosome entry site (IRES) elements from viral and cellular IRES were evaluated for foreign gene expression in CHO-S cells. We constructed eleven fusing plasmids containing different IRES sequences downstream of the enhanced green fluorescent protein (EGFP) gene. EGFP expre ...[more]

PMID: 29703950

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Project description:BackgroundIn red blood cells, protein 4.1 (4.1R) is an 80 kDa protein that stabilizes the spectrin-actin network and anchors it to the plasma membrane through its FERM domain. While the expression pattern of 4.1R in mature red cells is relatively simple, a rather complex array of 4.1R protein isoforms varying in N-terminal extensions, internal sequences and subcellular locations has been identified in nucleated cells. Among these, 135 kDa and 80 kDa isoforms have different N-terminal extensions and are expressed either from AUG1- or AUG2-containing mRNAs, respectively. These two types of mRNAs, varying solely by presence/absence of 17 nucleotides (nt) which contain the AUG1 codon, are produced by alternative splicing of the 4.1R pre-mRNA. It is unknown whether the 699 nt region comprised between AUG1 and AUG2, kept as a 5' untranslated region in AUG2-containing mRNAs, plays a role on 4.1R mRNA translation.ResultsBy analyzing the in vitro expression of a panel of naturally occurring 4.1R cDNAs, we observed that all AUG1/AUG2-containing cDNAs gave rise to both long, 135 kDa, and short, 80 kDa, 4.1R isoforms. More importantly, similar results were also observed in cells transfected with this set of 4.1R cDNAs. Mutational studies indicated that the short isoforms were not proteolytic products of the long isoforms but products synthesized from AUG2. The presence of a cryptic promoter in the 4.1R cDNA sequence was also discounted. When a 583 nt sequence comprised between AUG1 and AUG2 was introduced into bicistronic vectors it directed protein expression from the second cistron. This was also the case when ribosome scanning was abolished by introduction of a stable hairpin at the 5' region of the first cistron. Deletion analysis of the 583 nt sequence indicated that nucleotides 170 to 368 are essential for expression of the second cistron. The polypyrimidine tract-binding protein bound to the 583 nt active sequence but not to an inactive 3'-fragment of 149 nucleotides.ConclusionOur study is the first demonstration of an internal ribosome entry site as a mechanism ensuring the production of 80 kDa isoforms of protein 4.1R. This mechanism might also account for the generation of 60 kDa isoforms of 4.1R from a downstream AUG3. Our results reveal an additional level of control to 4.1R gene expression pathways and will contribute to the understanding of the biology of proteins 4.1R and their homologues, comprising an ample family of proteins involved in cytoskeletal organization.

Project description:In recent years, hepatitis C virus (HCV)-related viruses were identified in several species, including dogs, horses, bats, and rodents. In addition, a novel virus of the genus Hepacivirus has been discovered in bovine samples and was termed bovine hepacivirus (BovHepV). Prediction of the BovHepV internal ribosome entry site (IRES) structure revealed strong similarities to the HCV IRES structure comprising domains II, IIIabcde, pseudoknot IIIf, and IV with the initiation codon AUG. Unlike HCV, only one microRNA-122 (miR-122) binding site could be identified in the BovHepV 5' nontranslated region. In this study, we analyzed the necessity of BovHepV IRES domains to initiate translation and investigated possible interactions between the IRES and core coding sequences by using a dual luciferase reporter assay. Our results suggest that such long-range interactions within the viral genome can affect IRES-driven translation. Moreover, the significance of a possible miR-122 binding to the BovHepV IRES was investigated. When analyzing translation in human Huh-7 cells with large amounts of endogenous miR-122, introduction of point mutations to the miR-122 binding site resulted in reduced translation efficiency. Similar results were observed in HeLa cells after substitution of miR-122. Nevertheless, the absence of pronounced effects in a bovine hepatocyte cell line expressing hardly any miR-122 as well suggests additional functions of this host factor in virus replication.IMPORTANCE Several members of the family Flaviviridae, including HCV, have adapted cap-independent translation strategies to overcome canonical eukaryotic translation pathways and use cis-acting RNA-elements, designated viral internal ribosome entry sites (IRES), to initiate translation. Although novel hepaciviruses have been identified in different animal species, only limited information is available on their biology on molecular level. Therefore, our aim was a fundamental analysis of BovHepV IRES functions. The findings which show that functional IRES elements are also crucial for BovHepV translation expand our knowledge on molecular mechanism of hepacivirus propagation. We also studied the possible effects of one major host factor implicated in HCV pathogenesis, miR-122. The results of mutational analyses suggested that miR-122 enhances virus translation mediated by BovHepV IRES.

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Human rhinovirus internal ribosome entry site element enhances transgene expression in transfected CHO-S cells.

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Human rhinovirus internal ribosome entry site element enhances transgene expression in transfected CHO-S cells.

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