Expression of RNA virus proteins by RNA polymerase II dependent expression plasmids is hindered at multiple steps
© Ternette et al; licensee BioMed Central Ltd. 2007
Received: 08 March 2007
Accepted: 05 June 2007
Published: 05 June 2007
Proteins of human and animal viruses are frequently expressed from RNA polymerase II dependent expression cassettes to study protein function and to develop gene-based vaccines. Initial attempts to express the G protein of vesicular stomatitis virus (VSV) and the F protein of respiratory syncytial virus (RSV) by eukaryotic promoters revealed restrictions at several steps of gene expression.
Insertion of an intron flanked by exonic sequences 5'-terminal to the open reading frames (ORF) of VSV-G and RSV-F led to detectable cytoplasmic mRNA levels of both genes. While the exonic sequences were sufficient to stabilise the VSV-G mRNA, cytoplasmic mRNA levels of RSV-F were dependent on the presence of a functional intron. Cytoplasmic VSV-G mRNA levels led to readily detectable levels of VSV-G protein, whereas RSV-F protein expression remained undetectable. However, RSV-F expression was observed after mutating two of four consensus sites for polyadenylation present in the RSV-F ORF. Expression levels could be further enhanced by codon optimisation.
Insufficient cytoplasmic mRNA levels and premature polyadenylation prevent expression of RSV-F by RNA polymerase II dependent expression plasmids. Since RSV replicates in the cytoplasm, the presence of premature polyadenylation sites and elements leading to nuclear instability should not interfere with RSV-F expression during virus replication. The molecular mechanisms responsible for the destabilisation of the RSV-F and VSV-G mRNAs and the different requirements for their rescue by insertion of an intron remain to be defined.
Eukaryotic cells differ from prokaryotic cells by increased compartmentalisation of the intracellular environment to facilitate complex enzymatic reactions required for efficient protein expression and modification, cell metabolism and/or cell division. Adaptation to the host cell and particularly to its expression machinery is the key requirement for the replication of any virus. Several RNA viruses only replicate in the cytoplasm of their eukaryotic host cell. These viruses possess their own transcription machinery involving a viral RNA-dependent RNA polymerase which allows cytoplasmic mRNA synthesis from the viral genomic RNA. Therefore, these viruses are not adapted to the complex nuclear milieu of the eukaryotic host cell. Inefficient expression of genes from RNA viruses by RNA polymerase II (Pol II) dependent cellular promoters might be explained by lack of critical elements required for pre-mRNA stabilisation, mRNA processing and/or nuclear export. However, problems that occur during Pol II dependent expression of RNA virus proteins can be overcome by changing the codons of viral genes to those most frequently used by the genes of the host cells [1–3]. Since the codon optimised genes should also lack defined RNA elements directing mRNA processing and/or transport, the nucleotide sequence or composition of the viral wild type sequences might actually be inhibitory in nature or be targeted by innate viral defence mechanisms.
The precise reason why genes of RNA viruses are inefficiently expressed is still poorly understood. For lentiviruses, which were studied in more detail, expression of viral structural genes is regulated at the level of nuclear export and these viruses have a regulatory protein (Rev) involved in shuttling the mRNA for the structural proteins from the nucleus to the cytoplasm . Retention of these lentiviral mRNAs in the nucleus has been attributed to cis-repressive sequences or regions of instability but these sequences could not be narrowed down to well-defined nucleotide motifs. The unusual low GC content has also been reported to be responsible for the nuclear instability of lentiviral structural mRNAs . Whether similar mechanisms govern the fate of recombinant Pol II mRNAs of viruses replicating in the cytoplasm is unclear.
Instead of using cellular RNA polymerases for expression of viral proteins in eukaryotic cells, cytoplasmic expression systems based on RNA polymerases from vaccinia viruses, alpha-viruses or phages have been developed. The latter are also used for generation of recombinant vesicular stomatitis virus (VSV) [6, 7] and respiratory syncytial virus (RSV)  by reverse genetics. These systems are based on cytoplasmic transcription of viral cDNA by coexpression of phage T7 RNA polymerase. Recovery of infectious viruses was achieved by cotransfection of T7 RNA polymerase dependent expression plasmids for full-length antigenomic RNA and viral helper proteins which are necessary and sufficient for both RNA-replication and transcription. Expression of these viral helper proteins and/or the antigenomic RNA transcripts by eukaryotic promoters might facilitate and improve strategies for production of such recombinant viruses.
Additionally, the lack of eukaryotic expression systems not depending on coexpressed cytoplasmic polymerases hampered DNA vaccine development for several RNA viruses. This is a particular problem for the development of RSV vaccines, since immunisation with whole inactivated virus particles led to enhancement of RSV disease in children not protected from RSV infection [9, 10]. An aberrant T-helper cell type 2 response to the G protein of RSV and excessive CD4+ and CD8+ T cell responses to the F protein of RSV might be responsible for the enhanced airway inflammation underlying the detrimental effect of vaccination . Expression of a single viral protein by a DNA vaccine triggering T-helper cell type 1 responses might overcome vaccine-induced enhancement of RSV disease.
The potential of DNA vaccines and techniques used for reverse genetics has sparked our interest to better understand the requirements for expression of heterologous genes not adapted to the nuclear environment. Using the open reading frames of the G protein of VSV and the F protein of RSV as representatives of the rhabdovirus and paramyxovirus family, respectively, we analysed expression efficiency on mRNA and protein levels. We also attempted to rescue expression of these viral ORF by more subtle changes than codon optimisation to get hints on mechanisms responsible for inefficient expression of these viral genes.
Expression of the VSV-G protein can be rescued by insertion of the CMV-IE 5'-untranslated region independent of splicing
Inserting the first intron of CMV-IE gene including exonic flanking regions restored VSV-G expression from the wild type ORF to levels comparable to those obtained by the codon optimised expression plasmid. Despite a lower transfection efficiency, as evident from the Northern blot analysis (Fig. 1B), VSV-G mRNA expression was clearly detectable (pIGwt in Fig. 1B). Protein expression levels were comparable to those obtained with the codon optimised expression plasmid (pIGwt vs pGsyn in Fig. 1C, left panel). However, splicing was not required for this rescue, since a DNA expression plasmid, in which the CMV intron had been deleted by fusing the splice sites and retaining the exonic sequences also led to efficient expression of the protein (compare pIGwt to pIΔIGwt in Fig. 1C, right panel). Thus, correctly fused exons were sufficient to enhance VSV-G expression levels.
Further deletion analyses revealed that the first 106 nucleotides of the 5'-exon are mediating most of the effect (data not shown).
Expression of the RSV-F mRNA is dependent on splicing
Pol II mediated expression of the wild type RSV-F ORF results in premature polyadenylation
Undetectable levels of RSV-F protein in the presence of cytoplasmic RSV-F mRNA suggested an additional block at the translational level. We noticed that the mRNA species detected in the Northern blot analysis (Fig. 2B) migrated faster than the viral RSV-F mRNA, although they should be slightly larger due to the extended 5'- and 3'-UTR.
However, inspection of the RSV-F sequence revealed four potential polyadenylation consensus signals (AATAAA)  within the coding region (Fig. 3A). Using a PCR approach with an antisense primer anchored at the poly(A) tail (Oligo(dT)Add-a, Fig. 3A) and a pcDNA3.1+ specific sense primer at the 5'-UTR of the transcript (5' UTR-s, Fig. 3A), the entire mRNA transcript was reverse transcribed and amplified. Size and sequence analysis revealed that the second consensus poly(A) signal was used in 9 of 10 clones analysed (Fig. 3C, D) resulting in a mRNA with an RSV-F ORF truncated at position 1295. Since the absence of a stop codon has been shown to lead to degradation of such prematurely terminated proteins by cellular quality control pathways [15, 16], this might explain the absence of detectable levels of a truncated protein.
Deletion of the poly(A) consensus signal rescues RSV-F expression
Virus infection does not enhance Pol II dependent RSV-F expression
Chimeric ORF of RSV-F revealed strong dependency of protein expression on codon usage
The results demonstrate striking differences in the requirements for expression of genes of cytoplasmic RNA viruses by DNA expression plasmids. The use of codon optimised expression plasmids allowed exclusion of the possibility that protein instabilities or degradation is responsible for undetectable levels of the respective viral proteins. Insertion of intron A of the CMV-IE gene resulted in mRNA levels comparable to those obtained by codon optimised expression plasmids in case of VSV-G or by those obtained in natural infection for RSV. This was not surprising since splicing has been repeatedly shown to enhance expression levels [17, 18]. However, in case of VSV-G splicing was not the critical factor, since simple addition of the exonic sequences of the CMV-IE gene were sufficient to rescue VSV-G expression even at the protein level. This suggests that the exonic sequences somehow stabilise and/or contribute to nuclear export of the VSV-G mRNA. The same exonic sequences were not sufficient to rescue expression of RSV-F mRNA. Including the intron, however, allowed cytoplasmic expression of the RSV-F mRNA. Similar findings have been reported for mRNAs of the Simian Virus 40, where intronless RNA was retained and degraded in the nucleus, while the same transcript generated by splicing reached the cytoplasm . For other genes this has been attributed to recognition of the pre-mRNA by the exon junction complex (EJC), which has been found to be linked to nuclear export by direct binding to the heterodimer transport protein Tap-Nxt [20–22]. It is therefore likely that similar mechanisms are responsible for the rescue of cytoplasmic RSV-F mRNA levels by the first intron of the CMV-IE gene.
Another block to RSV-F protein synthesis was found to be premature polyadenylation. The second of the four consensus sites initiated the predominant premature polyadenylation of the RSV-F mRNA. The lack of a stop codon preventing an accurate translation termination results in synthesis of defective ribosomal products (DRiPs) which enter a pathway of proteasomal or other cytosolic decay mechanisms coupled to MHC class I presentation [23, 24]. This might explain why DNA vaccines encoding the wild type RSV-F ORF induced immune responses, although expression of full length protein was probably not very efficient [25–29]. The small amount of protein which could be detected despite that (Fig. 4D), might be the result of a rare skipping of poly(A) signals.
Mutagenesis of the recognised consensus sequence for polyadenylation led to usage of the last downstream consensus signal. However, even after mutagenising both used poly(A) signals, protein expression was around 50-fold lower than expression from codon optimised plasmids despite substantial amounts of correctly processed RSV-F mRNA in the cytoplasm. The fact that expression of other viral proteins by superinfection of the transfected cell with RSV did not increase RSV-F expression from the plasmid suggests that poor expression levels are not the consequence of a repressive RSV-F specific regulatory RNA element that can be overcome by an activating second viral factor. It rather seems that reducing the AU content of the RSV-F mRNA in general contributes to increased expression levels. Consistently, replacement of a third of the wild type nucleotide sequence by the codon optimised fragment resulted in intermediate expression levels and not in an all or none phenomenon. In summary, these findings indicate that premature polyadenylation is the major mechanism responsible for failure of protein expression from the original RSV-F wild type construct and that codon optimisation can further enhance expression of RSV-F.
Polyadenylation consensus signals were not only found in a single RSV strain but could be detected in all RSV-F sequences deposited in GenBank database. Other members of the paramyxovirus family, such as measles virus and parainfluenzaviruses, also harbour such consensus sites for polyadenylation. Since these consensus poly(A) signals are not expected to be of any functional relevance for the viruses due to their cytoplasmic replication, they are probably just the accidental result of the unusual high AU content of the viral genomes. The latter fact also leads to the presence of potential U-rich downstream elements that are also required for polyadenylation [30, 31].
Expression of genes of RNA viruses by Pol II dependent expression plasmids can be impaired at several steps. For VSV-G, a splicing-independent mechanism can lead to stabilisation of Pol II transcribed VSV-G mRNA, while splicing seems to be necessary for Pol II dependent expression of RSV-F mRNA. Premature polyadenylation is a second major block to expression of RSV-F protein from the wild type ORF. All these restrictions were efficiently overcome by codon optimisation providing a straightforward approach for the generation of Pol II dependent expression cassettes needed for development and production of antiviral vaccines and recombinant RNA viruses.
Viruses and infection
RSV based on the A2 long strain was kindly provided by B. Schweiger from the Robert Koch Institute, Berlin, Germany. GFP expressing recombinant rgRSV  was obtained by M. E. Peeples and P. L. Collins, Maryland, USA. RSV was passaged on Hep2 cells and stored at -80°C. Hep2 or 293T cells were infected at an MOI of 10 by adding RSV containing cell supernatant. Two hours following addition of the virus, supernatants were removed and cells were supplied with DMEM medium containing 0,5% FCS and 100 μg/ml penicillin G and streptomycin sulphate.
The ORF of VSV-G was amplified from pHIT-G  and cloned into pcDNA3.1 (Invitrogen, Karlsruhe, Germany) via BamHI/EcoRI (pGwt, kindly provided by R. Wagner, Regensburg). A Kozak consensus sequence (gccgccacc)  was inserted directly upstream of the start codon. For codon optimisation, viral codons were replaced by those most frequently used in human cells . Synthesis of the optimised VSV-G encoding nucleotide sequence was performed by Geneart (Regensburg, Germany) based on the amino acid sequence of GenBank database entry J02428 (pGsyn). Amino acid 57 and 96 were mutated from L to I and H to Q, respectively, to match the amino acid sequence of the wild type VSV-G precisely. The CMV-IE intron A was added into both vectors by inserting the SnaBI/HindIII fragment (GenBank database entry BK000394, nt 174903–173696) of the VSV-G expression plasmid pHIT-G resulting in pIGwt and pIGsyn.
Deletion of the 828 nt intron and exact fusion of the exon boundaries was achieved by replacement of a SacII/HindIII fragment by the annealed oligonucleotides (Sigma, Munich, Germany) Is (5'-gg ccgggaacggtgcattggaacgcggattccccgtgccaagagtgactcaccgtccttgacacga) and Ia (5'-agctt cgtgtcaaggacggtgagtcactcttggcacggggaatccgcgttccaatgcaccgttcccggccgc) resulting in pIΔIGwt. Nucleotides involved in generation of restriction sites are printed bold. VSV-G expression analyses included studies on the functional incorporation of VSV-G into lentiviral vector particles. Therefore, lentiviral gag-pol expression plasmids Hgpsyn  for HIV-1 gag-pol and SgpΔ2  for SIV gag-pol were cotransfected with VSV-G expression plasmids and the lentiviral vector construct VICGΔBH. VICGΔBH is based on the lentiviral vector VIGΔBH , containing a murine leukemia virus promoter driven GFP expression cassette. This cassette was excised via BglII/XhoI and replaced by the BamHI/XhoI fragment of HIV-CS-CG  containing a CMV-GFP expression cassette.
For the construction of the RSV-F expression plasmids, viral RNA was isolated from RSV containing cell supernatants using the QIAamp® viral RNA Mini Kit. After reverse transcription (ThermoScript™ RT-PCR System, Invitrogen) the RSV-F cDNA was amplified by PCR (Primers (Sigma): sense: 5'-gatccaagctt ccaccatggagttgccaatcctcaaa; antisense: 5'-tcgacctcgag ttagttactaaatgcaatattatttatacc) using the Platinum® Taq DNA-polymerase (Invitrogen). The 1.7 kb fragment including a Kozak sequence upstream of the ORF (ccacc) was subcloned into pcDNA3.1 (Invitrogen, Karlsruhe, Germany), or used to replace the VSV-G sequence pIGwt and pIΔI by digestion with HindIII/XhoI. Codon optimisation of the wild type ORF was performed by Geneart. The codon optimised ORF (GenBank database entry EF566942), also including a Kozak sequence (gccacc), was subcloned into pcDNA3.1 (Invitrogen) and pI vector by HindIII/XhoI restriction.
Deletion of the stop codon of the RSV-F ORF was achieved by PCR-directed mutagenesis. The RSV-Fsyn ORF without the stop codon was then subcloned into the pcDNA3.1(+) vector and the myc-tag was fused to the C-terminus of RSV-F by ligating annealed primers (Sigma) mycTAAs: 5'-tcgag gaacaaaaactcatctcagaagaggatctgtaat and mycTAAa: 5'-ctaga ttacagatcctcttctgagatgagtttttgttcc into the expression plasmid containing the RSV-F ORF lacking the stop codon via XhoI and XbaI sites.
Point mutations were introduced to the RSV-F ORF by overlap extension PCR and ligation of PpuMI/XhoI fragments.
Chimeric ORFs were produced by amplification of portions of the synthetic ORF and subcloning via HindIII/PpuMI or BsaBI/XhoI into the wild type expression vector pIFwt. All plasmids were confirmed by sequence analysis (Genterprise, Mainz, Germany).
Cells and transfection
293T and HEp2 cells were cultured in Dulbecco s modified Eagle s medium (Invitrogen) supplemented with 10% fetal calf serum (Invitrogen), penicillin G and streptomycin sulphate in a final concentration of 100 μg/ml each. Cells were transfected in 25 cm2 flasks with 5 μg plasmid-DNA by the calcium phosphate coprecipitation method as described elsewhere .
Control of transfection efficiency
To control transfection levels and guarantee comparable amounts of protein in lysates of transfected cells, plasmids for expression of reporter proteins were transfected additionally to VSV-G and RSV-F expression plasmids. In case of VSV-G expression analyses, cotransfection of lentiviral gag-pol expression plasmids Hgpsyn  for HIV-1 gag-pol and SgpΔ2  for SIV gag-pol served as control in Western and Northern blot analyses, respectively. Cotransfection of the lentiviral vector VICG3ΔBH containing a GFP-expression cassette directly monitored transfection efficiency in treated cells. For RSV-F expression analyses cotransfection of an EGFP expression plasmid (pEGFP-C1, BD Biosciences Clontech, Heidelberg, Germany) and quantitative measurement of fluorescence activity in cell lysates guaranteed similar transfection efficiency. In transfected cells subsequently infected with rgRSV, transfection efficiency was controlled by transfection of an expression plasmid for Gaussia luciferase (pCMV-GLuc1; Targeting Systems, Santee, USA) and measurement of its activity in cell supernatants.
Western blot analysis
Transfected 293T cells were lysed 48 h following transfection. Equal amounts of total protein measured by Bradford-Assay (Biorad, Munich, Germany) were loaded on sodium dodecyl sulphate 8–12% polyacrylamide gels in reducing (500 mM TrisHCl pH 6,8; SDS; 20 v/v β-mercaptoethanol; 40 v/v Glycerin; 0,04% (w/v) PyroninY) or non reducing (without β-mercaptoethanol) Laemmli buffer. After protein separation and blotting on nitrocellulose membrane, proteins were incubated at 4°C over night with monoclonal antibody against either VSV-G (P5D4, Sigma-Aldrich, Munich, Germany), HIV-p24 (AIDS Research and Reference Reagent Program, Dr. Jonathan Allan ), RSV-F (18F12 ) or the myc-tag (9E10 ). After washing, the membrane was incubated with horseradish peroxydase-linked goat-anti-mouse-Fc antibody (SantaCruz, Heidelberg, Germany) and detected proteins were visualised by enhanced chemiluminescence reaction (Chemiglow®, Biozym, Hamburg, Germany).
Northern blot analysis
Total or cytoplasmic RNA was isolated from transfected 293T cells by RNeasy® Mini Kit (Qiagen, Hilden, Germany), mRNA was isolated by Fast Track 2.0 kit (Invitrogen, Karlsruhe, Germany). Concentration of purified RNA was determined by measuring absorbance at 260 nm. Five μg RNA was separated on an 1% agarose gel and blotted on nylon membrane. DIG-labelled probes where synthesised by PCR using the DIG synthesis kit (Roche, Mannheim, Germany). Transcripts were detected by hybridisation to a probe directed to either the BGH-poly(A) signal of the pcDNA3.1(+) (length: 130 bp; Primers: BGHs: 5'-gagtctagagggcccgtttaa; BGHa: 5'-aggaaaggacagtgggagtg) or the RSV-F ORF (length: 780 bp bp; Primers: RSV-Fis: 5'-ggtcctgcacttagaaggag; RSV-Fia: 5'-catgacacaatggctcctag). Oligonucleotides for probe synthesis PCR were derived from Sigma.
DIG-labelled nucleic acids were visualised by an alkaline phosphatase coupled anti-DIG antibody and CSPD substrate (Roche).
We thank B. Schweiger from the Robert Koch Institute (Berlin, Germany) for the RSV A2 strain. Recombinant GFP expressing RSV was generously provided by M. E. Peeples and P. L. Collins (NIH, Maryland, USA). R. Wagner (University of Regensburg, Germany) kindly provided pGwt and pGsyn expression plasmids. NT was granted a scholarship from the "Allgemeines Promotionskolleg" of the Ruhr-Universität Bochum. The study was supported by "FoRUM" grant F467-2005 of the Ruhr-Universität Bochum.
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