Structural and molecular basis for Cardiovirus 2A protein as a viral gene expression switch.

Programmed -1 ribosomal frameshifting (PRF) in cardioviruses is activated by the 2A protein, a multi-functional virulence factor that also inhibits cap-dependent translational initiation. Here we present the X-ray crystal structure of 2A and show that it selectively binds to a pseudoknot-like conformation of the PRF stimulatory RNA element in the viral genome. Using optical tweezers, we demonstrate that 2A stabilises this RNA element, likely explaining the increase in PRF efficiency in the presence of 2A. Next, we demonstrate a strong interaction between 2A and the small ribosomal subunit and present a cryo-EM structure of 2A bound to initiated 70S ribosomes. Multiple copies of 2A bind to the 16S rRNA where they may compete for binding with initiation and elongation factors. Together, these results define the structural basis for RNA recognition by 2A, show how 2A-mediated stabilisation of an RNA pseudoknot promotes PRF, and reveal how 2A accumulation may shut down translation during virus infection.

An analysis by metabolic labelling of the encephalomyocarditis virus ribosomal frameshifting efficiency and stimulators.

Programmed -1 ribosomal frameshifting is a mechanism of gene expression whereby specific signals within messenger RNAs direct a proportion of ribosomes to shift -1 nt and continue translating in the new reading frame. Such frameshifting normally depends on an RNA structure stimulator 3'-adjacent to a 'slippery' heptanucleotide shift site sequence. Recently we identified an unusual frameshifting mechanism in encephalomyocarditis virus, where the stimulator involves a trans-acting virus protein. Thus, in contrast to other examples of -1 frameshifting, the efficiency of frameshifting in encephalomyocarditis virus is best studied in the context of virus infection. Here we use metabolic labelling to analyse the frameshifting efficiency of wild-type and mutant viruses. Confirming previous results, frameshifting depends on a G_GUU_UUU shift site sequence and a 3'-adjacent stem-loop structure, but is not appreciably affected by the 'StopGo' sequence present ~30 nt upstream. At late timepoints, frameshifting was estimated to be 46-76 % efficient.

Comparative analysis of internal ribosomal entry sites as molecular tools for bicistronic expression.

Internal ribosomal entry sites (IRESes) are sequences that drive cap-independent translation. They are found in some viral and cellular transcripts and they have been extensively used in both basic and applied research for the translation of two or more polypeptides from a single mRNA molecule in eukaryotic cells. Although the most widely used IRES comes from the encephalomyocarditis virus (EMCV), several other viral and cellular IRES elements have been identified and successfully used, including those of the human VCIP gene and the mouse Gtx gene. In this report we have compared the EMCV IRES with the VCIP and the Gtx IRESes, and we provide evidence that by using the EMCV IRES much higher levels of second cistron expression can be achieved.

NMR elucidation of the role of Mg2+ in the structure and stability of the conserved RNA motifs of the EMCV IRES element.

The three dimensional solution structures of a highly conserved 16mer RNA, endowed with a classic 'GNRA' tetraloop motif, and a 17mer RNA containing a cytosine-rich heptaloop which is predicted to be a potential receptor for the former RNA, of the I-domain of Encephalomyocarditis virus IRES have been determined by NMR spectroscopy. As Mg(2+) plays an important role in the activity of the IRES, the corresponding NMR structures of the Mg(2+) bound RNA complexes have also been determined. These RNA NMR structures, 16mer (21 constraints per residue), 16mer RNA/Mg(2+) (21 constraints per residue), 17mer (17 constraints per residue) and 17mer RNA/Mg(2+) (16 constraints per residue), were calculated to a high degree of precision with low RMSDs and low clash scores represent, to the best our knowledge, the first structures of a type II picornavirus IRES. Conformational analysis of the average structure showed that the RNAs and their Mg(2+) complexes adopt characteristic A-form helical structures, stabilised by canonical and non-canonical interactions in both the stem and loop regions. The GCGA tetraloop of the 16mer folds into a standard GNRA conformation, with the structural role of A550 being in the form of a G547.A550 sheared base-pair made up of two hydrogen bonds. Further, the previously uncharacterised AACCCCA heptaloop present in the 17mer forms a compact tertiary loop motif, held together by strong π-π interactions. Analysis of the NMR structures demonstrates that the role of Mg(2+) is principally to confer enhanced stability to the RNAs whereby the tetra- and heptaloops can achieve optimum conformation for any RNA-RNA interactions which are crucial for understanding the structure-function relationship of the IRES.

Presence of an encephalomyocarditis virus internal ribosome entry site sequence in avian infectious bronchitis virus defective RNAs abolishes rescue by helper virus.

Avian infectious bronchitis virus (IBV) defective RNAs (D-RNAs) have been used for the expression of heterologous genes in a helper-virus-dependent expression system. The heterologous genes were expressed under the control of an IBV transcription-associated sequence (TAS) derived from gene 5 of IBV Beaudette. However, coronavirus D-RNA expression vectors display an inherent instability following serial passage with helper virus, resulting in the eventual loss of the heterologous genes. The use of the picornavirus encephalomyocarditis virus (EMCV) internal ribosome entry site (IRES) sequence to initiate gene translation was investigated as an alternative method to the coronavirus-mediated TAS-controlled heterologous gene expression system. IBV D-RNAs containing the chloramphenicol acetyltransferase (CAT) reporter gene, under EMCV IRES control, were assessed for IRES-mediated CAT protein translation. CAT protein was detected from T7-derived IBV D-RNA transcripts in a cell-free protein synthesis system and in situ in avian chick kidney (CK) cells following T7-derived D-RNA synthesis from a recombinant fowlpox virus expressing the bacteriophage T7 DNA-dependent RNA polymerase. However, CAT protein was not detected in CK cells from IRES-containing IBV D-RNAs, in which the IRES-CAT construct was inserted at two different positions within the D-RNA, in the presence of helper IBV. Northern blot analysis demonstrated that the IRES-containing D-RNAs were not rescued on serial passage with helper virus, indicating that the EMCV IRES sequence had a detrimental effect on IBV D-RNA rescue.

Molecular analysis of the capsid coding region of a virulent encephalomyocarditis virus isolate after serial cell passages and assessment of its virulence.

EMCV has been recognized as a worldwide-shared pathogen in many host species with natural differences of virulence among strains. Differences of virulence were also demonstrated experimentally after serial passages of particular EMCV strains in cell culture. In order to evaluate the genetic variability of the CCR of an highly virulent Belgian EMCV isolate after 210 passages in cell culture, this region has been molecularly characterised by RT-PCR and sequencing. The analyses enlightened the stability of the isolate as only 3 mutations appeared within the CCR and this was observed between passage 200 and 210. One of the mutations is adjacent to the amino acid described as the differentiation amino acid between diabetogenic and non-diabetogenic variants of EMCV. An experimental inoculation of pigs with the passage 210 of the virus enlightened its attenuation and the challenged pigs were protected against the wild-type virus. Results confirm the ability of EMCV to become attenuated after passages in cell culture.

Three-color flow cytometry analysis of tricistronic expression of eBFP, eGFP, and eYFP using EMCV-IRES linkages.

BACKGROUND: The ability to quickly analyze and sort double or triple fluorescent reporter constructs using simultaneous analysis provides significant flexibility in the solution of analytical and process-related questions in biotechnology. METHODS: Bicistronic eBFP/eGFP and eBFP/eYFP constructs were made on two mammalian episomal plasmids using an internal ribosomal entry sequence from encephalomyocarditis virus (EMCV-IRES) to link two GFP expressions. Simultaneous two-color flow cytometry (FCM) analysis was accomplished using a dual Argon-laser multi-line configuration set at excitation wavelengths of 360 and 488 nm. Blue fluorescence emission (440 nm) and green fluorescence emission (507 nm) were detected using 405/20 (FL4) and 510/20 (FL1) bandpass filters. Dual eBFP/eYFP and three-color simultaneous analysis of eBFP/eGFP/eYFP was accomplished using the dual-laser configuration but also using a short-pass (525-nm) dichroic mirror and 550/30 bandpass filter configuration to detect yellow fluorescence emission (527 nm) in a third channel (FL2). RESULTS: Human 293 cells transfected with the bicistronic construct of eBFP-IRES-eGFP were easily detected using simultaneous analysis, and the signals were well separated with a mean blue fluorescent intensity (MFI) in the 2nd-log decade (FL4) and green MFI in the 4th-log decade (FL1). Likewise, eBFP-IRES-eYFP transfected cells were as easily detected and also demonstrated very good signal separation. A tricistronic construct of eBFP-IRES-eGFP-IRES-eYFP was also made and transfected into 293 cells. Triple-color fluorescent cells were easily detected using the cytometer configuration for simultaneous analysis. All three signals separated with only moderate compensation required for green and yellow emission spectra. The respective MFI for each of the fluorescent proteins was correlative to what had been observed with the separate bicistronic constructs. CONCLUSIONS: Our results demonstrate that we have developed a novel fluorescent flow cytometry method that can be used as a powerful tool to differentiate and analyze three colors simultaneously from either a dual or a triple cistronic construct which has been transfected into living cells.

Antibodies against EMC virus RNA-VPg recognize Tyr-(5'P-->O)-pU and immunostain infected cells.

Covalent complexes of nucleic acids and proteins are widespread among viruses. Covalent complexes of RNA and proteins are proposed to exist in eukaryotic cells. The goal of this work was to obtain specific antibodies to the covalent linkage unit (CLU) between virus RNA and protein to search cellular RNA-protein complexes. Antibodies were generated by direct immunization of a rabbit with the BSA-coupled EMC virus RNA-VPg complex. By a dot-blot immunoassay and immunofluorescent microscopy it was found that the antibodies specifically recognize both EMC virus RNA-VPg and synthetic CLU-containing compounds. Thus, a fraction of the antibodies was directed to CLU.

Expression and purification of recombinant, glycosylated human interferon alpha 2b in murine myeloma NSo cells.

We have expressed recombinant human interferon-alpha 2b in mammalian cells and isolated cell lines constitutively secreting very high levels of biologically active protein. The expression system takes advantage of the strong human cytomegalovirus immediate early promoter in mouse myeloma NSo cells and glutamine synthetase as a selectable marker; spontaneous mutants with amplified gene copy numbers were selected by growth of primary transfectants in the presence of methionine sulfoximine. Using this procedure, we have isolated a recombinant NSo cell line which secretes human interferon at the rate of 20 micrograms/10(6) cells/24 h and accumulates up to 120 micrograms/ml (approximately 2.4 x 10(7) U/ml) following prolonged undiluted culture. The interferon (IFN) could be efficiently purified on a polyclonal bovine anti-human IFN alpha specific antibody column and the glycosylation pattern was found to be similar to that of nonrecombinant IFN alpha 2b purified from virus-induced human Namalwa cells. The biological activity of the recombinant material was indistinguishable from that of natural IFN from Namalwa cells, and the specific antiviral activity, as assayed on human HeLa cells challenged with encephalomyocarditis virus, was 2 x 10(8) IU/mg, similar to that of nonrecombinant IFN preparations. This represents the highest reported level of glycosylated, recombinant IFN expression in a stable mammalian system and is a significant advance in the large-scale production of these clinically important cytokines.

Differences in the two-dimension-gel electrophoresis protein patterns of the lethal K and benign B variants of encephalomyocarditis virus.

Variants of encephalomyocarditis virus (EMCV) are indistinguishable by hyperimmune serum. In spite of their antigenic similarity, they produce different disease syndromes in susceptible strains of mice. To understand the basis for the diversity in pathogenicity, studies have been initiated to characterize each of the virus variants. In this study, two-dimensional gel electrophoresis was used to compare the proteins produced by the benign EMCV-B with those produced by lethal EMCV-K. The data show that (i) the replication cycle of each of the virus variants is characteristic of picornaviruses, (ii) the VP1 of EMCV-K is more basic than that of EMCV-B, and (iii) three proteins, one a major component of VP1, the other two with molecular weight of about 12,000, are present in EMCV-K but not in EMCV-B.

Characterization of encephalomyocarditis virus isolated from aborted swine fetuses.

Characteristics of 2 encephalomyocarditis virus (EMCV) isolates (MN-25 and MN-30) recovered from aborted swine fetuses were examined along with 2 other swine isolates (NVSL-MDV and NVSL-PR) and a reference ATCC strain (VR-129). All 5 EMCV isolates were found to be serologically related by cross testing, using serum neutralization and fluorescent antibody assays. Hemagglutination (HA) properties of the 5 isolates were compared, using 5 diluents. The MN-25 and NVSL-MDV strains had HA activity with guinea pig RBC in all 5 diluents, whereas MN-30, NVSL-PR, and VR-129 had HA activity only in KCl-borate buffer. The HA ability with RBC of various animal species was examined, using KCl-borate diluent. All virus isolates had high HA titer (1:512 to 1:2,048) with guinea pig, rat, and horse RBC and lower HA titer (1:16 to 1:64) with sheep RBC. The MN-25 and NVSL-MDV isolates agglutinated dogs RBC, whereas MN-30, NVSL-PR, and VR-129 strains did not. Viral replication was evident in 8 of 10 cell lines tested, although infectivity titers of each virus varied by cell line used. Plaque-forming ability was similar for all 5 isolates, but plaque size was different by virus and cell culture used. Virus isolates were found to be stable after being heated at 56 C and subjected to a wide range of pH. A viral polypeptide pattern difference for all 5 isolates was not found by use of sodium dodecyl sulfate-polyacrylamide gel electrophoresis.(ABSTRACT TRUNCATED AT 250 WORDS)