We have investigated the mechanism of the translation of the second open reading frame (ORF) of the respiratory syncytial virus M2 transcript that uses a novel coupled translation process requiring prior translation of the upstream ORF. The second M2-2 ORF sequences play no role in the coupling process and can be replaced with other gene sequences. Surprisingly, the overlap region of the two ORFs alone was not sufficient for coupled translation to occur. An analysis of the sequences required for the coupling process showed that portions of the transcript located along the length of the first ORF M2-1, upstream of the ORF overlap region, were essential for coupled translation to occur. A critically important region for this process was centered ϳ150 nucleotides upstream of the ORF2 initiation codons. This region was shown to contain a significant degree of secondary structure, and mutation of this sequence to remove predicted areas of base pairing significantly reduced coupled translation, confirming that the secondary structure was important for the coupling process. Additional sequences further upstream increased the efficiency of the coupled translation process. These data indicate that upstream sequences act in conjunction with the M2-1/M2-2 overlap region to promote coupled translation.

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Coupled translation, first described in the M2 gene of pneumovirus respiratory syncytial virus (RSV), is an alternative mechanism of translational initiation in which the ribosomes which translate the first (M2-1) open reading frame (ORF) move a short distance upstream after termination and reinitiate translation from a second (M2-2) overlapping ORF. Here, we show that the same mechanism occurs in two closely related viruses, avian pneumovirus (APV) and pneumonia virus of mice (PVM), although with markedly different efficiencies. To identify the reasons for the variation in efficiency of coupled expression between RSV and APV, we used chimeric M2-1 genes containing different lengths of the M2-1 ORF from each virus. An essential component allowing coupled expression in the chimeras was a segment from the RSV M2-1 coding region containing a high degree of secondary structure. Additional sequences at the 5 end of the RSV M2-1 ORF also promoted coupled translation when the region with high levels of secondary structure was present. These data indicate that at least two distant parts of the mRNA transcript, together with a suitable overlapping region, are involved in the coupling process. Replacement of the last 102 nucleotides of the RSV M2-1 ORF with the equivalent APV sequence showed identical levels of coupled translation. Thus, the overlapping region can direct the ribosome back onto the start codon of the second ORF while the upstream coding sequence of the M2-1 ORF determines the levels of coupled expression.

The live attenuated influenza vaccine FluMist® was withdrawn in the USA by the Centers for Disease Control and Prevention after its failure to provide adequate protective immunity during 2013–2016. The vaccine uses attenuated core type A and type B viruses, reconfigured each year to express the two major surface antigens of the currently circulating viruses. Here Fluenz™ Tetra, the European version of this vaccine, was examined directly for defective-interfering (DI) viral RNAs. DI RNAs are deleted versions of the infectious virus genome, and have powerful biological properties including attenuation of infection, reduction of infectious virus yield, and stimulation of some immune responses. Reverse transcription polymerase chain reaction followed by cloning and sequencing showed that Fluenz™ vaccine contains unexpected and substantial amounts of DI RNA arising from both its influenza A and influenza B components, with 87 different DI RNA sequences identified. Flu A DI RNAs from segment 3 replaced the majority of the genomic full-length segment 3, thus compromising its infectivity. DI RNAs arise during vaccine production and non-infectious DI virus replaces infectious virus pro rata so that fewer doses of the vaccine can be made. Instead the vaccine carries a large amount of non-infectious but biologically active DI virus. The presence of DI RNAs could significantly reduce the multiplication in the respiratory tract of the vaccine leading to reduced immunizing efficacy and could also stimulate the host antiviral responses, further depressing vaccine multiplication. The role of DI viruses in the performance of this and other vaccines requires further investigation.

The protocol described here is an adapted version of the toeprinting assay in which the oligonucleotide used to prime the reverse transcription step is labeled with a fluorescent dye instead of 32P. By using a fluorescent dye, the results of the assay are obtained within one hour by direct electrophoresis of the samples on an automated sequencing machine. This eliminates the need to cast and run polyacrylamide gels and to wait to expose dried gels. We show that an identical toeprint was found for the chloramphenicol acetyltransferase transcript using this nonradioactive method, which is in agreement with the previously published 32P-labeled method. Furthermore, in addition to being a faster and safer method, a larger region of sequence can be analyzed with one primer in a single experiment.

Many bacteria possess 2 or more genes for the chaperonin GroEL and the cochaperonin GroES. In particular, rhizobial species often have multiple groEL and groES genes, with a high degree of amino-acid similarity, in their genomes. The Rhizobium leguminosarum strain A34 has 3 complete groE operons, which we have named cpn.1, cpn.2 and cpn.3. Previously we have shown the cpn.1 operon to be essential for growth, but the two other cpn operons to be dispensable. Here, we have investigated the extent to which loss of the essential GroEL homologue Cpn60.1 can be compensated for by expression of the other two GroEL homologues (Cnp60.2 and Cpn60.3). Cpn60.2 could not be overexpressed to high levels in R. leguminosarum, and was unable to replace Cpn60.1. A strain that overexpressed Cpn60.3 grew in the absence of Cpn60.1, but the complemented strain displayed a temperature-sensitive phenotype. Cpn60.1 and Cpn60.3, when coexpressed in Escherichia coli, preferentially selfassembled rather than forming mixed heteroligomers. We conclude that, despite their high amino acid similarity, the GroEL homologues of R. leguminosarum are not functionally equivalent in vivo.

Abstract-Preeclampsia is a hypertensive disorder of pregnancy caused by abnormal placental function, partly because of chronic hypoxia at the utero-placental junction. The increase in levels of soluble vascular endothelial growth factor receptor 1, an antiangiogenic agent known to inhibit placental vascularization, is an important cellular factor implicated in the onset of preeclampsia. We investigated the ligand urotensin II (U-II), a potent endogenous vasoconstrictor and proangiogenic agent, for which levels have been reported to increase in patients with preeclampsia. We hypothesized that an increased sensitivity to U-II in preeclampsia might be achieved by upregulation of placental U-II receptors. We further investigated the role of U-II receptor stimulation on soluble vascular endothelial growth factor receptor 1 release in placental explants from diseased and normal patients. Immunohistochemistry, real-time PCR, and Western blotting analysis revealed that U-II receptor expression was significantly upregulated in preeclampsia placentas compared with controls (PϽ0.01). Cellular models of syncytiotrophoblast and vascular endothelial cells subjected to hypoxic conditions revealed an increase in U-II receptor levels in the syncytiotrophoblast model. This induction is regulated by the transcriptional activator hypoxia-inducible factor 1␣. U-II treatment is associated with increased secretion of soluble vascular endothelial growth factor receptor 1 only in preeclamptic placental explants under hypoxia but not in control conditions. Interestingly, normal placental explants did not respond to U-II stimulation. (Hypertension. 2010;56:172-178.)