Stephen C. Inglis

Characterization of an efficient coronavirus ribosomal frameshifting signal: Requirement for an RNA pseudoknot

Abstract The genomic RNA of the coronavirus IBV contains an efficient ribosomal frameshifting signal at the junction of two overlapping open reading frames. We have defined by deletion analysis an 86 nucleotide sequence encompassing the overlap region which is sufficient to allow frameshifting in a heterologous context. The upstream boundary of the signal consists of the sequence UUUAAAC, which is the likely site of ribosomal slippage. We show by creation of complementary nucleotide changes that the RNA downstream of this “slippery” sequence folds into a tertiary structure termed a pseudoknot, the formation of which is essential for efficient frameshifting.

Ribosomal pausing during translation of an RNA pseudoknot.

The genomic RNA of the coronavirus infectious bronchitis virus contains an efficient ribosomal frameshift signal which comprises a heptanucleotide slippery sequence followed by an RNA pseudoknot structure. The presence of the pseudoknot is essential for high-efficiency frameshifting, and it has been suggested that its function may be to slow or stall the ribosome in the vicinity of the slippery sequence. To test this possibility, we have studied translational elongation in vitro on mRNAs engineered to contain a well-defined pseudoknot-forming sequence. Insertion of the pseudoknot at a specific location within the influenza virus PB1 mRNA resulted in the production of a new translational intermediate corresponding to the size expected for ribosomal arrest at the pseudoknot. The appearance of this protein was transient, indicating that it was a true paused intermediate rather than a dead-end product, and mutational analysis confirmed that its appearance was dependent on the presence of a pseudoknot structure within the mRNA. These observations raise the possibility that a pause is required for the frameshift process. The extent of pausing at the pseudoknot was compared with that observed at a sequence designed to form a simple stem-loop structure with the same base pairs as the pseudoknot. This structure proved to be a less effective barrier to the elongating ribosome than the pseudoknot and in addition was unable to direct efficient ribosomal frameshifting, as would be expected if pausing plays an important role in frameshifting. However, the stem-loop was still able to induce significant pausing, and so this effect alone may be insufficient to account for the contribution of the pseudoknot to frameshifting.

Mutational analysis of the "slippery-sequence" component of a coronavirus ribosomal frameshifting signal.

Abstract The ribosomal frameshift signal in the genomic RNA of the coronavirus IBV is composed of two elements, a heptanucleotide “slippery-sequence” and a downstream RNA pseudoknot. We have investigated the kinds of slippery sequence that can function at the IBV frameshift site by analysing the frameshifting properties of a series of slippery-sequence mutants. We firstly confirmed that the site of frameshifting in IBV was at the heptanucleotide stretch UUUAAAC, and then used our knowledge of the pseudoknot structure and a suitable reporter gene to prepare an expression construct that allowed both the magnitude and direction of ribosomal frameshifting to be determined for candidate slippery sequences. Our results show that in almost all of the sequences tested, frameshifting is strictly into the −1 reading frame. Monotonous runs of nucleotides, however, gave detectable levels of a −2 +1 frameshift product, and U stretches in particular gave significant levels (2% to 21%). Preliminary evidence suggests that the RNA pseudoknot may play a role in influencing frameshift direction. The spectrum of slip-sequences tested in this analysis included all those known or suspected to be utilized in vivo. Our results indicate that triplets of A, C, G and U are functional when decoded in the ribosomal P-site following slippage ( XXX YYYN) although C triplets were the least effective. In the A-site (XXY YYY N), triplets of C and G were non-functional. The identity of the nucleotide at position 7 of the slippery sequence (XXXYYY N ) was found to be a critical determinant of frameshift efficiency and we show that a hierarchy of frameshifting exists for A-site codons. These observations lead us to suggest that ribosomal frameshifting at a particular site is determined, at least in part, by the strength of the interaction of normal cellular tRNAs with the A-site codon and does not necessarily involve specialized “shifty” tRNAs.

Polypeptides specified by the influenza virus genome: I. Evidence for eight distinct gene products specified by fowl plague virus

The structural polypeptides of fowl plague virus (influenza A) and those synthesized in fowl plague virus-infected chick embryo fibroblasts have been analyzed by high resolution polyacrylamide gel electrophoresis. We detected eight distinct virus gene products: three polymerase-associated polypeptides (P1, P2, P3), hemagglutinin (HA), nucleoprotein (NP), neuraminidase (NA), membrane polypeptide (M), and a nonstructural polypeptide (NS). The molecular weights of these polypeptides correlate closely with the molecular weights of the eight genome RNA species found in fowl plague virus. The three high molecular weight polypeptides, P1, P2, and P3, were detected both in virions and infected cells, and their separate identity established by a two-dimensional tryptic peptide mapping procedure. An active RNA polymerase enzyme complex isolated from virions and virus-infected cells contained all three P proteins together with the NP protein. The nonstructural polypeptide (NS), together with the P proteins and the NP, appeared early in the infectious cycle, while the M protein and HA protein appeared later in infection. The NS and M polypeptides, which have similar molecular weights, were separated on SDS-polyacrylamide gels and shown to be distinct by tryptic peptide mapping.

Mutational analysis of the RNA pseudoknot component of a coronavirus ribosomal frameshifting signal

Abstract The genomic RNA of the coronavirus IBV contains an efficient ribosomal frameshift signal at the junction of the overlapping 1a and 1b open reading frames. The signal is comprised of two elements, a heptanucleotide “slip-site” and a downstream tertiary RNA structure in the form of an RNA pseudoknot. We have investigated the structure of the pseudoknot and its contribution to the frameshift process by analysing the frameshifting properties of a series of pseudoknot mutants. Our results show that the pseudoknot structure closely resembles that which can be predicted from current building rules, although base-pair formation at the region where the two pseudoknot stems are thought to stack co-axially is not a pre-requisite for efficient frameshifting. The stems, however, must be in close proximity to generate a functional structure. In general, the removal of a single base-pair contact in either stem is sufficient to reduce or abolish frameshifting. No primary sequence determinants in the stems or loops appear to be involved in the frameshift process; as long as the overall structure is maintained, frameshifting is highly efficient. Thus, small insertions into the pseudoknot loops and a deletion in loop 2 that reduced its length to the predicted functional minimum did not influence frameshifting. However, a large insertion (467 nucleotides) into loop 2 abolished frameshifting. A simple stem-loop structure with a base-paired stem of the same length and nucleotide composition as the stacked stems of the pseudoknot could not functionally replace the pseudoknot, suggesting that some particular conformational feature of the pseudoknot determines its ability to promote frameshifting.

The Mutation Rate and Variability of Eukaryotic Viruses: An Analytical Review

Perhaps the most important factor to limit the effectiveness of vaccines against virus infections is that of virus variation. Successful vaccines have been developed against viruses such as those causing smallpox, measles, yellow fever and poliomyelitis, and they are effective against most circulating virus strains. However, with some viruses vaccination has been much less successful either because numerous antigenically distinct strains co-circulate, as is the case for rhinoviruses, or because new strains are continually emerging, as in the case of influenza virus. Despite the importance of virus variability, little is known about the factors that influence it and that are responsible for the dramatically different patterns of variation displayed by different viruses. The primary source of variation is obviously mutation, and it has been suggested in several recent papers that the extreme variability of some viruses may be a consequence of an unusually high rate of mutation (Holland et al., 1982; Reanney, 1984; Domingo et al., 1985; Saitou & Nei, 1986).

Association of the infectious bronchitis virus 3c protein with the virion envelope.

Abstract A highly purified radiolabeled preparation of the coronavirus infectious bronchitis virus (IBV) was analyzed, by immunoprecipitation with monospecific antisera, for the presence of a series of small virus proteins recently identified as the products of I BV mRNAs 3 and 5. One of these, 3c, a 12.4K protein encoded by the third open reading frame of the tricistronic mRNA3 was clearly detectable and was found to cofractionate with virion envelope proteins on detergent disruption of virus particles. These results, together with the hydrophobic nature of 3c and its previously demonstrated association with the membranes of infected cells, suggest strongly that 3c represents a new virion envelope protein, which may have counterparts in other coronaviruses.

A polycistronic mRNA specified by the coronavirus infectious bronchitis virus

Abstract The third largest of the nested set of subgenomic mRNAs (mRNA3) from the coronavirus infectious bronchitis virus (IBV) contains three separate open reading frames (3a, 3b, and 3c) which are not present on the next smallest of the mRNAs, suggesting that this mRNA may be functionally polycistronic. However, although a protein product has been identified from the 3c open reading frame, to date the coding function of 3a and 3b has not been established. We present nucleotide sequence data suggesting that each of the three open reading frames is conserved in a variety of different IBV strains and further show, through the preparation of monospecific antisera against bacterial fusion proteins, that IBV-infected cells contain small amounts of the products of these ORFs. In vitro translation studies using synthetic mRNAs containing the 3a, 3b, and 3c open reading frames suggest strongly that all three proteins can be translated from a single molecular species, and expression studies carried out in intact cells support this conclusion. Thus mRNA3 of IBV appears to be functionally tricistronic.

Complex formation between influenza virus polymerase proteins expressed in Xenopus oocytes.

Abstract All three influenza virus polymerase (P) proteins were expressed in Xenopus oocytes from microinjected in vitro transcribed mRNA analogs, with yields of up to 100 ng per oocyte. To examine the functional state of the Xenopus-expressed P proteins, the polypeptides were tested for their ability to form stable complexes with each other. As seen in virus-infected cells, all three P proteins associated into an immunoprecipitable complex, suggesting that the system has considerable promise for the reconstruction of an active influenza RNA polymerase. Examination of the ability of paired combinations of the P proteins to associate indicated that PB1 contained independent binding sites for PB2 and PA, and so probably formed the backbone of the complex. Sedimentation analysis of free and complexed P proteins indicated that PB1 and PB2 did not exist as free monomers, and that similarly, complexes of all three P proteins did not simply consist of one copy of each protein. The heterodisperse sedimentation rate seen for complexes of all three P proteins did not appear to result from their binding to RNA, suggesting the incorporation of additional polypeptides polymerase complex.

Polypeptides specified by the influenza virus genome 2. Assignment of protein coding functions to individual genome segments by in vitro translation

Abstract Cytoplasmic RNA extracted from chick embryo fibroblasts infected with influenza A (fowl plague) virus (FPV) was translated in a wheat germ cell-free protein-synthesizing system. Polypeptides which comigrated during SDS-polyacrylamide gel electrophoresis with marker virus-specific polypeptides P 1 , P 2 , P3, NP, M, and NS were synthesized in vitro . The NP, M, and NS polypeptides were positively identified by tryptic peptide mapping. The polypeptide component of the virus glycoprotein HA was also synthesized in vitro , and was identified by tryptic peptide mapping. RNA extracted from purified FPV (vRNA) did not direct the synthesis of any recognizable virus-specific polypeptides in vitro , either as a total preparation, or as individual RNA genome segments. The protein coding functions of the vRNA segments were identified by hybridization of individual segments to a preparation of infected cell cytoplasmic RNA. On subsequent translation of the RNA in vitro , synthesis of the virus-specific polypeptide corresponding to the hybridized vRNA segment was specifically reduced. We conclude that, for FPV, virion RNA segments 1–3 code for the three P polypeptides and segments 4, 5, 6, 7, and 8 code for polypeptides HA, NP, NA, M, and NS, respectively.