Abstract
BackgroundThe genus Alphavirus includes several potentially lethal human viruses. Additionally, species such as Sindbis virus and Semliki Forest virus are important vectors for gene therapy, vaccination and cancer research, and important models for virion assembly and structural analyses. The genome encodes nine known proteins, including the small '6K' protein. 6K appears to be involved in envelope protein processing, membrane permeabilization, virion assembly and virus budding. In protein gels, 6K migrates as a doublet – a result that, to date, has been attributed to differing degrees of acylation. Nonetheless, despite many years of research, its role is still relatively poorly understood.ResultsWe report that ribosomal -1 frameshifting, with an estimated efficiency of ~10–18%, occurs at a conserved UUUUUUA motif within the sequence encoding 6K, resulting in the synthesis of an additional protein, termed TF (TransFrame protein; ~8 kDa), in which the C-terminal amino acids are encoded by the -1 frame. The presence of TF in the Semliki Forest virion was confirmed by mass spectrometry. The expression patterns of TF and 6K were studied by pulse-chase labelling, immunoprecipitation and immunofluorescence, using both wild-type virus and a TF knockout mutant. We show that it is predominantly TF that is incorporated into the virion, not 6K as previously believed. Investigation of the 3' stimulatory signals responsible for efficient frameshifting at the UUUUUUA motif revealed a remarkable diversity of signals between different alphavirus species.ConclusionOur results provide a surprising new explanation for the 6K doublet, demand a fundamental reinterpretation of existing data on the alphavirus 6K protein, and open the way for future progress in the further characterization of the 6K and TF proteins. The results have implications for alphavirus biology, virion structure, viroporins, ribosomal frameshifting, and bioinformatic identification of novel frameshift-expressed genes, both in viruses and in cellular organisms.
Highlights
The genus Alphavirus includes several potentially lethal human viruses
Key observations include: (i) a number of anomalies in the old data that were inconsistent with the old explanation for the 6K doublet, are perfectly consistent with the new frameshifting explanation; (ii) after adjusting for the Sindbis virus (SINV) TF:6K Cys ratio being 9:5, the frameshifting efficiency in SINV-infected cells may be calculated from the old data, and ranges from 10–18%; (iii) TF appears to be much more heavily palmitoylated than 6K (~1 fatty acid on 6K and ~5–7 on TF for SINV; fewer fatty acids on TF for Semliki Forest virus (SFV)); and (iv) in SINV and SFV, TF but not 6K is present in the virion
We have demonstrated the existence of a ribosomal -1 frameshift site in the alphavirus structural polyprotein, that gives rise to the transframe protein TF, and demonstrated that it is primarily TF, rather than 6K, that is incorporated into the virion
Summary
The genus Alphavirus includes several potentially lethal human viruses. Species such as Sindbis virus and Semliki Forest virus are important vectors for gene therapy, vaccination and cancer research, and important models for virion assembly and structural analyses. The single-stranded genomic RNA is positive sense and about 11–12 kb long. It contains two long open reading frames (ORFs) separated by a short non-coding sequence (Figure 1). The 3'-proximal ORF codes for an ~140 kDa structural polyprotein (C-E3-E2-6K-E1) that is translated from a subgenomic RNA (26S sgRNA) and cleaved autocatalytically (to generate the capsid protein C) and by cellular proteases (to yield the envelope glycoproteins E1, E2 and E3). E3 is present in the virion of some (e.g. SFV) but not all (e.g. SINV) alphaviruses
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