Characterization of Ribosomal Frameshifting in Theiler's Murine Encephalomyelitis Virus

Abstract

Theiler's murine encephalomyelitis virus (TMEV) is a member of the genus Cardiovirus in the Picornaviridae, a family of positive-sense single-stranded RNA viruses. Previously, we demonstrated that in the related cardiovirus, Encephalomyocarditis virus, a programmed −1 ribosomal frameshift (−1 PRF) occurs at a conserved G_GUU_UUU sequence within the 2B-encoding region of the polyprotein open reading frame (ORF). Here we show that −1 PRF occurs at a similar site during translation of the TMEV genome. In addition, we demonstrate that a predicted 3′ RNA stem-loop structure at a noncanonical spacing downstream of the shift site is required for efficient frameshifting in TMEV and that frameshifting also requires virus infection. Mutating the G_GUU_UUU shift site to inhibit frameshifting results in an attenuated virus with reduced growth kinetics and a small-plaque phenotype. Frameshifting in the virus context was found to be extremely efficient at 74 to 82%, which, to our knowledge, is the highest frameshifting efficiency recorded to date for any virus. We propose that highly efficient −1 PRF in TMEV provides a mechanism to escape the confines of equimolar expression normally inherent in the single-polyprotein expression strategy of picornaviruses.IMPORTANCE Many viruses utilize programmed −1 ribosomal frameshifting (−1 PRF) to produce different protein products at a defined ratio, or to translate overlapping ORFs to increase coding capacity. With few exceptions, −1 PRF occurs on specific “slippery” heptanucleotide sequences and is stimulated by RNA structure beginning 5 to 9 nucleotides (nt) downstream of the slippery site. Here we describe an unusual case of −1 PRF in Theiler's murine encephalomyelitis virus (TMEV) that is extraordinarily efficient (74 to 82% of ribosomes shift into the alternative reading frame) and, in stark contrast to other examples of −1 PRF, is dependent upon a stem-loop structure beginning 14 nt downstream of the slippery site. Furthermore, in TMEV-based reporter constructs in transfected cells, efficient frameshifting is critically dependent upon virus infection. We suggest that TMEV evolved frameshifting as a novel mechanism for removing ribosomes from the message (a “ribosome sink”) to downregulate synthesis of the 3′-encoded replication proteins.

Ribosomal frameshifting and transcriptional slippage: From genetic steganography and cryptography to adventitious use

“…Whether the nascent peptide context required for StopGo serves to exclude eEF5 prior to facilitating hydrolysis of the ester linkage required for liberation of the upstream encoded protein is unknown. Under the conditions tested, which do not include altered polyamine levels, StopGo has not been shown to influence the frameshifting (58,59). However, whether naturally there is some influence of polyamine levels remains to be determined.…”

“…The codons directly 5 of human antizyme 1 ORF1 stop specify NLGPGPRWCS (where S is encoded by the shift codon UCC, see Table 2 just above). This is similar to the sequence, NPGPVQS, directly 5 of the tandem −1 frameshift shift site utilized in expression of Theiler's murine encephalomyelitis and Saffold cardioviruses (58,59). The first part of this sequence can be written NPG/P to signify that the G is the C-terminal amino acid of the upstream-encoded protein that is liberated by the action of StopGo.…”

Non-canonical Translation in Plant RNA Viruses

“…This recoding event can occur in the + or − direction relative to the normal 0 frame of mRNA translation by shifting the ribosome in one or two nucleotides forward or backward. Productive frameshifting normally competes poorly with standard decoding, so the efficiency of frameshifting in viruses varies from 1% in BYDV to 82% in cardioviruses (Barry and Miller, 2002;Finch et al, 2015). Thus far, most frameshifting by plant viruses is in the -1 direction.…”

A [Cu]rious Ribosomal Profiling Pattern Leads to the Discovery of Ribosomal Frameshifting in the Synthesis of a Copper Chaperone

“…The best known example is in decoding of the E. coli dnaX gene where 50% efficient frameshifting yields a second DNA polymerase subunit in a 1:1 stoichiometric ratio with the product of standard decoding. Other examples include a component of a Cyanobacterial organelle-like carboxysome (Chaijarasphong et al, 2016), and in the 2B gene of a Cardiovirus (Loughran et al, 2011;Finch et al, 2015).…”