Viewpoint Cite This: Biochemistry XXXX, XXX, XXX−XXX
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Scaring Ribosomes Shiftless Jonathan D. Dinman* Department of Cell Biology and Molecular Genetics, University of Maryland, College Park, Maryland 20742, United States
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production of Gag-pol and its cleavage products, knocked down reverse transcriptase activity, and abrogated HIV-1 propagation in infected cells. Critically, siRNA knockdown of c19orf66 reversed all of these effects. On the basis of the ability of this protein to inhibit −1 PRF, the authors christened the gene shiftless (SFL). Follow-up experiments revealed that SFL inhibited −1 PRF directed by sequences derived from 5 additional different retroviruses (Rous sarcoma virus, human T-cell leukemia virus, mouse mammary tumor virus, HIV-2, and simian immunodeficiency virus), a member of the alphavirus family (sindbis virus), and two cellular mRNAs (PEG10 and CCR5). SFL appears to be specific to −1 PRF; it did not alter the efficiency of +1 PRF directed by the OAZ1 gene nor did it affect readthrough of a termination codon programmed by an element found in mouse leukemia virus. A series of cofractionation experiments were performed to investigate the nature of this specificity. These revealed that SFL interacts with the ribosome by directly binding the ribosomal proteins uL5 and eS31. These are located on the large and small subunits, respectively, and are brought closer to one another when ribosomes are in the pretranslocation “rotated” state (Figure 1). Importantly, a hyper-rotated conformation has been shown to be associated with ribosomes paused at a −1 PRF signal,4 which brings uL5 and eS31 into close proximity. SFL was also shown to specifically interact with a −1 PRF signal containing mRNA but not with a negative control. While the authors did not quantitatively characterize the interactions between SFL and these ribosomal proteins, the apparently low affinity of SFL for each of these proteins suggests cooperative binding, in which SFL is able to bind both uL5 and eS31 in the hyper-rotated ribosomes, delivering it to ribosomes stalled by −1 PRF signals. Presumably, it then interacts with the −1 PRF signal itself, causing the ribosome to stall in a conformation that is able to recruit the eRF1/eRF3 release factor complex (rather than the NoGo mRNA decay apparatus) to terminate translation. Intriguingly, mass spectroscopic analysis of the terminated HIV-1 derived protein product(s) revealed that their C-termini are encoded by the 0-frame codons in the HIV-1 slippery site. This suggests that termination occurs because SFL may irretrievably stabilize ribosomes in a state that can neither elongate nor frameshift. Additionally, since release factor recruitment requires an empty A-site, some sort of pseudotranslocation step may be involved. This work makes an important and novel contribution that will serve as a springboard to broadening our understanding of the biological significance of how and why −1 PRF may be regulated. It also opens up an entirely new avenue for discovery
xceptions to rules provide opportunities to illuminate otherwise opaque phenomena. While the three base “codon” is a universal feature of the genetic code, examples of elements that direct translating ribosomes to shift reading frame have impacted a variety of fields, including translational control, ribosome structural biology, and virology (reviewed in ref 1). This class of molecular mechanism is generally called programmed ribosomal frameshifting (PRF). Specific cis-acting mRNA elements can direct ribosomes to slip at specific sequences with defined frequencies. The most well understood is −1 PRF, in which a combination of a special “slippery” sequences paired with strong proximal downstream mRNA structural elements targets ribosomes to slip backward by one base in the 5′ or −1 direction. Less well explored +1 PRF elements direct ribosomes to slide in the opposite or +1 direction. In eukaryotic mRNAs, −1 PRF appears to be used to negatively regulate gene expression by directing mRNAs to degradation pathways. In viruses, −1 PRF is widely exploited to expand the limited coding capacities of viral genomes, ensure production of the stoichiometrically appropriate ratios of viral proteins, and control the programming of their genetic subroutines. Critically, altering the frequency of viral −1 PRF events can negatively impact these functions, with negative consequences on virus assembly and replication. This has made −1 PRF an attractive target for antiviral intervention. The 5′ half of the HIV-1 genome encodes two open reading frames (ORFs). The 5′ ORF (the gag gene) encodes the Gag polyprotein, which is cleaved into structural proteins by viral protease after immature viruses bud from the plasma membrane of infected cells. The 3′ pol ORF partially overlaps with and is in the −1 reading frame with respect to gag, and a −1 PRF event produces the Gag-pol fusion protein.2 Cleavage of this produces not only the gag-encoded structural proteins but also the reverse transcriptase/RNase H, protease, and integrase enzymes that are critical for cDNA synthesis, chromosomal integration, and virion maturation. Genetic mutations and drugs that alter −1 PRF rates negatively impact virus replication by changing the critical stoichiometric ratios among these proteins. It is also known that type-1 interferon (IFN) stimulates the expression of multiple interferon stimulated genes (ISGs), some of which inhibit HIV-1 replication at various steps of the viral replication cycle. With this in mind, Wang et al.3 hypothesized that IFN may stimulate production of a protein that may have antiviral effects by inhibiting −1 PRF. A preliminary proof-of-principle experiment revealed that IFN treatment of HIV-1 infected cells reduced the expression of Gag-pol relative to Gag alone, leading them to screen a library of 99 human ISGs for −1 PRF inhibitory activity. One of these, a previously unnamed ORF called c19orf66, strongly inhibited HIV-1 directed −1 PRF. Extensive follow-up experiments using multiple cell lines and HIV-1 variants revealed that expression of c19orf66 inhibited © XXXX American Chemical Society
Received: February 26, 2019
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DOI: 10.1021/acs.biochem.9b00162 Biochemistry XXXX, XXX, XXX−XXX
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Biochemistry
Figure 1. Modeling inhibition of −1 programmed ribosomal frameshifting by shiftless. A translating ribosome pauses at a −1 PRF signal in the unrotated state with its peptidyl- and aminoacyl-tRNAs in the “classic” P/P and A/A states, respectively. The −1 PRF signal consists of a heptameric x xxy yyz “slippery site” followed by a strong mRNA secondary structure. Upon peptidyltransfer, the tRNAs assume the hybrid P/E and A/P states, and the large and small subunits (LSU and SSU) rotate relative to one another in a motion that includes the SSU head swivel (white arrow). Translational frameshifting results in the tRNAs pairing to the XXX and YYY codons and induces a hyper-rotated conformation that brings uL5 and eS31 into close proximity with one another. This is proposed to recruit SFL, which physically interacts with uL5, eS31, and the −1 PRF signal. Release of the truncated peptide is effected by recruitment of the eRF1/3 complex. In order for the release factor complex to do this, it should theoretically be preceded by a translocation of the peptidyl-tRNA to the P-site.
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and design of antiviral therapeutics. Obviously, more detailed biochemical and biophysical measurements are in order. These might include pairwise and in ensemble quantitative measurements of the interactions between SFL and uL5, eS31, and various PRF signals. The ability of SFL to dimerize raises questions about its biophysical interactions with the preframeshift complex. Molecular genetics approaches can be used as a first approach to these questions, to be followed up with more sophisticated cryo-EM- and smFRET-based inquiries. The potential impact of atomic resolution structural analyses of SFL on a the frameshift complex could be tremendous. There is also an alternatively spliced short form of SFL, called SFLS, which does not have antiframeshifting activity. Did this evolve as a way to regulate SFL activity in cells? If so, how is the alternative splicing regulated and what is its biological significance? Lastly, the authors note that SFL expression enhances production of the terminated form of CCR5. Notably, the −1 PRF signal is located at the same position as the CCR5Δ32 allele, which is well-known for its ability to confer resistance to HIV-1 infection. One hypothesis is that a CCR5-tropic retrovirus such as HIV-1 may have acted to drive selection for this element sometime in our ancestral history. However, it is not likely that a general inhibitor of −1 PRF would have evolved solely to interfere with a specific virus/ receptor interaction. Instead, a hint may come from computational studies suggesting that −1 PRF is also widely used to control the expression of cellular mRNAs. A recent report demonstrated that (a) downregulation of CCR5 after a stroke and traumatic brain injury induces motor recovery and improves cognition, (b) human CCR5Δ32 carriers have better poststroke outcomes, and (c) inhibition of C−C chemokine receptor 5 (CCR5) signaling enhances learning, memory, and plasticity processes in hippocampal and cortical circuits.5 It is tempting to speculate that, by driving ribosomes to prematurely terminate translation of CCR5 at its −1 PRF signal, SFL may play a role in learning and recovery from brain injury. This would provide a nonviral example of an organismal function of SFL and could be exploited to search for examples where regulation of −1 PRF in other cellular mRNAs by SFL may have biological and biomedical significance.
AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. ORCID
Jonathan D. Dinman: 0000-0002-2402-9698 Notes
The author declares no competing financial interest.
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ACKNOWLEDGMENTS This work was partially supported by grants to the author by the National Institutes of Health (R01 HL119439 and R01 GM117177).
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REFERENCES
(1) Dever, T. E., Dinman, J. D., and Green, R. (2018) Translation Elongation and Recoding in Eukaryotes. Cold Spring Harbor Perspect. Biol. 10, a032649. (2) Jacks, T., Power, M. D., Masiarz, F. R., Luciw, P. A., Barr, P. J., and Varmus, H. E. (1988) Characterization of ribosomal frameshifting in HIV-1 gag-pol expression. Nature 331, 280−283. (3) Wang, X., Xuan, Y., Han, Y., Ding, X., Ye, K., Yang, F., Gao, P., Goff, S. P., and Gao, G. (2019) Regulation of HIV-1 Gag-Pol Expression by Shiftless, an Inhibitor of Programmed −1 Ribosomal Frameshifting. Cell 176, 625−635. (4) Chen, J., Petrov, A., Johansson, M., Tsai, A., O’Leary, S. E., and Puglisi, J. D. (2014) Dynamic pathways of −1 translational frameshifting. Nature 512, 328−332. (5) Joy, M. T., Ben Assayag, E., Shabashov-Stone, D., LirazZaltsman, S., Mazzitelli, J., Arenas, M., Abduljawad, N., Kliper, E., Korczyn, A. D., Thareja, N. S., Kesner, E. L., Zhou, M., Huang, S., Silva, T. K., Katz, N., Bornstein, N. M., Silva, A. J., Shohami, E., and Carmichael, S. T. (2019) CCR5 is a therapeutic target for recovery after stroke and trumatic brian injury. Cell 176, 1143−1157.e13.
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DOI: 10.1021/acs.biochem.9b00162 Biochemistry XXXX, XXX, XXX−XXX
