Translatomics Insights on SARS-CoV-2 Mechanisms
Viruses, lacking their own cellular organelles, necessarily hijack its host’s translational machinery, namely the ribosome, to produce the proteins required for replication and propagation. Additionally, many viruses utilise the recoding phenomenon of frameshifting to significantly increase the genomic information within their small genomes. As such, translatomics is a key area of study in the field of molecular virology, and is especially pertinent with the emergence of SARS-CoV-2, the pathogen responsible for the recent global pandemic. Here, we bring together five impactful research works on SARS-CoV-2 translation, providing an ideal point of access into this fascinating subject.
The coding capacity of SARS-CoV-2
Nature, Sept 2020; 589(7840), pp.125-130
Finkel, Y., Mizrahi, O., Nachshon, A., Weingarten-Gabbay, S., Morgenstern, D., Yahalom-Ronen, Y., Tamir, H., Achdout et al.
SARS-CoV-2 is a single-stranded RNA virus, with a genome of approximately 30kb. Within this genome, two ORFs are known to be translated, ORF1a and ORF1b, which overlap and are distinct due to a frameshifting site prior to the ORF1a stop codon. The amino acid sequences translated from these ORFs are then cleaved post-translationally to give rise to a number of protein products. Some of these introduce alternative splicing of the RNA transcript (giving subgenomic RNAs), which later give rise to other canonical ORFs. Prior to this work, the inferred coding capacity of this virus was based on homology to related viruses (particularly SARS-CoV-1) and computationally generated predictions. Here however, the authors utilise ribosome profiling of initiating and elongating ribosome assays on this virus, allowing for direct identification of true canonical ORF expression, as well as the interrogation of this genome for unannotated/unpredicted ORFs.
- For most canonical ORFs, transcript abundance correlated strongly with ribosome footprint densities, indicating similar translational efficiencies, which was attributed to their common 5’ leader. Exceptions to this included ORF1a and ORF1b, which displayed reduced footprint densities compared to their transcript abundances, possibly due to distinct 5’ UTR features.
- 23 novel ORFs were uncovered, including upstream ORFs and several in-frame and out-of-frame ORFs embedded within previously identified ORFs, resulting in truncated products and novel polypeptides.
- Ribosome profiling was able to detect the expression of two canonical ORFs not picked up with mass spectroscopy, highlighting the advantage of its usage in parallel to other protein detection techniques.
This work gives the most accurate map of coding regions of the SARS-CoV-2 virus, and as such opens up access to potential protein targets that may be used to treat the infection. It also reveals mechanisms on how some of these targets are produced at a translational level, opening up further avenues for investigative research. Furthermore, it gives insights into the mechanisms of propagation used in this and other coronaviruses, contrasting it with other viral families, and as such, deepens knowledge which may prove useful in designing virus-specific therapeutics.
SARS-CoV-2 Disrupts Splicing, Translation, and Protein Trafficking to Suppress Host Defences
Cell, Nov 2020; 183(5), pp.1325-1339
Banerjee, A.K., Blanco, M.R., Bruce, E.A., Honson, D.D., Chen, L.M., Chow, A. et al.
While the Finkel et al., (2020) looked into the coding capacity of SARS-CoV-2, Banerjee et al., (2020) are among the first to investigate how these protein products mechanistically promote viral pathogenesis. These proteins can be broken down into three categories; structural proteins (such as nucleocapsid and spike proteins), non-structural proteins/NSPs (which can have a variety of functions, such as helicases or polymerases), and accessory (whose function remains comparatively uncharacterised). Given SARS-CoV-2 is an RNA virus, with many predicted RNA-interacting features in its proteins, the authors reasoned many of these proteins may interact with host mRNAs, thus dictating pathological progression. Thus, they carry out analysis of 27 SARS-CoV-2 proteins and host mRNAs, alongside RNA UV cross-linking analysis to determine viral protein-host mRNA interactions.
- Through this comprehensive analysis, it was found that 10 of the 27 proteins tested were able to bind host mRNAs. These interactions were highly specific for each protein, and binding occurred at specific sites on each mRNA in question.
- NSP16 binds to the pre-mRNA recognition domains of both U1 and U2 snRNAs (more specifically, the 5’ splice recognition site and branchpoint recognition site respectively). These snRNAs are involved in the splicing of pre-mRNAs, to ultimately form mature mRNA. It was demonstrated that the presence of NSP16 suppresses global mRNA spicing in HEK293T cells.
- NSP1 was found to bind exclusively to 18S rRNA (part of the 40S ribosome subunit), at helix 18, adjacent to the mRNA entry channel. Further assays demonstrated that this protein, when expressed individually in mammalian cells, drastically supresses overall translation, whereas other SARS-CoV-2 proteins had no effect. Interestingly, the first stem loop in the 5’ leader of SARS-CoV-2 is sufficient to circumvent this translational inhibition, thus allowing viral mRNA expression.
- Two viral proteins, NSP8 and NSP9, bind at distinct sites of the signal recognition particle (SRP) complex. This complex associates with the ribosome and binds a signal sequence within the nascent chain, which then targets it to the ER for proper folding and trafficking. NSP8 and NSP9 expression was demonstrated to reduce proteins trafficked to the membrane.
This paper was particularly revealing and comprehensive in the field of SARS-CoV-2 study, in its analysis of SARS-CoV-2 proteins interactions with host mRNA , thus allowing a granular view into specific roles. The combination of its screening, alongside the analysis of functionality of the associated mRNAs, make it an excellent example in terms of study designs, ideal as a framework for future studies into viral infection.
The short isoform of the host antiviral protein ZAP acts as an inhibitor of SARS-CoV-2 programmed ribosomal frameshifting
Nature Communications, Dec 2021; 12(1), p.7193
Zimmer, M.M., Kibe, A., Rand, U., Pekarek, L., Ye, L., Buck, S. et al.
-1 programmed ribosomal frameshifting is essential for the propagation of SARS-CoV-2, a mechanism also utilised by a number of other pathological viruses. Such frameshifting is necessary for the expression of the full repertoire of proteins this virus possesses, enabling its growth. Much study has been directed to the cis-acting factors in driving frameshifting efficiency, such as nucleotide sequence and secondary structures. However, questions remain about the trans-acting factors, such as RNA-binding proteins, that may influence this efficiency. Here, the authors utilise RNA affinity and mass spectroscopy screenings, in infected and uninfected Calu-3 and HEK293 cells, to identify factors that interact with the SARS-CoV-2 frameshifting sequence, thus deepening the knowledge of this process.
- Using RNA pulldown assays, the authors were able to identify at least 100 proteins that were over two-fold enriched in compared to a standardised, non-structural positive control. Of these, 9 were found to be enriched in all assays tested (Calu-2, HEK293, infected, un-infected). The functions of a majority of these proteins related to involvement in the processes of translation.
- Of 20 selected interactors, 3 were found to majorly decrease ribosomal frameshifting efficiency, these being HNRNPH1, HNRNPH2 and ZAP-S, as uncovered through dual-luciferase assays.
- HNRNPH1 and HNRNPH2 expression was not significantly altered following infection, although expression of ZAP-S was enriched 6-fold. Furthermore, in vitro studies found that overexpression of this protein in infected cells drastically reduced viral replication. Interestingly, ZAP-S expression was only influential on SARS-CoV-1 and SARS-CoV-2 frameshifting, with no impact on this process in HIV-1, CHIKV, MERS-CoV or West Nile Virus, among others.
- Utilising RNA-protein binding assays and truncated pseudoknot variants, it was determined that ZAP-S is likely to bind directly to the second and third stem loops (SL2 and SL3) of the frameshift stimulating pseudoknot. The presence of ZAP-S had no impact on the unfolding of this pseudoknot structure, but strongly inhibited its refolding, likely impacting on its frameshifting stimulating properties.
This paper delves deeply into the secondary structures required for frameshifting in SARS-CoV-2, and identifies host trans factors that can be crucial in inhibiting it. Interestingly, it identifies ZAP-S as a potent inhibitor of such frameshifting, and its specificity in targeting this virus and the related SARS-CoV-1, but not others, which may be suggestive of other virus-specific host factors targeting this process.
Ribosome-profiling reveals restricted post transcriptional expression of antiviral cytokines and transcription factors during SARS-CoV-2 infection
International Journal of Molecular Sciences, Mar 2021; 22(7), p.3392
Alexander, M.R., Brice, A.M., Jansen van Vuren, P., Rootes, C.L., Tribolet, L., Cowled, C., Bean, A.G et al.
Ribosome profiling is a technique which can allow for detailed insights into regulation at a translational level. It is a particularly important technique in viral research, as viruses, lacking their own translational machinery, are required to highjack host ribosomes in order to produce their own proteins. One of the first things viruses often attempt to do upon infection is to to dampen immunological responses prior to their replication and propagation. While the Finkel et al. looked at the complete coding capacity of the viral genome, here Alexander et al. have a greater focus on its impact on host translation in the early stages of infection.
- By developing an infection model in Calu-3 cells, the transcriptional and translational changes in gene expression at 24 h post SARS-CoV-2 infection were explored.
- Transcriptionally, 229 genes were altered in expression (166 upregulated vs 63 downregulated). Expectedly, anti-viral defence featured prominently among those genes which were enriched (for example, IFIT1 and HERC5).
- While genes annotated as belonging the innate immunity category were similarly upregulated at a translational level as they were at a transcriptional level, genes from the categories of transcription factors and cytokines were typically translationally downregulated, despite their own transcriptional upregulation, although this phenomenon was not universal for all genes in these groups.
- Unstable mRNAs were found to be more likely to be translationally downregulated. This was hypothesised to relate to poorer ribosome occupancy and subsequently limited closed-loop assisted reinitiation, leading to more susceptibility to inhibition of scanning initiation mechanisms by this virus.
- SARS-CoV-1, inactivating eIF2A and thus triggering the integrated stress response upon infection, leads to preferential translation of genes with upstream ORFs. However, this contrasts with the findings of this study, where SARS-CoV-2-infected cells display no preference for such genes, thus indicating its inhibitory translation effects are likely to lie outside of such responses.
This study gives illuminating insights and suggestions relating to the early stages of SARS-Cov-2 infection, and the mechanisms by which this virus aims to overcome the initial stages of host response. It details differences between it and the closely related SARS-CoV-1, and should prove useful to the scientific community in exploring some of the first events following infection.
Structural basis of ribosomal frameshifting during translation of the SARS-CoV-2 RNA genome
Science, May 2021; 372(6548), pp.1306-1313
Bhatt, P.R., Scaiola, A., Loughran, G., Leibundgut, M., Kratzel, A., Meurs, R., Dreos, R et al.
Genomic SARS-CoV-2 RNA has two principal ORFs, ORF1a and ORF1b. These ORFs overlap, with a “slippery sequence” ribosomal frameshifting site prior to the stop codon of ORF1a. This -1 programmed frameshifting is evolutionarily conserved in all coronaviruses, and is necessary for the production of RNA polymerase, and thus is crucial for replication. Because of its necessity, and the comparative lack of similar frameshifting structures in humans, this presents an ideal target for potential therapeutics. Here, the authors attempt to interrogate the functional states of ribosomes across this frameshifting site, in order to reveal the mechanistic and structural features of this frameshifting event.
- Cryo-electron microscopy revealed that a pseudoknot structure lodges in the mRNA entry channel when the ribosome was centred on the slippery sequence. Disome profiling also demonstrated that this structure promoted ribosome pausing.
- This pseudoknot structure was further shown to bind to the ribosome protein uS3, and further given structural support by uS5 AND eS30.
- The position of the ORF1a stop codon, which is five codons downstream of the frameshifting site, is important in frameshifting efficiency. Removal of the stop codon entirely reduced the frameshifting efficiency to 50% of wild type. It is suggested that a stop codon here, leading to termination, allows time and space for the pseudoknot to refold, allowing trailing ribosomes to encounter this properly folded structure, heightening efficiency.
- The nascent chain emerging at the point of frameshifting was demonstrated to interact with the exit tunnel. Furthermore, mutational analysis, changing only 2 amino acids, lead to an increase of 30% in frameshifting efficiency, highlighting the role nascent chains may play in regulation of such events.
The depth of cryo-EM analysis demonstrated here has vastly advanced the knowledge of ribosome structures, with this research revealing the most detailed picture of the mammalian 80S to date. Furthermore, it unveils multiple mRNA- and nascent chain-ribosome interactions, which may serve as useful targets in the search for drugs against SARS-CoV-2, or indeed any related -1 frameshifting-utilising pathogens.
