Understanding Viral-Host Interactions with Translatomics
Translation within the context of viral-host interaction is a crucially important factor in determining the propagation of the invading pathogen. Due to their lack of cellular organelles, it is a requirement upon most viruses to hijack host machinery, in particular the translational apparatus such as the ribosomes. This feature of viral infection means ribosome profiling can be an especially useful technique in determining the gene expression and subsequent actions of these pathogens, as well as cellular host response. Furthermore, due to the high density of information embedded within small viral genomes, the insights gained through ribosome profiling, such as the identification of frameshifting and ribosome pausing sites, are particularly valuable in deciphering the coding potential of these pathogens.
The following papers display the vast potential ribosome profiling has in uncovering crucial insights into the interaction between viruses and their human hosts.
Decoding human cytomegalovirus
Science, 2012; 338(6110):1088-93
Stern-Ginossar, N., Weisburd, B., Michalski, A., Le, V.T.K., Hein, M.Y., Huang, S.X., Ma, M., Shen, B., Qian, S.B., Hengel, H. and Mann, M.
Human cytomegalovirus (HCMV) is particularly prevalent, infecting roughly 65% of the populations of developed countries, and nearly 100% in developing countries. While typically unnoticed in healthy individuals, it can lead to severe symptoms, and even death, in those who are immunocompromised. While transcriptome analysis had been completed, insights into the translatome had previously remained elusive. Here, researchers probed the intricacies of this complex virus using ribosome profiling, providing greater detail in gene expression.
- In addition to confirming previously identified ORFs, researchers utilising ribosome profiling uncovered hundreds of previously unidentified ORFs, such as novel internal ORFs and out-of-frame ORFs.
- A significant proportion of these newly discovered ORFs were particularly short in length, and typically found upstream of longer ORFs, suggesting a regulatory function. However, 120 ORFs encoding for proteins longer than 80 amino acids were also uncovered.
- Researchers were able to use the reads generated by this technology to identify alternative start codons among certain ORFs at different time periods, resulting in production of differentially truncated proteins according to differential temporal timescales.
This work was one of the first to utilise ribosome profiling to analyse a complex DNA virus, yielding a significant haul of novel ORFs, as well as laying out an experimental framework for the investigation of such viruses. It provided a much more detailed landscape of the coding potential of this pathogen.
KSHV 2.0: a comprehensive annotation of the Kaposi’s sarcoma-associated herpesvirus genome using next-generation sequencing reveals novel genomic and functional features
PLoS pathogens, 10(1), p.e1003847
Arias, C., Weisburd, B., Stern-Ginossar, N., Mercier, A., Madrid, A.S., Bellare, P., Holdorf, M., Weissman, J.S. and Ganem, D.
Kaposi’s sarcoma-associated herpesvirus (KSHV) is a recently discovered virus, only being detailed since 1994 in Kaposi’s sarcoma lesions from AIDS patients. It is an oncogenic virus, targeting immunocompromised patients. While the genome of this virus had been sequenced and analysed (largely from in silico analysis), a detailed map of its expression profiles and biological functions of gene products remained unknown. Here, researchers utilised ribosome footprinting, along with other sequencing techniques, to provide a high-resolution view of gene expression and regulation in this virus.
- While previously identified ORFs were confirmed, a further 63 novel ORFs were detected, representing an increase of around 45% in the annotated coding capacity of this virus. Of these, a majority encoded peptides of <100 amino acids.
- Researchers found that ribosomes accumulated at multiple translation initiation sites within existing ORFs, strongly suggesting the presence of alternate splicing events and potentially expanding the known coding capacity of this virus.
- In contrast to the previous hypothesis of PAN RNA (the most abundant RNA transcript in cells infected with this virus) being restricted to the nucleus of the cell acting as a non-coding RNA, researchers found footprints of this transcript in Ribo-Seq data, suggesting that it is both present outside the nucleus and is actively translated. Additionally, 3 short ORFs were identified within this transcript.
- Temporal analysis revealed time-period-specific transcription and translation, with only four messages being transcribed during latency, with this number steadily increasing up to 72 hours following reactivation.
This study greatly expanded the known repertoire of KSHV, while also highlighting the limitations of in silico analysis alone in investigating gene expression. It also gave insights into temporal gene expression in this species. The detailed nature of this analysis makes KSHV an ideal model candidate to study herpesviruses at large.
The coding capacity of SARS-CoV-2
Nature, 2021, 589(7840), pp.125-130.
Finkel, Y., Mizrahi, O., Nachshon, A., Weingarten-Gabbay, S., Morgenstern, D., Yahalom-Ronen, Y., Tamir, H., Achdout, H., Stein, D., Israeli, O. and Beth-Din, A.
This paper analysed the coding capacity of SARS-CoV-2, or more commonly known as COVID-19, responsible for the recent worldwide pandemic. Previous research on the coding regions of COVID-19 had been garnered from computational predictions, relying on homology between it and related coronaviruses. However, this research is the first to utilise ribosome profiling techniques in order to directly determine coding regions of this virus, as well as making a determination on their expression levels.
- The authors uncovered 23 novel ORFs, including upstream ORFs and several in-frame and out-of-frame ORFs embedded within previously identified ORFs, resulting in truncated products and novel polypeptides respectively.
- It was found that viral mRNA is not translated any more efficiently than host mRNA, but dominates due to higher levels of transcription. Interestingly, this correlates with previous work done in the related Murine coronavirus, but contrasts with what is found in HSV-1, a member of the Herpesviridae family. This contrasting information across families may indicate different methods of propagation employed by different types of viruses.
- Ribosome profiling was able to detect the expression of two canonical ORFs not picked up through 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 COVID-19 virus, and as such opens up access to potential therapeutic targets that may be used to treat the infection. It also 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 therapeutics.
High-Resolution Analysis of Coronavirus Gene Expression by RNA Sequencing and Ribosome Profiling
PLoS pathogens, 2016, 12(2), p.e1005473.
Irigoyen, N., Firth, A.E., Jones, J.D., Chung, B.Y.W., Siddell, S.G. and Brierley, I.
In this paper, the authors carried out the first ribosome profiling analysis of an RNA virus, in this case Murine coronavirus, a model organism belonging to the Coronaviridae family, and as such, is closely related to SARS-CoV-2.
- Researchers found that viral mRNA is not translated more efficiently than host mRNA, but simply dominates the translational machinery due to high levels of viral mRNA transcripts formed at later timepoints.
- The authors uncovered several short ORFs either upstream or embedded within existing larger ORFs.
- While ribosome pausing sites were identified within the ORF pp1a, they were found to not significantly associate with ribosome frameshifting sites, casting doubt on the role of ribosome pausing within the function of ribosome frameshifting.
As the first paper to analyse ribosome sequencing data from an RNA virus, it gave particular insights into the mechanisms by which these viruses tend to dominate host translation. It also uncovered a number of previously unannotated uORFs, which give a greater depth of understanding in viral transcriptional regulation.
Widespread disruption of host transcription termination in HSV-1 infection
Nature communications, 2015, 6(1), pp.1-15.
Rutkowski, A.J., Erhard, F., L’Hernault, A., Bonfert, T., Schilhabel, M., Crump, C., Rosenstiel, P., Efstathiou, S., Zimmer, R., Friedel, C.C. and Dölken, L
Herpes simplex virus-1 (HSV-1) is a large DNA virus which replicates within neurons, and is responsible for the common cold sore. However, it can have more severe effects in immunocompromised and immunodeficient individuals. Upon infection and lytic reactivation, HSV-1 quickly initiates a repression of host gene expression. However, with the advent of more powerful technologies, researchers were able to probe the mechanism of this process in greater detail. In this study, the researchers utilised 4sU-Seq and ribosome profiling to gain temporal and translational insights into HSV-1 infection.
- Despite viral HSV-1 mRNA only accounting for 27% of total RNA at 8 hours post infection, viral genes accounted for ~80% of translated mRNAs at the same timepoint, indicating a significant usurpation of host translational machinery.
- 75% of cellular genes were transcriptionally downregulated, with 5.8% upregulated. However, even considering this, only 0.34% of genes were actually translationally upregulated. There were particularly high levels of long intergenic non-coding RNAs (lincRNAs).
- Sequencing data from 4sU-Seq revealed that HSV-1 infection induces an inhibition of transcription termination, resulting in significant “read-in” transcription of downstream genes. It was also found that the previously noted accumulation of introns in HSV-1 infection is due to this “read-in” phenomenon, as opposed to an inhibition of splicing.
- It was uncovered that neither the viral host shutoff (vhs) protein or ICP27 is required for the inhibition of transcription termination, although the latter was suggested to be involved in aberrant splicing within HSV-1 infection.
This study uncovered a fascinating mechanism by which HSV-1 disrupts host cellular homeostasis. Importantly, it challenged a previous hypothesis which suggested that HSV-1 induces an accumulation of introns via a general inhibition of splicing, instead revealing this is largely due to an inhibition of transcription termination. It also set forth an ideal mechanistic framework for investigating gene expression in viruses with particularly short replication cycles.
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