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Lost in Translation; Cricket Paralysis Virus initiates translation internally

Most of us have heard the term "Central Dogma" in a Biology class at some point in school. DNA is first transcribed into mRNA which is then translated into proteins to make all that you and I consist of.

 Over the years, the Central Dogma has been revisited and new information has come to light, such as the processing of eukaryotic transcripts. This processing includes the addition of a 5' cap. After the RNA has been processed, then translation of the RNA strand into a functional polypeptide can begin.

Figure 1: Eukaryotic translation initiation
Translation begins by the recruitment of various molecules to the 5' cap, including numerous initiation factors, the small ribosomal subunit, and Met-tRNA, which translates a specific "start codon" into methionine, the first amino acid in the polypeptide chain. This assembly of various enzymes and factors then scans the length of the RNA until it reaches the start codon, which signals for translation to begin by binding the large ribosomal subunit and releasing the initiation factors.


Next, the tRNA carrying the amino acid of the adjacent corresponding codon can bind in a spot in the ribosome complex called the A spot, next to the met-tRNA (which is bound in the P spot). The RNA can be translated into a polypeptide by moving down the length, codon by codon, and recruiting t-RNA's to the A spot to elongate the polypeptide.
Viral mRNA often lacks processing and as a result may not have a 5' cap or the same AUG-met start codon that eukaryotic transcripts have, yet they use the transcription machinery of the host to transcribe their viral proteins. Nevertheless, the ribosomal subunits are recruited and transcription is initiated in eukaryotic hosts in spite of viral mRNA lacking the initiation methods eukaryotes use.


Figure 2: Four types of IRES and the required initiation factors 
One strategy used by viruses depends on structured sequences in their mRNA that allow them to bypass many or all aspects of traditional eukaryotic initiation. These structured RNA sequences, called IRES (Internal Ribosomal Entry Site), can be categorized according to their degree of dependence on Initiation Factors. 

Perhaps one of the most impressive of the viral IRES is the Cricket Paralysis Virus, CrPV IRES, which requires no initiation factors and bypasses the need for a 5' cap and AUG start codon to initiate translation. In 2016, Murray et al. at MRC Laboratory of Molecular Biology, Cambridge, United Kingdom, used cryo-EM to resolve the detailed structure of the CrPV IRES bound to the small ribosomal subunit and discover how it recruits the large subunit to construct a functionally competent ribosome complex.

 They were able to observe the functionally significant regions of the IRES responsible for its binding to the small ribosomal subunit (40S). These were the stem loop regions IV and V (SL-IV&SL-V). They were also able to observe pseudoknot I in the decoding center of the ribosomal complex. This pseudoknot I, or PK-I, is a section of the IRES that mimics the met-tRNA in traditional eukaryotic translation initiation. It tricks the small ribosome into believing that met-tRNA has bound and scanned to a start codon. It can then recruit the large ribosomal subunit to create a functional ribosome and begin translation.

 The video below shows the 3D structure of the small ribosome (40s head and body) with the pseudoknot I  (PK-I) bound.

Their research also provided information on the structure of the ribosome-IRES complex and the shift of PK-I of the IRES from the A site to the P site of the ribosome, which is brought about by a GTP-binding elongation factor, eEF2. This presented the first images of eEF2 docked on the ribosome with a tRNA or tRNA mimic such as the PK-I of CrPV IRES.
The video below shows the PK-I of the CrPV IRES bound in the A spot of the ribosome complex being shifted into the P spot by the elongation factor eEF2.




The ability of the Cricket Paralysis Virus to bypass traditional eukaryotic translation initiation methods allows the virus to replicate and produce viral proteins from its RNA without having undergone any modifications. This is especially impressive when we consider that initiation is accomplished without the aid of any enzymes and proteins, but with the viral mRNA alone! Continued research and studies on the CrPV IRES and other IRES's will increase our understanding of the versatility of RNA and may one day lead to new drug targets and treatments in medicine.


References

  1. Marintchev, A. and Wagner, G. (2004). Translation initiation: Structures, mechanisms, and evolution. Quarterly Reviews of Biophysics, 37(3/4), 214-215. http://dx.doi.org/10.1017/S0033583505004026. Retrieved from https://gwagner.med.harvard.edu/sites/gwagner.med.harvard.edu/files/marintch
  2. Murray, J., Savva, C. G., Shin, B.-S., Dever, T. E., Ramakrishnan, V., & Fernández, I. S. (2016). Structural characterization of ribosome recruitment and translocation by type IV IRES. eLife, 5, e13567. http://doi.org/10.7554/eLife.13567
  3. Kuo-Ming Lee, Chi-Jene Chen, & Shin-Ru Shih (2017). Regulation Mechanisms of Viral IRES-Driven Translation. Trends in Microbiology, July 2017, Vol. 25, No. 7. http://dx.doi.org/10.1016/j.tim.2017.01.010
  4. David A Costantino, Jennifer S Pfingsten, Robert P Rambo & Jeffrey S Kieft (2008). tRNA–mRNA mimicry drives translation initiation from a viral IRES. Nature Structural & Molecular Biology Vol. 15, No. 1, 57-63 http://www.nature.com/nsmb



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