Protecting Picornavirus Genomes from RNAse Degradation within Endosomes

Submitted by Winter Kemppainen

The research article, Picornavirus RNA is protected from cleavage by ribonuclease during virion uncoating and transfer across cellular and model membranes,1 outlines the results from a study that investigated how the RNA of Picornaviruses is transferred from the virion into the host cell’s cytoplasm without degradation by ribonucleases. The research focuses specifically on the well-known poliovirus, belonging to the enterovirus genus. This summary includes background information regarding Picornaviruses and their entry into host cells, a review of the questions and models posed by the study, and a discussion of the results presented in the article.

Picornaviruses belong to the family, Picornaviridae, that contains six different genera. Picornaviruses are small, icosahedral viruses. Their virion diameters measure 18-30 nm in length. The viruses are non-enveloped, meaning the virion capsids are not enclosed in a protective membrane. The genomes of Picornaviruses are made up of single-stranded, positive-sense RNA (+ssRNA). The +ssRNA acts as mRNA in host cells to encode a single polyprotein that later gets cleaved into smaller, functional proteins after transcription.2

Non-enveloped Picornaviruses, such as poliovirus, enter their host cell via receptor-mediated endocytosis. This process occurs by virion attachment at specific receptor proteins located on the cell membranes of the host cells. Polioviruses bind to CD155 Poliovirus Receptors (PVR) of their host cells. It is important to mention that the PVRs alter the size of the virions from 160S to 135S after attachment, resulting in a 4% enlargement.1 Once bound to the outer cell membrane of the host, the virion is brought into the cell by an inversion of the cell membrane to create a vesicle for the virion, termed an “endosome” by the authors.

During the endocytotic process, the virion is not the only particle brought into the host cell. The serum within which the virion was travelling through before host cell attachment, and enzymes within the serum also get enveloped by the endosome and delivered into the cell. One of the enzymes that typically included is ribonuclease A (RNase A). Ribonucleases are enzymes that catalyze the breaking up and degradation of RNA molecules.3 The role of RNases are to regulate mRNA expression and protect cells from foreign nucleic acids.

It is important to note the significance of the RNase A as it gets brought into host cells along with viruses carrying RNA. This study aims to answer four questions involving the possible interactions between the endosome, virion, and RNases during RNA transfer to the cytoplasm:

“a) is the RNA released from random positions on the particle, only a random <10% of which are adjacent to the membrane?

  1. b) does the attachment process induce a polarization of the particle so that RNA is only released from a position adjacent to the membrane?
  2. c) can RNA released into the endosomal lumen traverse the membrane to reach the cytoplasm?
  3. d) is the RNA protected during transmission across the membrane from RNases that might be present in the endosomal lumen?”1

The answers of these questions have the potential to shed light on the unclear mechanism of how the Picornavirus viral RNA enters host cell cytoplasm without degradation by ribonucleases.

The article poses three possibilities for RNA translocation. The first possible model involves disruption of the endosomal membrane to release the contents of the vesicle into the cell cytoplasm. The second model involves the disruption of the endosomal membrane caused by the insertion of viral peptides. The third model suggests peptides interact with the endosome membrane and the virion to create a channel through which the RNA travels through.1 The results of this study suggest the first two models are improbable, while the third model provides a probable explanation for Picornavirus RNA transfer.

The study first tested the ability of viral RNA to be translocated into a vesicle by virus uncoating. Cryoelectron tomographic imagery was used to capture viral +ssRNA insertion into an in vitro liposome (mimicking an in vivo endosome).1

The liposomes contained poliovirus receptors on their outer membranes to facilitate virus attachment. The entry of viral RNA into the receptor-decorated liposomes supports the idea that RNA is able to travel through channels or pores, and that membrane disruption is not a necessary step in RNA translocation.

The research study also found that the translocated RNA in the liposomes was insensitive to RNase A.1 YoPro-1 fluorescence was used to monitor nucleic acid binding inside and outside of the liposomes, while RNase A was added only to the outside of the liposomes. Fluorescence microscopy was used to observe RNA survival in the presence of RNase A.

The preservation of the viral RNA across the liposome membrane in the presence of RNase A further supports that polioviruses are able to transfer their genomes across a lipid membrane without degradation by ribonucleases. The channels through which viral RNA travel are able to protect the nucleic acid from potentially fatal environments.

The study expanded the research by attempting in vivo experiments to determine whether or not viral RNA was translocated successfully in cultured cells and protected from RNase A in similar ways observed in the in vitro experiments. HeLa Ohio cells were infected with poliovirus in the presence of RNase A. The extracellular fluid was marked with dextrans, a red fluorescent marker. The polioviruses were marked with fluorescent dye Cy2 that fluoresces green. When overlapped, the two markers fluoresce yellow. The results of the experiment were viewed with fluorescent microscopy.

The results of the in vivo experiment indicate that extracellular fluid and material is taken into the cell along with the poliovirus during endocytosis. Assuming RNases could be present in the extracellular fluid and could be taken into the cell with the viruses, the possibility of a protected gateway through which viral RNA can travel through to avoid degradation is even more likely.

Finally, the infectivity of poliovirus was tested while the virus particles were covalently linked to RNase A.1 Linkage of the virus to the ribonuclease ensured that the RNase would be taken into the host cell with the virus. The poliovirus was fluorescently labeled with Cy2 marker. The RNase was fluorescently labeled with DyLight-594 marker. Fluorescence microscopy was used to show the interaction between the poliovirus and RNase A enzymes inside a host cell. The results indicate that the polioviruses were unaffected by the RNase A and were still able to infect the host cell. This result further supports the hypothesis that viral RNA insertion is protected from RNase A degradation.

The results of the study suggest plausible answers to the four questions posed regarding RNA translocation. The experiment suggests that RNA release may have certain directionality, of which could be a result of the altered virus particle interacting biochemically with the cellular membrane. Questions a) and b) cannot be fully answered according to these results, but they provide a strong foundation for further research. The answer to question c) is that RNA can travel through endosomal lumen, but in the presence of ribonucleases, needs to be protected. The answer to question d) is yes, RNA is protected during transmission to the cytoplasm. The experiments indicate that while RNase A is present and able to catalyze the degradation of RNA, the poliovirus remains viable within the host cell and continues with infection. At this point, infection can only be possible if the viral RNA is protected from the RNases.

While the results of the study were not capable of answering all the questions posed in full clarity, the model in which Picornaviruses insert their +ssRNA into the host cell’s cytoplasm was narrowed down quite clearly to the third model: that channels are formed to allow the viral RNA to translocate safely to the cytoplasm.1 Figure 1 outlines a simplified prediction of this channel-formation model.

Polio genome protection.jpg

Fig. 1. Simplified model of Poliovirus RNA translocation inside host cell. As poliovirus binds to the CD155 Poliovirus Receptor on the cell membrane, endocytosis takes in the virus along with RNase As within the extracellular serum. A channel forms between the endosome membrane and the virus particle to allow the +ssRNA to leave the endosome and enter the cytoplasm.

The other two models involving membrane disruption may be ruled out in this case, because the results show that RNase A is capable of degrading viral RNA. If membrane disruption were to occur, the RNases would be released along with the viral RNA into the cytoplasm, and there would degrade the RNA before transcription could proceed. Based on the results from this study, picornavirus RNA must be protected at all times from RNase A to successfully infect its cellular hosts.

The study admits that there is still debate on how protein channels could form between virus particles and the endosomal membrane. However, this research provides some clarity on how Picornaviruses translocate their +ssRNA to infect their hosts. This study has laid the foundation for further research to be done to try to determine the mechanisms of viral RNA protection as it gets transferred to the host cell cytoplasm.



  1. Groppelli E, Levy HC, Sun E, Strauss M, Nicol C, Gold S, et al. (2017). Picornavirus RNA is protected from cleavage by ribonuclease during virion uncoating and transfer across cellular and model membranes. PLoS Pathog 13(2): e1006197.
  2. Collier L, Kellam P, Oxford J.(2016). Picornaviruses: Polio, hepatitis A, enterovirus, and common cold. In Human Virology(5th ed., pp. 82-91). Oxford, UK: Oxford University Press.
  3. (n.d.). Ribonucleases (RNases) | Thermo Fisher Scientific – US. Retrieved September 22, 2018, from

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