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Destroying RNA of viruses using Ribonuclease

Destroying RNA of viruses using Ribonuclease


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I wonder if it is possible to design some Ribonuclease to destroy only specific RNAs (like those of viruses). Then, if virus tries to infect, his RNA will be cut.

Or, instead of creating Ribonuclease, we can design suitable mRNA and let the cell do the rest.

Is this possible?


The human body basically does this on its own. We express various RNAses, some of which appear to have specific antiviral or bactericidal roles.

(This appears to be due to both RNAse activity and to biochemical properties of the proteins independent of RNA digestion)


CRISPR/cas is used for exactly this purpose in bacteria. The CRISPR array contains sequences from bacteriophages, which will prime various cas-nucleases to cleave either the DNA or the RNA of the virus.


Expression of an extracellular ribonuclease gene increases resistance to Cucumber mosaic virus in tobacco

The apoplast plays an important role in plant defense against pathogens. Some extracellular PR-4 proteins possess ribonuclease activity and may directly inhibit the growth of pathogenic fungi. It is likely that extracellular RNases can also protect plants against some viruses with RNA genomes. However, many plant RNases are multifunctional and the direct link between their ribonucleolytic activity and antiviral defense still needs to be clarified. In this study, we evaluated the resistance of Nicotiana tabacum plants expressing a non-plant single-strand-specific extracellular RNase against Cucumber mosaic virus.

Results

Severe mosaic symptoms and shrinkage were observed in the control non-transgenic plants 10 days after inoculation with Cucumber mosaic virus (CMV), whereas such disease symptoms were suppressed in the transgenic plants expressing the RNase gene. In a Western blot analysis, viral proliferation was observed in the uninoculated upper leaves of control plants, whereas virus levels were very low in those of transgenic plants. These results suggest that resistance against CMV was increased by the expression of the heterologous RNase gene.

Conclusion

We have previously shown that tobacco plants expressing heterologous RNases are characterized by high resistance to Tobacco mosaic virus. In this study, we demonstrated that elevated levels of extracellular RNase activity resulted in increased resistance to a virus with a different genome organization and life cycle. Thus, we conclude that the pathogen-induced expression of plant apoplastic RNases may increase non-specific resistance against viruses with RNA genomes.


Sources of RNase Contamination

RNases are found in all cell types and organisms from prokaryotes to eukaryotes. These enzymes generally have very high specific activity, meaning miniscule amounts of contamination in an RNA sample is sufficient to destroy the RNA. The major sources of RNase contamination in a typical laboratory include:

  • Aqueous solutions, reagents used in experiments
  • Exposure to RNase from environmental sources (lab surfaces, aerosols from pipetting, ungloved hands, etc.)
  • Contaminated reagents

Picornavirus RNA is protected from cleavage by ribonuclease during virion uncoating and transfer across cellular and model membranes

Picornaviruses are non-enveloped RNA viruses that enter cells via receptor-mediated endocytosis. Because they lack an envelope, picornaviruses face the challenge of delivering their RNA genomes across the membrane of the endocytic vesicle into the cytoplasm to initiate infection. Currently, the mechanism of genome release and translocation across membranes remains poorly understood. Within the enterovirus genus, poliovirus, rhinovirus 2, and rhinovirus 16 have been proposed to release their genomes across intact endosomal membranes through virally induced pores, whereas one study has proposed that rhinovirus 14 releases its RNA following disruption of endosomal membranes. For the more distantly related aphthovirus genus (e.g. foot-and-mouth disease viruses and equine rhinitis A virus) acidification of endosomes results in the disassembly of the virion into pentamers and in the release of the viral RNA into the lumen of the endosome, but no details have been elucidated as how the RNA crosses the vesicle membrane. However, more recent studies suggest aphthovirus RNA is released from intact particles and the dissociation to pentamers may be a late event. In this study we have investigated the RNase A sensitivity of genome translocation of poliovirus using a receptor-decorated-liposome model and the sensitivity of infection of poliovirus and equine-rhinitis A virus to co-internalized RNase A. We show that poliovirus genome translocation is insensitive to RNase A and results in little or no release into the medium in the liposome model. We also show that infectivity is not reduced by co-internalized RNase A for poliovirus and equine rhinitis A virus. Additionally, we show that all poliovirus genomes that are internalized into cells, not just those resulting in infection, are protected from RNase A. These results support a finely coordinated, directional model of viral RNA delivery that involves viral proteins and cellular membranes.

Conflict of interest statement

I have read the journal's policy and the authors of this manuscript have the following competing interests: Eileen Sun now works for Aspyrian Therapeutics.

Figures

Fig 1. Section through a subtomograms from…

Fig 1. Section through a subtomograms from a cryoelectron tomographic reconstruction of a warmed virus-receptor-…

Fig 2. Receptor-decorated liposomes containing fluorescent dye…

Fig 2. Receptor-decorated liposomes containing fluorescent dye detect PV RNA release.

Fig 3. PV infectivity and RNA integrity…

Fig 3. PV infectivity and RNA integrity are not affected by the presence of high…

Fig 4. PV infectivity is not affected…

Fig 4. PV infectivity is not affected by covalent linkage of RNase A to the…

Fig 5. ERAV is co-internalized with RNase…

Fig 5. ERAV is co-internalized with RNase A but infectivity is not compromised.


Programmable Inhibition and Detection of RNA Viruses Using Cas13

The CRISPR effector Cas13 could be an effective antiviral for single-stranded RNA (ssRNA) viruses because it programmably cleaves RNAs complementary to its CRISPR RNA (crRNA). Here, we computationally identify thousands of potential Cas13 crRNA target sites in hundreds of ssRNA viral species that can potentially infect humans. We experimentally demonstrate Cas13's potent activity against three distinct ssRNA viruses: lymphocytic choriomeningitis virus (LCMV) influenza A virus (IAV) and vesicular stomatitis virus (VSV). Combining this antiviral activity with Cas13-based diagnostics, we develop Cas13-assisted restriction of viral expression and readout (CARVER), an end-to-end platform that uses Cas13 to detect and destroy viral RNA. We further screen hundreds of crRNAs along the LCMV genome to evaluate how conservation and target RNA nucleotide content influence Cas13's antiviral activity. Our results demonstrate that Cas13 can be harnessed to target a wide range of ssRNA viruses and CARVER's potential broad utility for rapid diagnostic and antiviral drug development.

Keywords: Arenavirus CRISPR Cas13 Cas13-based detection RNA interference RNA viruses antiviral crRNA design influenza virus multiplexing.

Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.

Figures

Figure 1.. Potential Cas13 target sites are…

Figure 1.. Potential Cas13 target sites are abundant in viral genomes

Figure 2.. Cas13 efficiently reduces levels of…

Figure 2.. Cas13 efficiently reduces levels of viral RNA in mammalian cells

Figure 3.. Identification of Cas13 target sites…

Figure 3.. Identification of Cas13 target sites and crRNA design criteria

Figure 4.. Enhanced Cas13-based antivirals through multiplexing…

Figure 4.. Enhanced Cas13-based antivirals through multiplexing and paired diagnostics

(A) Normalized vRNA levels comparing…

Figure 5.. Additional considerations for improving Cas13-based…

Figure 5.. Additional considerations for improving Cas13-based antivirals


Author information

Affiliations

Department of Genetics, Cell Biology and Development, University of Minnesota, St. Paul, MN, USA

Evan E. Ellison, Maria Elena Gamo & Daniel F. Voytas

Center for Precision Plant Genomics, University of Minnesota, St. Paul, MN, USA

Evan E. Ellison, Maria Elena Gamo & Daniel F. Voytas

Center for Genome Engineering, University of Minnesota, St. Paul, MN, USA

Evan E. Ellison, Maria Elena Gamo & Daniel F. Voytas

Plant and Microbial Biology Graduate Program, University of Minnesota, St. Paul, MN, USA

Department of Plant Biology and The Genome Center, College of Biological Sciences, University of California, Davis, Davis, CA, USA

Ugrappa Nagalakshmi, Pin-jui Huang & Savithramma Dinesh-Kumar

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Contributions

E.E.E. designed the research, performed experiments with assistance from M.E.G., analysed data and wrote the manuscript. The work with tRNAs was carried out by U.N. and P.-J.H. D.F.V. and S.D.-K. supervised the research and helped write the manuscript.

Corresponding author


THE ANGIOGENINS

Similar to the eosinophil RNases, angiogenin had been identified and substantially characterized prior to its identification as a member of the RNase A gene family [67]. Once its amino acid sequence was determined, it was clear that angiogenin (also known as RNase 5) shared the two-histidine, one-lysine catalytic triad, signature CKXXNTF motif, and paired cysteines typical of members of the enlarging RNase A gene superfamily. The angiogenin lineage has also been the subject of rapid diversification [68], and recently, based on evidence from lower vertebrate sequences, Cho and colleagues [41] have suggested that angiogenin, with three as opposed to four paired cysteines, represents the more ancient of the RNase A RNase lineages.

There is one functional angiogenin gene in the human genome and six in the mouse. Of the six mouse angiogenin genes, Hooper and colleagues [20] found that mouse angiogenin 4 was expressed in Paneth cells and had the unexpected property of having bactericidal activity against specific intestinal microbes (Fig. 5). Upon further evaluation, the authors found that mouse angiogenin 1 and human angiogenin also displayed antimicrobial activity, with 100-fold reductions of colony counts of S. pneumoniae and Candida albicans observed in response to low micromolar concentrations of recombinant protein. Interestingly, Avdeeva and colleagues [21] recently reported that a commercial preparation of recombinant human angiogenin was no more effective than an albumin control at inhibiting the growth of S. pneumoniae or C. albicans. The reason for this remarkable discrepancy is not immediately apparent and will require further experimental clarification, although it might be noted that the pathogen in question, S. pneumoniae, is routinely identified by its sensitivity to detergent-mediated lysis (bile solubility test or 2% sodium deoxycholate), and its growth in culture can be inhibited by remarkably small amounts of detergent contaminants.


How RNA viruses copy themselves: Hijack cellular enzyme to create viral replication factories on cell membranes

Nihal Altan-Bonnet, assistant professor of cell biology, Rutgers University in Newark, and her research team have made a significant new discovery about RNA (ribonucleic acid) viruses and how they replicate themselves.

Certain RNA viruses -- poliovirus, hepatitis C virus and coxsackievirus -- and possibly many other families of viruses copy themselves by seizing an enzyme from their host cell to create replication factories enriched in a specific lipid, explains Altan-Bonnet. Minus that lipid -- phosphatidylinositol-4-phosphate (Pl4P) -- these RNA viruses are not able to synthesize their viral RNA and replicate. The key structural components on cell membranes, lipids often serve as signaling molecules and docking sites for proteins.

Viral replication is the process by which virus particles make new copies of themselves within a host cell. Those copies then can go on to infect other cells. An RNA virus is a virus that has RNA, rather than DNA, as its genetic material. Many human pathogens are RNA viruses, including SARS virus, West Nile virus, HIV, and the ones Altan-Bonnet has been studying.

As reported in the May 28, 2010 issue of Cell, Altan-Bonnet and her co-researchers for the first time have uncovered that certain RNA viruses take control of a cellular enzyme to design a replication compartment on the cell's membrane filled with PI4P lipids. Those lipids, in turn, allow the RNA viruses to attract and stimulate the enzymes they need for replication. In uninfected cells, the levels of PI4P lipids are kept low, but in virally infected cells those levels increase dramatically. The findings by Altan-Bonnet and her colleagues not only open several possibilities for preventing the spread of various viral infections, but also may help to shed new light on the regulation of RNA synthesis at the cellular level and potentially on how some cancers develop.

"The goal of the virus is to replicate itself," notes Altan-Bonnet. "For its replication machines to work, the virus needs to create an ideal lipid environment which it does by hijacking a key enzyme from its host cell."

Altan-Bonnet and her team also were able to identify the viral protein (the so-called 3A protein in poliovirus and coxsackievirus infections) that captures and recruits the cellular enzyme (phosphatidylinositol-4-kinase III beta). Additionally, her lab was able to impede the replication process by administering a drug that blocked the activity of the cellular enzyme once it had been hijacked. Drug therapies to prevent viral replication potentially also could be targeted to prevent the hijacking of the enzyme.

Once that enzyme is hijacked, cells are prevented from normally operating their secretory pathway, the process by which they move proteins to the outside of the cell. In many cases, the impeding of that process can result in the slow death of the cell, leading to such problems as cardiac and vascular complications in those infected with the coxsackievirus and neurological damage in those with poliovirus.

Utilizing their recent findings, Altan-Bonnet and her team now plan to investigate PI4P dependence in other viruses as well as the role other lipids may play in different virus families. For example, the SARS virus also requires a lipid-rich environment for its replication, so her lab now is working with SARS researchers on determining what lipid is necessary for that virus's replication. In addition, they will be examining the role of lipids in regulating RNA synthesis in cells, potentially providing new insight into some of the cellular mutations that occur in cancer.

"Given that a lot of what we know about cellular processes historically comes from the study of viruses, our studies may provide insight into the novel roles lipids play in regulating the expression of genetic material in cells," notes Altan-Bonnet.

Altan-Bonnet's research into RNA replication is supported with grants from the National Science Foundation and the Busch Foundation.

Story Source:

Materials provided by Rutgers University. Note: Content may be edited for style and length.


Sources of RNase Contamination

RNases are found in all cell types and organisms from prokaryotes to eukaryotes. These enzymes generally have very high specific activity, meaning miniscule amounts of contamination in an RNA sample is sufficient to destroy the RNA. The major sources of RNase contamination in a typical laboratory include:

  • Aqueous solutions, reagents used in experiments
  • Exposure to RNase from environmental sources (lab surfaces, aerosols from pipetting, ungloved hands, etc.)
  • Contaminated reagents

Scientists Program CRISPR to Fight Viruses in Human Cells

CRISPR is usually thought of as a laboratory tool to edit DNA in order to fix genetic defects or enhance certain traits&mdashbut the mechanism originally evolved in bacteria as a way to fend off viruses called bacteriophages. Now scientists have found a way to adapt this ability to fight viruses in human cells.

In a recent study, Catherine Freije, Cameron Myhrvold and Pardis Sabeti at the Broad Institute of the Massachusetts Institute of Technology and Harvard University, and their colleagues programmed a CRISPR-related enzyme to target three different single-stranded RNA viruses in human embryonic kidney cells (as well as human lung cancer cells and dog kidney cells) grown in vitro and chop them up, rendering them largely unable to infect additional cells. If further experiments show this process works in living animals, it could eventually lead to new antiviral therapies for diseases such as Ebola or Zika in humans.

Viruses come in many forms, including DNA and RNA, double-stranded and single-stranded. About two thirds of the ones that infect humans are RNA viruses, and many have no approved treatment. Existing therapies often use a small molecule that interferes with viral replication&mdashbut this approach does not work for newly emerging viruses or ones that are evolving rapidly.

&ldquoCRISPR&rdquo refers to a series of DNA sequences in bacterial genomes that were left behind from previous bacteriophage infections. When the bacteria encounter these pathogens again, enzymes called CRISPR-associated (Cas) proteins recognize and bind to these sequences in the virus and destroy them. In recent years, researchers (including study co-author Feng Zhang) have reengineered one such enzyme, called Cas9, to cut and paste DNA in human cells. The enzyme binds to a short genetic tag called a guide RNA, which directs the enzyme to a particular part of the genome to make cuts. Previous studies have used Cas9 to prevent replication of double-stranded DNA viruses or of single-stranded RNA viruses that produce DNA in an intermediate step during replication. Only a small fraction of RNA viruses that infect humans produce such DNA intermediates&mdashbut another CRISPR enzyme, called Cas13, can be programmed to cleave single-stranded RNA viruses.

&ldquoThe nice thing about CRISPR systems and systems like Cas13 is that their initial purpose in bacteria was to defend against viral infection of bacteria, and so we sort of wanted to bring Cas13 back to its original function&mdashand apply this to mammalian viruses in mammalian cells,&rdquo says Freije, who is a doctoral student in virology at Harvard. &ldquoBecause CRISPR systems rely on guide RNAs to specifically guide the CRISPR protein to a target, we saw this as a great opportunity to use it as a programmable antiviral.&rdquo

Freije and her colleagues programmed Cas13 to target three different viruses: lymphocytic choriomeningitis virus (LCMV), influenza A virus (IAV) and vesicular stomatitis virus (VSV). LCMV is an RNA virus that mostly infects mice&mdashbut it is in the same family as the virus that causes Lassa fever, which is found in West Africa and is much more dangerous to study in the lab. IAV is a flu virus although some antiviral medications for flu already exist, such viruses evolve rapidly, so there is a need for better options. Finally, VSV is a model for many other single-stranded RNA viruses.

To determine how effective Cas13 was at destroying the viruses, the researchers also used it as a diagnostic tool to see how much viral RNA was being released from infected cells. They saw a twofold to 44-fold reduction in RNA, depending on which virus they were looking at and the time point. They also looked at how well the released RNA was able to go on and infect new cells. In most cases, they saw a 100-fold reduction in infectivity&mdashand in some cases, more than 300-fold&mdashaccording to Freije. The findings were published online on October 10 in Molecular Cell.

&ldquoThe results are very impressive,&rdquo says Chen Liang, a professor at the Lady Davis Institute at Jewish General Hospital and the department of microbiology and immunology at McGill University in Montreal, who was not involved in the study. His own laboratory has used the Cas9 enzyme to deactivate DNA viruses. The concept is very similar, but Cas13 has a few advantages, he says. For one, Cas13 can be used to target one virus using several guide RNAs, making it difficult for the virus to &ldquoescape.&rdquo Secondly, the new study also used Cas13 to detect how much viral RNA was left over to infect cells. The amount of viral knockdown the group achieved is &ldquovery significant,&rdquo Liang says. &ldquoIf you can target and inactivate all three [of these] viruses, in principle, you can inactivate any virus.&rdquo

As with any approach, there are limitations. One is the question of how to deliver the Cas13 to target a virus in a living person, Liang notes, and the researchers have not yet done any animal studies. Another is the fact that viruses will eventually develop resistance. But Cas13 has an advantage here: when Cas9 cuts viral DNA, mammalian cells repair it and can cause mutations that make the virus more resistant. Yet with Cas13, these cells do not have the mechanism to repair the RNA and introduce errors that would help the virus escape being destroyed. Even if a virus does evolve resistance, or if a new virus is encountered, the method could be quickly adapted.

&ldquoOne of the things that&rsquos most exciting about this approach is the programmability,&rdquo says Myhrvold, a postdoctoral fellow at Harvard. &ldquoOnce you figure out how to do this well for one virus it&rsquos not that hard to design sequences against another virus&mdashor another one. Furthermore, if the virus changes its own sequence&mdashas viruses are known to do, just during an outbreak or in response to therapy&mdashyou can very easily update the CRISPR RNA sequence and keep up with the virus.&rdquo

Freije agrees. &ldquoWe are definitely excited about future prospects of optimizing the system and trying it out in mouse models,&rdquo she says. Beyond therapeutics, the team hopes to understand more about how viruses operate&mdashhow they replicate and what parts of their genomes are most important. Using approaches like this, &ldquoyou can really start to get a better picture of what parts of these viruses are and, most importantly, what really makes them tick.&rdquo



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