Use of Crispr/Cas9 to Investigate CAR Isoform Function in AdV Infection

Paper: Readler, J. M., AlKahlout, A. S., Sharma, P., & Excoffon, K. J. (2019). Isoform specific editing of the coxsackievirus and adenovirus receptor. Virology, 536, 20-26.

Coxsackievirus infects a range of hosts, including humans, and is linked to a number of diseases such as myocarditis and pancreatic inflammation [2]. Of a different viral family, adenovirus also infects humans, inducing mild diseases such as gastroenteritis and upper respiratory tract infection, in addition to more severe conditions when paired with host immunosuppression. Coxsackievirus is made up of single-stranded RNA, while adenovirus is comprised of double-stranded DNA. Despite having different genomic structures, both coxsackievirus and adenovirus utilize a cell-adhesion protein encoded by the CXADR gene as their primary receptor [2]. This transmembrane receptor exists in two isoforms that are dependent on alternative splicing, a gene expression regulatory process that differentially includes or excludes particular exons of a transcript. The two isoforms, Car^Ex7 and Car^Ex8, have distinct C-terminal ends that lead to different localization in polarized epithelial cells. These polarized cells, as depicted in the image below, are characterized by apical and basal faces that are distinct from one another in structure and function at sites of cell-cell contact [4]. The Car^Ex7  isoform encodes the first seven exons of CXADR and is localized at the basolateral cell surface, while Car^Ex8 includes an additional eighth exon and is localized at the apical cell surface.

Epithelial Cell Polarity: Apical and basolateral surfaces are associated with distinct structures and locations within the cell.
Image source: Figure 4-1. "The Polarity of Epithelial Cells." Adapted from Pearson Education Inc., 2015, p. 116.

            In this study, Readler et al. aimed to better illuminate the functional differences between these two isoforms of the coxsackievirus and adenovirus receptor (CAR). Specifically, researchers chose to focus on the importance of the Car^Ex8 isoform to adenovirus entry at apical cell surfaces. Two key components of this investigation were the Madin Darby Canine Kidney (MDCK) cell line and Crispr/Cas9 gene editing technology. Readler et al. previously created the MDCK cells to stably express human Car^Ex8 cDNA with a promoter that is inducible with the antibiotic doxycycline. With this model cell line established to manipulate the induction of Car^Ex8 expression, researchers then utilized Crispr/Cas9 to knockdown endogenous expression of Car^Ex8 in the MDCK cells. This novel knockdown cell-line thereby isolated the functionality of the Car^Ex8 receptor to a level never achieved before.

           

            To ensure that they successfully targeted the eighth exon of the MDCK CXADR gene, researchers first performed a screen using fluorescence-activated cell sorting and PCR. From this screen, one clone was identified to be a successful knockout, as PCR primers set to recognize the inside of the eighth exon sequence produced no bands. Furthermore, sequencing this clone, renamed JR1-Car^Ex8-KO, verified that the intended deletions were indeed present when compared to MDCK-Car^Ex8 parental cells. After successfully completing this exonic knockout, researchers then investigated total CAR and Car^Ex8  expression levels within the JR1-Car^Ex8-KO cells via western blot analysis. As shown in Figure 3 below, these cells exhibited significantly less expression of Car^Ex8, while showcasing partially reduced levels of total CAR. In both cases, treatment with the antibiotic (Dox), which induced expression of human Car^Ex8 in this cell line, restored expression levels to that of the parental cells. This demonstrated the role of the Car^Ex8 knockdown on the decrease of CAR expression levels, as well as that the insert of human Car^Ex8 was not impacted by the endogenous MDCK Car^Ex8 deletion.

 

Figure 3. CAR expression in JR1-Car^Ex8-KO cells as compared to parental cells. Western blots were performed using polyclonal antibodies detecting A) total CAR expression and B) Car^Ex8 expression alone. Total CAR expression partially decreased while Car^Ex8 reduced significantly in knockout cells (KO) as compared to parental cells (Par). With treatment of 200 ng/mL doxycycline (Dox), KO showed expression restored to the level of Par.

Another important finding of this research was that the less pronounced impact of the knockdown on total CAR expression is likely due to the endogenous levels of Car^Ex7 still maintained in the cell line. As the specificity of CRISPR editing targeted the intronic regions on either side of the eighth exon alone, the seventh exon of CXADR remained intact to generate the majority of CAR expression levels still seen. Another component of this figure worth highlighting is the slight expression of Car^Ex8, despite a seemingly complete knockdown, that was detected via western blot. As compared to the PCR screen (shown below) that showed no amplification of exon 8, the presence of this protein expression in Figure 3B above raises questions as to the true completeness of this knockout. In addressing this discrepancy, Readler et al. offer the possibility that one allele remains, although with primer recognition sites altered, as an explanation for the lack of this Car^Ex8 presence in their PCR analysis. Additionally, they consider non-specific binding within the western blot as a possible reason the fully complete knockdown of Car^Ex8 was not demonstrated within the protein expression levels seen.

 

Figure 1C. PCR knockout screening using primers inside the exon 8 cut site. JR1-Car^Ex8-KO cells (KO) exhibited no band as compared to untreated parental cells (Par) and two haploid knockouts (Hap 1 and 2).

            Despite this inconsistency in their results, Readler et al. determined their knockdown of Car^Ex8 significant enough to investigate the receptor isoform’s importance to adenovirus infection. Using a virus reporter gene, β-galactosidase, researchers quantified the levels of viral-induced luminescence per milligram of protein for MDCK-Car^Ex8 parental and knockout cells. In knockout cells, viral transduction was subjected to a two-fold decrease, suggesting the importance of Car^Ex8 to adenovirus entry. This is supported by Car^Ex8’s known tendency to localize the CAR receptor at apical cell surfaces that are exposed to cellular lumen   where adenovirus is able infect host cells [3]. Further, to look at this apical entry alone, researchers targeted infection to the apical surface of fully polarized MDCK cells and measured genomic levels of adenovirus via qPCR. As shown in Figure 4C below, at 24 hours post-infection a three-fold reduction in adenovirus genome was seen in Car^Ex8-knockouts as compared to parental cells. This difference was restored with the reestablishment of Car^Ex8 induced through Dox antibiotic treatment. This greater fold-change in adenovirus infection when targeted to the apical cell surface supports the notion that Car^Ex8 is essential for the mediation of viral entry at this polarized cell surface, a function that was less pronounced when Car^Ex7 was still allowing adenovirus entry at the basolateral cell surface in these knockouts.

Figure 4C. Cell associated adenovirus genomes in MDCK-Car^Ex8 parental and JR1-Car^Ex8-KO cells. Detected via qPCR, KO cells exhibited a significant reduction in cell-associated viral genomes, as compared to parental and KO cells treated with 200 ng/mL doxycycline.

            Ultimately, this novel Car^Ex8 knockout cell line revealed the importance of this receptor isoform to adenovirus entry at the apical surface of polarized cells. With attenuated expression of Car^Ex8, MDCK cells showed reduced levels of adenovirus transduction and genome levels, which were restored with antibiotic-induced Car^Ex8 expression from a gene insert. The methods utilized here offer a model system for future studies to elucidate further functionalities of the Car^Ex8 receptor using this knockout cell line. Additionally, this methodology could be applied to other genes, such as CD46, which has 14 different receptor isoforms [1]. The use of Crispr/Cas9 editing to create exonic knockouts of this receptor could showcase the interaction between specific CD46 isoforms and the entry of measles or herpesvirus strains [1]. In conclusion, the most notable result of this research is the clinical relevance this method holds in narrowing down receptor isoform correlations to viral entry, as regulation of particular exonic expression could work to inhibit certain strains of viral infection.

References

[1] Excoffon, K. J., Bowers, J. R., & Sharma, P. (2014). 1. Alternative splicing of viral receptors: A review of the diverse morphologies and physiologies of adenoviral receptors. Recent research developments in virology, 9, 1.

[2] He, Y., Chipman, P. R., Howitt, J., Bator, C. M., Whitt, M. A., Baker, T. S., ... & Rossmann, M. G. (2001). Interaction of coxsackievirus B3 with the full length coxsackievirus-adenovirus receptor. Nature Structural & Molecular Biology, 8(10), 874.

[3] Kotha, P. L., Sharma, P., Kolawole, A. O., Yan, R., Alghamri, M. S., Brockman, T. L., ... & Excoffon, K.J. (2015). Adenovirus entry from the apical surface of polarized epithelia is facilitated by the host innate immune response. PLoS pathogens, 11(3), e1004696.

[4] Rappel, W. J., & Edelstein-Keshet, L. (2017). Mechanisms of cell polarization. Current opinion in systems biology, 3, 43-53.

[5] Readler, J. M., AlKahlout, A. S., Sharma, P., & Excoffon, K. J. (2019). Isoform specific editing of the coxsackievirus and adenovirus receptor. Virology, 536, 20-26.

Cardiotonic Steroids May Provide Avenue for the Treatment of Human Adenovirus

In response to: Saha, B., Varette, O., Stanford, W. L., Diallo, J. S., &Parks, R. J. (2019). Development of a novel screening platform for the identification of small molecule inhibitors of human adenovirus. Virology.

           

            Human adenovirus (HAdV) includes a broad group of DNA viruses that can cause infected patients to develop symptoms ranging from respiratory infections or conjunctivitis¹. Over eighty strains of this extremely prevalent nuclear virus have been identified². While this virus is quite prevalent, current anti-viral medications prescribed to people infected with the disease have only limited effectiveness against the disease. Improved anti-viral drug treatments for HAdV are needed because this virus can be fatal in immunocompromised hosts such as patients with AIDS, the elderly or infants³.

           One avenue for developing an effective treatment for human adenovirus involves the identification of small molecules that may disrupt the organization pathways that HAdV uses to infect a cell. When infecting a human host, HAdV must rely on proteins and molecular pathways native to the host cell to generate more viruses that are capable of infecting more host cells. The HAdV hijacks host cell mechanism to multiply and infect other cells in the human host. Small molecules such as selective estrogen receptor modulators have previously been shown to successfully disrupt the propagation of viruses like Ebola by interfering with the viruses’ ability to manipulate host cell proteins and pathways⁴. Until recently, however, very few studies have investigated the effectiveness of small molecules in disrupting the progression of HAdV, and those studies that have investigated the impact of small molecules on the disruption of HAdV have failed to perform a large enough exploration of small molecules⁵. Thus, the Parks laboratory at the Ottawa Hospital designed a new experimental method for visualizing the interaction between HAdV and small molecules that has broad application for testing purposes.

           Researchers at the Parks laboratory ultimately developed a fluoresce tagging protocol that fused a red fluorescent protein (RFP) with the HAdV’s major late promoter (MLP). The MLP is active during the end of HAdV replication process and, consequently, is expressed in greater quantities when the virus is about to spread to other cells. Greater expression of MLP in a cell indicates that the virus is forming more viral particles as the MLP is involved in the construction of structural proteins used for the new viruses to exit the host cell. By fusing a RFP with the MLP, the researchers could determine whether or not specific small molecules were able to disrupt HAdV by visualizing a reduction in MLP activation. HAdV’s that are unable to express MLP would be unable to reproduce effectively and thus the small molecule will have affectively disrupt the virus. 

           After manipulating the HAdV’s genome to include RFP, the virus was incubated with a human lung-derived cell line that was tagged with a green fluorescent protein and infection was allowed to occur. The two different fluorescent colors enabled the researchers to optimize their study and identification technique. The relative amount of green fluorescence permitted the researches to assess whether or not the cells utilized in the experiment were damaged. If the cells were damaged, a decrease in red fluoresce could be the result of a decrease in viable cells instead of a suppression of HAdV replication. Thus, the multiple fluorescent tags improved the validity of the Parks’ study. Researchers quantified the level of RFP in the virally infected cell line after twenty-four hours to determine the base level of RFP in HAdV infected human lung cell lines. 

Figure 1: Imagining of Human Lung Cells Infected with RFP Tagged HAdV. Live-cell images of human lung cells following 18, 21, or 24 respective hours following infection with RFP tagged HAdV. Images display the effectiveness of the tag to increase fluorescence following longer incubation periods in cells and thus higher presence of the virus. 

           After testing the validity of their viral construct through imaging seen in Figure 1, the researchers then used their newly developed fluorescent method to examine the effectiveness of 1200 FDA approved small molecules to inhibit the spread of HAdV. From their screen assays, the Parks laboratory determined that digoxin, digitoxigenin, and lanatoside C were the drugs that yielded the greatest reduction in RFP. All three drugs are cardiotonic steroids that inhibit the activity of Na+/K+ ATPases which are critical for the maintenance of cell membrane potential⁶. Through plaque assays that measure virally induced cell death, the experimenters also showed that exposure to dioxin, digitoxigenin, and lanatoside C lead to a significant reduction in HAdV pathogenicity after twenty-four hours of exposure. Cardiotonic steroids were previously shown in another study to effectively repress the infectious capabilities of HAdV⁷. The combination of the experimenters’ RFP and viral plaque assay findings and the previous work of Grosso et al. provides substantial support for the experimenters’ conclusion that cardiotonic steroids have the potential to be used as effective anti-viral drugs against HAdV. 

Figure 2: Impact of Cardiotonic Steroids on Expression of MLP in HAdV Infected Cells. As the concentration of the cardiotonic steroids (dioxin, digitoxigenin, and lanatoside C) increases in the cell culture there is a decrease in the intensity of RFP in the culture. The decrease in RFP is far greater in the cardiotonic steroid molecules than in the control small molecule (SAHA).

            Ultimately, the Parks’ study is important to the field of virology and the general public for two key reasons. First, the research group was able to construct and execute a new fluorescent technique that could be used to determine the impact of small molecules like cardiotonic steroids on the infectiousness of HAdV. The development of valid visualization methods to monitor HAdV is critically important for the development of effective anti-virals. The Parks’ fluorescence method could potentially be used to monitor the impact of other anti-HAdV treatments beyond small molecules to inhibit the production of late viral proteins in infect cells and thus inhibit the spread of the disease. Additionally, the research group was able to add to the body of literature which has suggested the cardiotonic steroid molecules could be used as an effective anti-viral therapy against HAdV. More research on the impact of cardiotonic steroid molecules on cell cultures infected with HAdV should be performed to determine whether or not these molecules could be used as an effective treatment for people infected with HAdV. Developing an effective anti-viral to HAdV could significantly improve the survival chances and quality of life of immunocompromised people with HAdV. 

1 Lion T. (2014). Adenovirus infections in immunocompetent and immunocompromised patients. Clinical microbiology reviews, 27(3), 441–462. doi:10.1128/CMR.00116-13

2 Binder, A. M., Biggs, H. M., Haynes, A. K., Chommanard, C., Lu, X., Erdman, D. D., … Gerber, S. I. (2017). Human Adenovirus Surveillance - United States, 2003-2016. MMWR. Morbidity and mortality weekly report, 66(39), 1039–1042. doi:10.15585/mmwr.mm6639a2

3 Bhatti, Z., & Dhamoon, A. (2017). Fatal adenovirus infection in an immunocompetent host. The American journal of emergency medicine, 35(7), 1034-e1.

4 Johansen, L. M., Brannan, J. M., Delos, S. E., Shoemaker, C. J., Stossel, A., Lear, C., ... & Lehár, J. (2013). FDA-approved selective estrogen receptor modulators inhibit Ebola virus infection. Science translational medicine, 5(190), 190ra79-190ra79.

5 Duffy, M. R., Parker, A. L., Kalkman, E. R., White, K., Kovalskyy, D., Kelly, S. M., & Baker, A. H. (2013). Identification of novel small molecule inhibitors of adenovirus gene transfer using a high throughput screening approach. Journal of controlled release, 170(1), 132-140.

6 Prassas, I., & Diamandis, E. P. (2008). Novel therapeutic applications of cardiac glycosides. Nature reviews Drug discovery, 7(11), 926.

7 Grosso, F., Stoilov, P., Lingwood, C., Brown, M., & Cochrane, A. (2017). Suppression of adenovirus replication by cardiotonic steroids. Journal of virology, 91(3), e01623-16.

Potentially Killing Two Viruses with One Drug

Based on this paper: Outlaw, V. K., Bottom-Tanzer, S., Krietler, D. F., … Moscona, A. (2019) Dual Inhibition of Human Parainfluenza Type 3 and Respiratory Syncytial Virus with a Single Agent

            Human Parainfluenza Virus Type 3 (HPIV3) and Respiratory Syncytial Virus (RSV) are two common viruses that often infect the respiratory systems of young children and, in extreme cases, can lead to death. There are currently no vaccines for either infection and, while there are some treatments being used for each, they are limited by the ability to distinguish between the similar immunologic responses caused by both viruses, making targeted treatment difficult. Recent research, such as conducted by this group, has been to find methods to inhibit fusion of viral membranes to cell membranes in order to prevent infections from ever taking hold. This study found that the HPIV3 C terminal heptad repeats (HRCs), the end section of the fusion protein, and their mutated derivatives were able to block the fusion of the viral protein with cell membranes in both HPIV3 virions and RSV virions.

            Previous research has demonstrated that during normal viral fusion, the F proteins act by extending the C terminal domain of their protein into the membrane of the host cell. Then, the protein folds back onto itself so the C terminus is adjacent to the N-terminus, the other side of the protein that is still stuck in the viral envelope. This interaction between the HRN and HRC is extremely stable because three proteins come together to form a 6-helix bundle (6HB).¹ The research group was interested to determine if derivatives of the HRC peptide, a smaller portions of the protein, of the HPIV3 virus would inhibit RSV viral fusion. They were specifically using the HRC domain of HPIV3 because previous studies had shown it to be a potent inhibitor of HPIV3 fusion and fusion of other paramyxoviruses.² Although RSV is not a paramyxovirus, researchers were hopeful that its clinical relatedness to HPIV3 may be indicative of similar fusion mechanisms.

Figure 1: Demonstration of viral fusion mechanism. Orange structures are HRN domains, red structures are insertion portions of the viral protein, and green structures are HRC domains. (D) shows the 6HB being formed. The goal of the research group was to make an inhibitor that would replace the green interactions in part (D) such that the HRC of the virus protein could not come together with the HRN of the virus protein.

            The first thing that the researchers needed to do was to find an inhibitor that would have the potential to obstruct viral fusion. Knowing that the HRC of HPIV3 needed to become bound the the HRN region for fusion, the research group made two mutants of the HRC, one with 2 amino acid residue changes (VI) and one with 5 amino acid residue changes (VIQKI). An amino acid residue is simply the building block unit of a protein and the letters are used to denote the changes of specific amino acids from the natural peptide sequence to the mutant sequence. These particular mutations were essential to maintain the ability of the inhibitor to form the aforementioned 6-helix bundle with the HRN. The researchers found that the VI mutant was ineffective at binding with the HRN of RSV, however, the VIQKI mutant had a strong binding affinity to create 6HB with the HRN of both HPIV3 and RSV.

            Next, the group conjugated (joined) the VIQKI mutant with lipid particles, which can insert into the cell membranes and virion envelopes, because lipid conjugation has previously been shown to increase inhibition³. VIQKI was conjugated with two different lengths of lipids and it was found that VIQKI-PEG₂₄-Chol, the longer lipid conjugate, was very effective at inhibiting both HPIV3 and RSV fusion in cell cultures of human airway epithelium, the native target cell of both viruses.

Figure 2: Demonstrates the different amount of inhibition of lipid conjugated VIQKI derivatives. VIQKI-PEG₂₄-Chol is the best inhibitor of both HPIV3 (A) and RSV (B) in cells. Viral infection is undetectable in human epithelial cells until Day 9 when treated with the artificially produced inhibitor.

            Later characterization of the structures that the VIQKI inhibitor formed with both the HPIV3 and RSV HRN peptides confirmed the 6HB that is reminiscent of the native HRC binding for the virion fusion to cell membranes. Interestingly, the opposite end of the mutant VIQKI inhibitor creates different structures in order to stabilize independently with HPIV3 and RSV. The research group then experimented with different derivatives of the mutant VIQKI in order to determine what amino acid sequence would form the most favorable interactions with both virion HRN domains. They found only one derivative that was able to minimally increase the stability of interactions between the VIQKI inhibitor derivative with both of the virus HRNs being examined.

            Overall, this study is an extremely important step in creating a therapeutic drug for both HPIV3 and RSV in young children. This study demonstrated that a peptide derivative of HPIV3, namely the VIQKI-PEG₂₄-Chol derivative, is highly effective at decreasing the cytopathic effects of both viruses by inhibiting viral fusion and entry into potentially infectable cells. Essential to the importance of this study is that a single agent can be used to inhibit both because it would allow for faster treatment because exact diagnosis would not be required prior to the start of treatment. This can hopefully lead to decreases in infant mortality as a result of infection by HPIV3 and RSV.

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1. Outlaw, V. K., Bottom-Tanzer, S., Krietler, D. F., … Moscona, A. (2019). Dual Inhibition of Human Parainfluenza Type 3 and Respiratory Syncytial Virus with a Single Agent. Journal of the American Chemical Society, 141, 12648-12656.
2. Chang, A & Dutch, R. E. (2012). Paramyxovirus Fusion and Entry: Multiple Paths to a Common End. Viruses, 4(4), 613-636.
3. Mathieu, C., Augusto, M. T., Niewiesk, S., Horvat, B., … Moscana, A. (2017). Broad spectrum antiviral activity for paramyxoviruses is modulated by biophysical properties of fusion inhibitory peptides. Scientific Reports, 7(43610), 1-15.
4. Porotto, M., Yokoyama, C. C., Palermo, L. M., Mungall, B., … Moscona, A. (2010). Viral entry inhibitors targeted to the membrane site of action. Journal of Virology, 84(13), 6760-6768.