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Summer Research Fellowship Programme of India's Science Academies

Study of host cell proteins in HCV biology

Vedatrayee Chattopadhyay

Amity Institute of Biotechnology Kolkata, Amity University, Major Arterial Road (South-East), Action Area II, Newtown, Kolkata, West Bengal 700135

Dr. Binay Chaubey

Department of Botany, University of Calcutta, Ballygunge Circular Road, Kolkata, West Bengal 700019

Abstract

Hepatitis C is a liver infection caused by the Hepatitis C virus (HCV) leading to serious liver damage. In this project, we have used the genome editing technique CRISPR/ cas9 using a LentiCRISPR construct to knock-out the La gene that encodes the La autoantigen protein. This
host protein interacts with Hepatitis C virus (HCV) internal ribosome entry site (IRES) and stimulates the replication and translation of HCV RNA. In this study, first we have ligated the gRNA oligos with the LentiCRISPR v2 vector which were then transformed into the Top10
E. coli cellsf that were made chemically competent previously. In the next step, we would have transfected the Huh 7.5 cells with the LentiCRISPR construct to achieve the knock-out of La gene in these cells, followed by screening and verification of the knocked-out product but due to time constraints, we were unable to complete our experiments. Hence, in the available time, we were only able to clone the gRNA oligos into our digested LentiCRISPR v2 vector and verify it using PCR and Agarose Gel Electrophoresis.

Keywords: Hepatitis C, Hepatitis C virus (HCV), Internal ribosome entry site (IRES), La autoantigen protein.

INTRODUCTION

HCV Biology

Hepatitis C is a liver disease caused by the hepatitis C virus (HCV), a blood borne virus that causes both acute and chronic hepatitis, ranging in severity from a mild illness lasting a few weeks to a serious, lifelong illness. According to a report by W.H.O., globally around 71 million people have chronic hepatitis C infections and are likely to develop cirrhosis or liver cancer and till now, there are no vaccines available for this disease. HCV causes chronic hepatitis, which may result in tissue damage, fibrosis, cirrhosis, and the eventual development of hepato-cellular carcinoma.

HCV, a small RNA virus belonging to the family Flaviviridae and genus hepacivirus, has a single-stranded RNA genome packaged by a core protein that is enveloped by a lipid bilayer containing two viral glycoproteins to form the virus particles or virion. The lifecycle of HCV begins with the attachment of a virion to its specific receptor on hepatocytes and after binding with its receptor complex, the virion is internalized and the nucleo-capsid is released into the cytoplasm. Once in the cytoplasm, the virus uncoats itself to free the genomic RNA which is then used for polyprotein translation and replication in the cytoplasm.(1) HCV replication takes place within the “replication complex” containing the viral non-structural proteins and cellular proteins and the HCV translation is mediated by an internal ribosome entry site (IRES).(3)

Human La autoantigen, a RNA-binding protein that is involved in initiation and termination of RNA polymerase III transcription (2), has been shown to influence the internal initiation of translation of hepatitis C virus (HCV) RNA (Pudi et al. Journal of Biological Chemistry, 2003 and 2004). RRM2, one of the RNA Recognition Motifs of La, interacts with HCV internal ribosome entry site (IRES) around the GCAC motif near the initiator AUG present in the stem region of stem-loop IV.(3)

Introduction to CRISPR/cas9

The CRISPR-cas9 is a gene-editing technology that originated from the type II CRISPR-Cas system in bacteria which provides them with adaptive immunity against foreign particles like viruses and plasmids. The CRISPR-associated protein, Cas9 is an endonuclease that uses a guide sequence within an RNA duplex, tracrRNA: crRNA, to form base pairs with DNA target sequences, enabling Cas9 to introduce a site-specific double-strand break in the DNA.(4)

The dual RNA duplex, tracrRNA: crRNA was then modified as a single guide RNA (sgRNA) which created a simple two-component system in which changes in the sequence of the sgRNA Cas9 can target any DNA sequence of interest. Due to the simplicity of CRISPR-Cas9 system, it can be used to precisely and efficiently target, edit, modify, regulate, and mark genomic loci of a wide array of cells and organisms.(5)

Since HCV interacts with some host cell factors like La protein to gain entry to the host cells, hence in this project, we tried to knock-out the gene encoding La protein using CRISPR-cas9 technology to understand its role in HCV biology and to observe the effects on the host cells.

LentiCRISPR v2 vector

fig1.PNG
    LentiCRISPR v2 vector

    In this project, we used the shuttle vector LentiCRISPR v2, a mammalian expression vector of lentiviral origin, to achieve the knock-out of the La protein. This vector has two antibiotic resistance genes, namely Ampicillin and puromycin which can be used as selectable markers for E. coli and the human cells respectively. This LentiCRISPR v2 vector has around 4200bp cas9 cassette, encoding cas9 from Streptococcus pyrogenes and also has a filler region for the guide oligos, under the U6 promoter. Different guide oligos targeting different genes can be designed by using CHOP-CHOP, a web-based bioinformatics tool.

    Objectives of the Project

    • Designing of gRNA oligos, specific against La protein
    • Designing of vector-specific primers for the PCR verification
    • Transformation of recombinant LentiCRISPR v2 vector in the competent E. coli Top10 cells
    • Transfection of recombinant LentiCRISPR v2 vector in human hepatocyte derived cellular carcinoma cell line (Huh 7.5 cell line).

    LITERATURE REVIEW

    Scientists found out that the human La autoantigen interacts with the internal ribosome entry site (IRES) of hepatitis C virus (HCV) (Pudi et al. (2003)) and binds at the GCAC Site near the initiator AUG and is implicated in the Internal Ribosome Entry Site-mediated Translation process of HCV (Pudi et al. (2004)). Later on they showed that a peptide derived from La protein has the potential to inhibit the internal initiation of HCV translation (Pudi et al. (2005)).

    METHODOLOGIES

    Concepts- Designing of La specific gRNA oligos

    For designing the La specific gRNA oligos, CHOPCHOP, an online web-based bioinformatics tool was used. On opening the webpage, a search box appears consisting of four drop-down menus.

    fig2.PNG
      CHOPCHOP Home Page

      1. In the first drop-down menu, we can enter the RefSeq id or the ENSEMBL id or the gene name or the genomic coordinates of the target sequence.

      2. In the second drop-down menu, we can select the reference genome of the organism on which we will be conducting the experiment.

      3. In the third drop-down menu, we have to select the type of gene-editing tool we will be using in our experiment and

      4. In the fourth drop-down menu, we have to select what kind of experiment we will be performing.

      For our experiments, in the first drop-down menu of the search box, we entered “SSB” as the target sequence. SSB is the gene that encodes the La autoantigen. Then we selected “Homo sapiens” as our reference genome from the second drop-down menu. From the third drop-down menu, we selected “CRISPR/cas9” as our gene-editing tool and finally, in the fourth drop-down menu, we entered “knock-out” as our experiment.

      We clicked on the “Find Target Sites” button after all the details are entered and then the result was displayed containing the details about different gRNA target sites. On clicking the target sites, primer pairs flanking the target site and the restriction sites are displayed. From the list, we selected the first target site as it has least off-target sites the gRNA can anneal to. After clicking on the first target site, a new page appeared that displayed the complete sequence and the suggestions for primers flanking the target sequence as well. From here, we came to know about our target sequence and we made the target sequence compatible with our vector by the addition of nucleotides at the end of the gRNA.

      fig3.PNG
        Results of CHOPCHOP.

        fig4.PNG
          Individual targets

          fig5.PNG
            Target Sequence Cloning Protocol

            fig6.PNG
              gRNA Oligos for La protein

              Methods

              Culturing of TOP10 E. coli cells: Primary Culture

              E. coli TOP10 cells containing the LentiCRISPR v2 vector were inoculated in four culture tubes, each of them having 5 ml of 2% Luria broth (LB) medium. Each tube was inoculated with a single bacterial colony. After inoculating the media, each of the four tubes were kept in the incubator-shaker overnight at 37° C.

              TOP10 cells are a strain of E. coli, which are used for cloning and plasmid preparation. They have high transformation efficiency and have the genes for recA (a recombinase) and endA (a DNAse) knocked out as these genes lowers plasmid yield. 

              Isolation of LentiCRISPR v2 vector

              Using the alkaline lysis method, we have isolated the plasmid vectors (LentiCRISPR v2) from the overnight culture of Top10 E. coli cells.

              For the alkaline lysis method, we prepared three solutions, Solution I, II and III.

              Composition of Solution I

              1. 50 mM glucose: Glucose maintains the osmotic pressure, preventing the cells from bursting out.

              2. 25 mM Tris.Cl (pH 8.0): Tris maintains the pH of the bacterial cells around the pH 8.

              3. 10 mM EDTA (pH 8.0): EDTA chelates the divalent ions and inhibits the action of nuclease enzymes.

              100 ml of Solution I was prepared, autoclaved at 10 psi for 15 minutes and was stored at -4°C.

              Composition of Solution II

              1. 0.2 N NaOH: Sodium hydroxide breaks open the cell wall and releases the cellular constituents like the plasmid DNA and sheared cellular DNA.

              2. 1% SDS: Sodium Dodecyl Sulfate or SDS is an anionic detergent which solubilizes the cell membrane and denatures most of the cellular proteins.

              Solution II was freshly prepared and was stored at room temperature.

              Composition of Solution III

              1. 5 M Potassium acetate 60 ml: Potassium acetate reduces the alkalinity of the solution enabling the single-stranded DNA to renature into its double-stranded form.

              2. Glacial acetic acid 11.5 ml: Glacial acetic acid neutralizes the pH of the solution which allows the single-stranded DNAs to renature.

              3. Water 28.5 ml

              Solution III was stored at -4°C.

              Procedure

              1. The four overnight culture tubes were taken out from the incubator-shaker, checked for the bacterial growth by observing the level of turbidity and were centrifuged at 6000g for 5 minutes at room temperature to obtain the pellets.

              2. The supernatant was discarded and the pellet was kept in the Eppendorf tubes.

              3. The pellet was re-suspended in 100ul of ice-cold Solution I by vortexing.

              4. Then, 200ul of Solution II (at room temperature) was added to the suspension and mixed by gently inverting the tubes. Then it was incubated on ice for 5 minutes.

              5. After 5 minutes incubation on ice, 150ul of ice-cold Solution III was added and mixed by gently inverting the tubes. This was then incubated at ice for 5 minutes.

              6. The mixture was then centrifuged at 12000g for 5 minutes at 4°C and the supernatant was carefully recovered in a fresh tube.

              7. Two volumes of isopropanol were added to the supernatant to precipitate out the plasmid DNA. After adding isopropanol, the tube was gently inverted for mixing thoroughly. This was centrifuged at 12000g for 5 minutes at 4°C.

              8. After centrifugation, the supernatant was discarded. The pellet was then washed with 70% ice-cold ethanol and air-dried till the ethanol got evaporated.

              9. After air-drying, the pellet was dissolved in pure autoclaved distilled water and was stored at -20°C.

              Preparation of competent E. coli TOP10 cells

              We have used E. coli TOP10 cells for our experiment. The Top10 cells are a strain of E. coli, which are used for cloning and plasmid preparation.  These have high transformation efficiency and have the genes for recA (a recombinase) and endA (a DNase) knocked out as these genes lower the plasmid yield.

              Procedure

              1. To a conical flask containing freshly prepared 99 ml of LB media, 1ml of overnight culture of E. coli TOP10 cells were added and this was incubated for 3-4 hours at 37°C in a shaker incubator.

              2. The reagents were freshly prepared and stored at 4°C. Even the disposable tubes and glassware were kept at 4°C.

              3. Then the culture was centrifuged at 2700rpm for 10 minutes at 4°C.

              4. The supernatant was discarded and the pellet was dissolved in ice-cold 20ml 100mM CaCl2. This was incubated on ice for 15 minutes and centrifuged at 2700rpm for 10 minutes at 4°C.

              5. Again the supernatant was discarded and the pellet was dissolved in ice-cold 20ml 100mM CaCl2. This was incubated on ice for 15 minutes and centrifuged at 2700rpm for 10 minutes at 4°C.

              6. Then, the supernatant was discarded and the pellet was re-suspended in 5ml of ice-cold 85mM CaCl2 (100mM CaCl2 containing 15% glycerol as antifreeze). This was then stored in 5 Eppendorf tubes at -80°C.

              Composition of 250ml of 100mM CaCl2

              1. 3.675g CaCl2.2H2O (Molecular weight: 147.02)

              2. 250ml autoclaved distilled water.

              Composition of 100ml of 85mM CaCl2

              1. 85ml of 100mM CaCl2

              2. 15ml Glycerol.

              Restriction digestion of LentiCRISPR v2 vector using the enzyme BsmBI

              LentiCRISPR v2 vector was digested with the restriction enzyme BsmBI. After digestion with the restriction enzyme, we observed two fragments having different molecular weights. One fragment was of around 12.8 kb and the other fragment was of 2 kb, which was observed on Agarose gel. The heavier fragment was the linearized part of the LentiCRISPR v2 vector that we will be using for the ligation reaction and the lighter fragment is the filler region.

              After the separation by Agarose gel electrophoresis, we extracted the larger fragment by electroelution. The purified larger fragment was treated with alkaline phosphatase to prevent it from getting re-circularized. We then purified the digested vector using Phenol: Chloroform: Isoamyl alcohol, followed by precipitation using 5M Sodium acetate and 100% ethanol. Then washing of the digested vector was done using 70% ethanol and finally the vector was dissolved in autoclaved distilled water and stored at -20°C for further use.

              Composition of the reaction mixture for restriction digestion (50 ul X 3)

              re digestion_1 table.PNG
                Composition of restriction digestion reaction mixture

                The reagents were added one by one in an Eppendorf tube and were incubated at 37° C for 1 hour. After that, the contents of all the three Eppendorf tubes were pulled into a single tube.

                Composition of the reaction mixture for alkaline phosphatase treatment

                alk p04atase_2 table.PNG
                  Composition of the reaction mixture for alkaline phosphatase treatment

                  The reagents were mixed in a tube and incubated at 37° C for 30 minutes after which they were heat inactivated at 65° C for 10 minutes.

                  Ligation of the LentiCRISPR v2 vector with gRNA oligos

                  The digested vector and the insert was ligated with T4 ligase, in the ratio of 1:5 (vector: insert) after the quantification was done using Nanodrop.

                  dna quanti_3 table.PNG
                    DNA quantification values

                    After quantification using Nanodrop, the reagents were diluted 50X for achieving the 1:5 ratios and was mixed in the following manner:

                    ligase_4 table.PNG
                      Ligation reaction mixture

                      After mixing the reagents, they were incubated overnight at 16° C.

                      Transformation of the competent E. coli TOP10 cells

                      Using the Heat-Shock method, we transformed the competent E. coli TOP10 cells with our recombinant LentiCRISPR v2 vector.

                      Procedure

                      1. The competent cells and the recombinant vector were thawed on ice.

                      2. Three micro-centrifuge tubes were taken and onto each 60 ul of competent cells were added, and no, 2 ul, and 4 ul of the recombinant plasmid were added into the tubes respectively. Then the tubes were incubated on ice for 30 minutes.

                      3. Then heat-shock was given by placing the tubes in water bath at 42°C for 90 seconds. The tubes were again incubated on ice for 20 minutes.

                      4. After 20 minutes, the contents of the three tubes were transferred to 3 culture tubes containing 1ml of LB media each. The culture tubes were incubated at 37°C for 1 hour in a shaker-incubator.

                      5. Earlier, five Petri-plates were prepared using 20ml of autoclaved LB-Agar media and freshly prepared 100ug/ml Ampicillin solution.

                      6. After 1 hour, the three culture tubes were taken out from the incubator and from the tube 1, 100ul culture was spread onto the plate 1. From the second tube, 100ul and 200ul culture was spread onto the plates 2 and 3 respectively. Finally, from the third tube, again 100ul and 200ul culture was spread onto the plates 4 and 5 respectively. The plates were incubated overnight at 37c.

                      Selection of recombinant colonies, plasmid isolation and insert verification via PCR

                      1. We inspected the overnight incubated plates the next day. Growth was seen on all the 4 plates, except the 1st one, which was the negative control.

                      2. Then we inoculated 4 culture tubes containing 5ml of freshly prepared LB-Amp media with random colonies from the 4th plate.

                      3. The 4 culture tubes were incubated at 37°C for 2 hours or till turbidity was seen.

                      4. After seeing the turbidity, one of the culture tubes was selected and from it, 1ml of culture was used to two conical flasks having 50 ml of LB-Amp media each. The conical flasks were incubated overnight at 37°C.

                      5. The next day, we used alkaline lysis method to isolate plasmids from the two conical flasks and this was used as a template for the PCR reaction, using LentiCRISPR v2 vector-specific and gRNA oligo-specific primers. This is done to validate the presence of the insert in the vector.

                      The PCR profile is as follows

                      ligase_4 table.PNG
                        PCR profile
                        fig7.PNG
                          PCR profile on-screen

                          RESULTS AND DISCUSSION

                          Isolation of LentiCRISPR v2 vector

                          Using the alkaline lysis method, we isolated our plasmid vector (LentiCRISPR v2) from the transformed E. coli TOP10 cells and qualitatively observed it by Agarose Gel Electrophoresis.

                          fig8.PNG
                            LentiCRISPR V2

                            The 1st lane contains previously isolated LentiCRISPR V2 vector, which is used as a control. The lanes from 6th to 11th contain our isolated vector.

                            Restriction Digestion of LentiCRISPR V2 vector

                            The plasmid vector (LentiCRISPR V2) that we isolated using alkaline lysis method was digested with the restriction enzyme BsmBI. The digested products were then visualized using Agarose Gel Electrophoresis.

                            fig9.PNG
                              Restriction Digestion

                              The 1st lane contains undigested LentiCRISPR V2 vector, which was used as a control. Our digested products were loaded onto the 2nd, 3rd and 4th lanes.

                              Whenever we incubated the vector with the restriction enzyme BsmBI for more than one hour, the vector started to get degraded which was seen as a smear on performing Agarose gel electrophoresis.

                              Ligation of the vector with gRNA oligos

                              The linearized digested LentiCRISPR v2 vector and the insert was ligated with T4 ligase, in the ratio of 1:5 (vector: insert) after the quantification was done using Nanodrop.

                              Transformation of the E .coli TOP10 cells with our recombinant vector

                              After the ligation experiment, we transformed the competent E. coli TOP10 cells with the recombinant plasmid (LentiCRISPR v2 vector ligated with the insert).

                              After performing transformation, we inoculated 5 plates containing LB-Agar-Amp solid media with the transformed cells, keeping the 1st plate as our negative control. Hence, colonies were only observed in the 2nd, 3rd, 4th and the 5th plates.

                              fig10.PNG
                                Plate 1: no colonies were observed

                                fig11.PNG
                                  Plate 2

                                   

                                  fig12.PNG
                                    Plate 3
                                    fig13.PNG
                                      Plate 4
                                      fig14.PNG
                                        Plate 5

                                        Plasmid isolation and insert verification via PCR

                                        After transformation of E. coli TOP10 cells with the insert, we selected individual colonies from the 4th plate and inoculated freshly prepared LB-Amp media in culture tubes. Then the culture tubes were incubated at 37° C overnight and the next day, using the alkaline lysis method, we isolated the plasmids and verified using PCR.

                                        For the verification of the insert, we performed two PCRs, using different primer pairs. For the 1st PCR program, we used the LentiCRISPR v2 vector-specific forward and reverse primer pairs and for the 2nd PCR program, we used the LentiCRISPR v2 vector-specific forward and oligo-specific (insert-specific) reverse primer pairs.

                                        fig15.PNG
                                          PCR 1

                                          This figure represents the 1st PCR experiment. We loaded the PCR product (345bp) in the 2nd and the 4th lane, the 1st lane represents the complete LentiCRISPR v2 vector and the 6th lane represents 500bp fragment as our marker.

                                          fig16.PNG
                                            PCR 2

                                            This figure represents the 2nd PCR experiment. We loaded the product of the 1st PCR experiment in the 2nd as a marker and in the 3rd and the 4th lanes; we loaded the product (270bp) of the 2nd PCR experiment.

                                            CONCLUSION AND RECOMMENDATIONS

                                            Conclusion

                                            Within the given time period, we could only complete our first three objectives. During this time, we gained an in-depth hands-on knowledge of proper research laboratory work and also came to know about the various real life constraints that can impede our works in a laboratory.

                                            A few experiments like “Transfection of recombinant LentiCRISPR V2 in Huh 7.5 cell line” and “Knock-out product confirmation using western blotting” are still needed to be done following our project, for a proper conclusion.

                                            Recom

                                            1. Incubation of the vector with the restriction enzyme BsmBI must not be done for more than one hour as this causes degradation of the vector.

                                            2. Also, instead of performing the Phenol distillation in laboratory, it is better to purchase the purified phenol as the process is dangerous and it causes painful burns on the skin.

                                            ACKNOWLEDGEMENTS

                                            I would like to express my special thanks of gratitude to my guide Dr. Binay Chaubey as well as to the IAS for for giving me this golden opportunity to do this wonderful project on the topic "Study of host cell proteins in HCV Biology", which helped me a lot by giving me hands-on experience of a Research laboratory and I came to know about so many new things. I am also really thankful to Dr. Anirban Ray for helping me get this internship. Secondly I would also like to thank my parents and friends who helped me a lot in finalizing this project within the limited time frame.

                                            CITATIONS

                                            (1) Li, H.C. and Lo, S.Y., 2015. Hepatitis C virus: Virology, diagnosis and treatment.World journal of hepatology,7(10), p.1377.

                                            (2) Craig, A.W., Svitkin, Y.V., Lee, H.S., Belsham, G.J. and Sonenberg, N., 1997. The La autoantigen contains a dimerization domain that is essential for enhancing translation.Molecular and cellular biology,17(1), pp.163-169.

                                            (3) Pudi, R., Srinivasan, P. and Das, S., 2004. La protein binding at the GCAC site near the initiator AUG facilitates the ribosomal assembly on the hepatitis C virus RNA to influence internal ribosome entry site-mediated translation.Journal of Biological Chemistry,279(29), pp.29879-29888.

                                            (4) Zeisel, M.B., Fofana, I., Fafi-Kremer, S. and Baumert, T.F., 2011. Hepatitis C virus entry into hepatocytes: molecular mechanisms and targets for antiviral therapies.Journal of hepatology,54(3), pp.566-576.

                                            (5) Doudna, J.A. and Charpentier, E., 2014. The new frontier of genome engineering with CRISPR-Cas9.Science,346(6213), p.1258096

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