key: cord-0864760-9d500apw authors: Abraham, Rachy; McPherson, Robert Lyle; Sreekumar, Easwaran; Leung, Anthony K. L.; Griffin, Diane E. title: Preparation of Recombinant Alphaviruses for Functional Studies of ADP-Ribosylation date: 2018-04-27 journal: ADP-ribosylation and NAD+ Utilizing Enzymes DOI: 10.1007/978-1-4939-8588-3_21 sha: 7bab422e4be46fff640eb4eefe7ca21a97c78122 doc_id: 864760 cord_uid: 9d500apw Recently we characterized the mono(ADP-ribosyl) hydrolase (MAR hydrolase) activity of the macrodomain of nonstructural protein 3 (nsP3(MD)) of chikungunya virus. Using recombinant viruses with targeted mutations in the macrodomain, we demonstrated that hydrolase function is important for viral replication in cultured neuronal cells and for neurovirulence in mice. Here, we describe the general cell culture and animal model infection protocols for alphaviruses and the technical details for biochemical characterization of the MAR hydrolase activity of nsP3(MD) mutants and the preparation of recombinant viruses incorporating those mutations through site-directed mutagenesis of an infectious cDNA virus clone. Alphaviruses are mosquito-borne plus-strand, enveloped RNA viruses that cause a variety of human diseases ranging from rash and arthritis by the "Old World" viruses to encephalitis by the "New World" viruses. The encephalitic alphaviruses, like Venezuelan, western, and eastern equine encephalitis viruses, are endemic in the Americas, and recently introduced arthritic alphaviruses like chikungunya virus (CHIKV) can also cause atypical complications like encephalitis [1, 2] . Alphaviruses have an approximately 11.7 kilobase (kb) positive-sense RNA genome with a 5′ 7-methylguanosine cap and 3′ poly-A tail that encodes four nonstructural proteins (nsP1-4) and six structural proteins (C, E3, E2, 6K/TF, and E1) that are expressed from a subgenomic RNA [3] . Recently, we have shown that the macrodomain of viral nonstructural protein 3 The original version of this chapter was revised. A correction to this chapter can be found at https://doi.org/10.1007/978-1-4939-8588-3_27 (nsP3) of CHIKV enzymatically reverses the protein modification mono(ADP-ribosyl)ation in vitro. Macrodomains are a conserved protein fold found from archaea to higher eukaryotes and are characterized by their binding to the small molecule ADP-ribose and its derivatives [4] . The viral macrodomain is part of the nonstructural protein in a subset of positive-strand RNA viruses, such as alphaviruses, coronaviruses, and hepatitis E virus [5, 6] . We identified and mutated residues in nsP3 critical for mono(ADP-ribosyl) binding and hydrolase (MAR hydrolase) activity, and using mutants with decreased MAR hydrolase activity, we demonstrated that reduction of this activity impairs viral replication in cultured mammalian and mosquito cells and virulence in neonatal mice [7] . Reverse genetic approaches have been extensively used in alphavirus research to understand virus biology and replication [8, 9] , vector competence [10] [11] [12] [13] , attenuation and virulence [14, 15] , and immunogenicity [16, 17] . The approach involves the construction of an infectious cDNA clone by incorporating a DNA copy of the viral RNA genome into a plasmid vector. With incorporation of the bacteriophage T7 or SP6 RNA polymerase promoters, the cDNA can be in vitro transcribed to produce full-length viral RNA transcripts. Transfection of the transcribed RNA into permissive cells results in production of infectious virus. To understand a loss or gain of gene function, variants or mutants are made in the infectious cDNA clone to produce mutant viruses in the same virus background for analysis. In our studies of the MAR binding and hydrolase activities of the nsP3 macrodomain, we introduced mutations into the macrodomain region of nsP3 in the infectious cDNA clone of the 181/clone 25, the live attenuated vaccine strain of CHIKV. The 181/25 virus was derived from the Southeast Asian strain AF15561 by 18 plaque-toplaque passages in MRC-5 human lung fibroblasts [18] . The infectious clone of 181/25 was prepared by employing site-directed mutagenesis to introduce five non-synonymous mutations into the parent clone 15,561 [10] . The parent clone 15,561 in a modified pSinRep5 plasmid was engineered to possess a SP6 RNA polymerase promoter at the 5′ terminus and a poly(A) tail, followed by a NotI restriction site at the 3'terminus [15] (Fig. 1) . Because 181/25 is a vaccine strain, we reversed the attenuating mutations I12T and R82G [15] in the E2 glycoprotein to generate a recombinant CHIKV strain (181/25 E2 I12T/R82G ) that is virulent for newborn mice. In this chapter, we describe the technical details of our strategies for biochemically characterizing the nsP3 MD mutants with MAR hydrolase assays and for incorporating the mutations into the viral genome by site-directed mutagenesis. Here, the general protocols for cell culture of alphaviruses, infection of mice, and generation of recombinant mutant viruses using chikungunya virus 181/25 infectious cDNA clone are explained. Variations in the methodology for other alphaviruses, particularly the prototype Sindbis virus, are also mentioned. A summary of the methodology is outlined in Fig. 2 MAR hydrolase assay involves automodifying the substrate PARP10 CD with radiolabeled 32 P-NAD + and incubating the substrate with recombinant proteins to be tested for MAR hydrolase activity followed by SDS electrophoresis and phosphoimaging. 1. Automodify desired amount of PARP10 CD (see Note 1) with 32 P-NAD + at a concentration of 0.25 μCi/μg of PARP10 CD in AM buffer at 30 °C for 30 min. 2. Add 1 mL of DM buffer to Bio-Rad Micro Bio-Spin column and allow gravity flow through the column. Repeat twice to equilibrate column in DM buffer. Add PARP10 CD automodification reaction to the top of the bed (100-150 μL total volume) and allow it to enter the column. Add 600 μL of DM buffer to the column and collect 2-drop fractions using the Geiger counter to determine when the 32 P-labeled PARP10 CD is eluted. Once the 32 P-labeled PARP10 CD begins to elute, collect a 500 μL fraction to maximize yield and desalting of unincorporated 32 P-NAD + . 3. For each reaction, make 1 μg aliquots of desalted 32 P-labeled PARP10 CD (see Note 2 about concentration of desalted sample from step 2 6. Rinse gel for 10 min with Milli-Q water followed by staining with Novex SimplyBlue Safe Stain for 1 h at room temperature. Once bands develop, rinse gel in Milli-Q water for 1 h at room temperature. 7. Image gel with a scanner to obtain total protein stain and expose the gel to a FujiFilm autoradiograph imaging plate overnight. 8. Image FujiFilm autoradiograph imaging plate on FujiFilm imager to acquire autoradiograph of gel. For quantification, open the autoradiography file in ImageJ and quantify the signal intensity of each lane using the GelAnalyzer tool (see Note 3). Quantify the intensity of the 32 P signal from automodified PARP10 CD and obtain the percentage of signal removed using the below equation: The infectious cDNA clone which is the double-stranded copy of the viral genome carried on a plasmid vector is modified for introducing mutation by PCR-based site-directed approach, followed by transformation to the E. coli cells. The mutant clone is then subjected to sequencing for checking the presence or absence of mutation. 1. Design oligonucleotide forward and reverse primers containing the desired mutation (see Note 4). 7. The plasmids are sequenced and compared with the sequence of the parent clone (see Note 6). In order to make the mutant viruses, the mutant full-length cDNA clones are linearized, purified, and in vitro transcribed to RNA. Linearize 3 μg of plasmid containing the desired mutation in the full-length cDNA with NotI enzyme in a 100 μL reaction (see Note 7). Incubate the reaction for 16-18 h at 37°C. The component volumes for the reaction are: 10× restriction buffer 10 μL Plasmid DNA 3 μg NotI (20 U/μL) 1.5 μL Nuclease-free water Make up to 100 μL Analyze 5 μL of the digested reaction by gel electrophoresis to ensure that the plasmid is fully linearized. Purify the RNA transcripts using the MEGAclear TM kit, and elute with 30 μL of RNase-free water (see Note 9). Generally, the expected concentration of the RNA synthesized from a standard 30 μL reaction starting from 1.5 μg of DNA is within a range 2-3 μg/μL. This section deals with the transfection of the CHIKV RNA transcript into permissive cell lines to recover the clone-derived virus. 1. Transfer BHK-21 cells in growth medium into a T150 cm 2 flask, and incubate for 18-24 h in 37 °C 5% CO 2 incubator so that the cells will be 80-90% confluent at the time of transfection (see Note 10). (Fig. 3) . Collect the viral supernatant fluid from the flasks into a sterile 50 mL tube, and centrifuge at 1000 rpm (216 rcf ) for 10 min at 4 °C. Virus should be stored as 1 mL aliquots in cryopreservation tubes at −80 °C (label as passage 0, P 0 ). The presence of the mutation in the clone-derived virus is verified by RNA isolation, followed by reverse transcriptase PCR, and finally sequencing. 6. Purify the PCR product to remove excess primers and other PCR reaction mix components. If there is only a single band present, purify the remaining PCR product directly using QIAquick® PCR purification kit. If there are additional bands, excise the correct band from the gel and purify using QIAquick® gel extraction kit. 7. The purified PCR product will be used as a template for sequencing to confirm the presence or absence of the mutation in the clone-derived virus. The purified PCR product can be stored at −20 °C (see Note 6 for the primer details). 1. Culture BHK-21 cells in growth medium in a T150 cm 2 flask the day before infection so that they are 80-90% confluent at the time of infection. 3. Collect supernatant containing the virus from the flasks into a sterile 50 mL tube, and centrifuge at 216 rcf for 10 min at 4 °C. Virus should be stored as 1 mL aliquots in cryopreservation tubes at −80 °C (label as passage 1, P 1 ). 4. Characterize the virus (P 1 ) by sequencing to confirm the presence of the desired mutation (follow Subheading 3.5), and proceed for virus quantitation by plaque assay titration. Detecting the viral load or viral titer is essential for any virologybased study. Plaque assay allows the quantification of infectious virus particles which rely on the ability of viruses to cause cytopathic effects on the host cells because of infection. This section describes the steps involved: infection of cell monolayers with tenfold dilutions of viral supernatant, followed by overlaying with media having high molecular weight substrate like Bacto agar, agarose, or carboxymethylcellulose, which will limit the viral spread. The plaques thus formed are visualized by staining cell monolayers with crystal violet or neutral red. FBS in a 6-well plate. A 100% confluent flask of Vero cells can be plated into five 6-well plates. Each plate will be for one sample; each well within this plate is for a tenfold dilution of that sample (Fig. 4) . Incubate the plates overnight at 37 °C, 5% CO 2 (see Note 13). Collect the supernatant at the desired time points and freeze at −80 °C. The supernatants can be further assayed for infectious virus titers using a plaque assay (follow Subheading 3.7) or for levels of secreted proteins such as cytokines by ELISA. The cells from each well can be collected either in buffer RLT (from Qiagen kit) for RNA isolation or in RIPA buffer for protein studies. Studies using mouse models to evaluate neurovirulence can be carried out by infecting the mice intracranially and monitoring them daily for clinical signs/mortality. The organs can be harvested to assess the viral replication by plaque assay or RNA quantification. 7. Restrain the pup manually on a solid surface by firmly placing your thumb and index finger on either side of the head just at the nape, and insert the needle having 10 μL of virus into the right side of the cranium approximately halfway between the eye and the ear just off-center from the midline (see Note 17). 8. Monitor the mice twice daily for weight gain/loss and morbidity/mortality. If any animal shows signs of being moribund or reaches experimental end points, euthanize immediately according to a method which has been approved by the ethics governing body. The 2-day old CD-1 mice infected with virulent strains of CHIKV will die without showing much evidence of illness, so clinical scoring is not possible for this model (see Note 18). 9 . At the end of the morbidity/mortality experiment, the surviving pups and the mother are generally sedated with isoflurane and euthanized according to a method which has been approved by the ethics governing body. 10 . For time course infection experiments to assess virus replication, at least three pups per group are euthanized at each time after infection, and blood and organs are collected. The pups are anesthetized using isoflurane, and blood is collected with a 1 mL syringe by cardiac puncture. The blood can be transferred to a microtainer tube and spun at 1500 rpm (200 rcf) for 15 min to collect serum. 11. Expose the heart, and perfuse with 20 mL ice cold PBS to remove the blood from the tissues before collecting the organs. When collecting the brain, one half brain can be homogenized in 1 mL QIAzol lysis reagent for RNA isolation using RNeasy lipid tissue mini kit. Genetic characterization of virus replicating in the brain is carried out by sequencing (see Subheading 3.5) . The other half of the brain can be homogenized in PBS (see Note 19) and used for assessing the infectious virus titer using plaque assay (see Subheading 3.7) . The viral titer is calculated using the formula: 1. We used 1 μg of automodified PARP10 CD per reaction but performed the automodification in bulk up to 100 μg. The automodification and desalting procedure can be applied to other MARylated substrates. 2. Desalting on the Bio-Rad Micro Bio-Spin 6 column causes sample dilution and loss. We automodified 85 μg of PARP10 CD in 100 μL for step 1 and recovered 50 μg of 32 P-labeled PARP10 CD in 500 μL after step 2. Concentration can be empirically determined by SDS-PAGE and coomassie blue staining with standards of known concentrations. 3. Extensive tutorials of this tool for quantifying band signals can be found online (e.g., http://lukemiller.org/index. p h p / 2 0 1 0 / 1 1 / a n a l y z i n g -g e l s -a n d -w e s t e r n -b l o t swith-image-j/). 10. Warm all cell culture media, PBS, and trypsin-EDTA in a water bath to 37 °C prior to adding to the cells. 11. The primers used for amplifying the nsP3 region of CHIKV in our study were: CHIKV nsP3 N1 forward primer (5′ AAGGCCGAATTCGGGCACCGTCGTACCGGGT AAAACGCA 3′) CHIKV nsP3 full reverse primer (5′ GGTACCCTCGAGTCATTACCCACCTGCCCTGT CTAGTC 3′) The PCR amplification with this primer set will yield an amplicon of 1.6 kb. The primers are designed to be highly specific and produce a single amplicon only. If other bands appear, further optimization can be carried out by gradient PCR or increasing the annealing temperature. Keep a nontemplate control for the PCR reaction. 12. The plaque assay for CHIKV uses Vero cells. For Sindbis virus, we use BHK-21 cells instead. The methodologies mentioned in steps 1 and 2 are the same. The plaques in BHK-21 are more clearly visible, when staining the live cells using 1:10 dilution of 0.33% neutral red in DPBS. Add 1 mL of diluted neutral red on the top of the agar overlay and incubate at 37 °C in a 5% CO 2 incubator for 2 h and count the plaques using a light box. For neutral red staining, the plaques should be counted immediately or/within a period of 2-3 h. 13. The cells should be 90-95% confluent with few spaces at the time of infection. Usually 1 × 10 5 cells were seeded overnight to obtain a monolayer the next day. The incubation time for culturing should not go beyond 24 h. 14. The 1.2% Bacto agar is autoclaved and stored at room temperature. The melted Bacto agar will solidify when cooled to room temperature. Therefore, take the overlay agar mixture from the 42 °C water bath just prior to use. 15. The mouse models differ for each strain of virus. To study the virulence of wild type versus mutants for CHIKV, we used 2-day old CD-1 pups. The same kind of virulence study for the TE strain of Sindbis virus can be done in 2-week old CD-1 mice. 16 . Verify that the mice are adequately anesthetized by gently pinching the toes. If the animal withdraws their paw, anesthetize for few more minutes till a deep plane of sleep has been attained. 17 . Place pups on a thermal mat to avoid a drop-in body temperature during anesthesia, and monitor until they recover. 18. Keep separate litters for morbidity/mortality studies and for a time course infection experiment involving the collection of organs and serum. 19 . We use lysing matrix tubes with lysing matrix A (orangecapped tube having garnet matrix and ¼ ceramic sphere) for homogenizing the tissues for protein and tubes with lysing matrix D (green-capped tubes having 1.4 mm ceramic spheres) for homogenizing the tissues for RNA in QIAzol reagent using a MP FastPrep-24 homogenizer. 20. Make sure to take out the BD OptEIA TMB substrate kit to room temperature, at least 30 min prior to use. Combine equal amounts of reagent A and reagent B no more than 15 min prior to adding to the plates. Biology and pathogenesis of chikungunya virus Chikungunya virus: an update on the biology and pathogenesis of this emerging pathogen The alphaviruses: gene expression, replication, and evolution Macrodomains: structure, function, evolution, and catalytic activities Putative papain-related thiol proteases of positive-strand RNA viruses. Identification of rubi-and aphthovirus proteases and delineation of a novel conserved domain associated with proteases of rubi-, alpha-and coronaviruses Computer-assisted assignment of functional domains in the nonstructural polyprotein of hepatitis E virus: delineation of an additional group of positive-strand RNA plant and animal viruses ADP-ribosylhydrolase activity of Chikungunya virus macrodomain is critical for virus replication and virulence Construction of an infectious Chikungunya virus cDNA clone and stable insertion of mCherry reporter genes at two different sites Characterization of synthetic Chikungunya viruses based on the consensus sequence of recent E1-226V isolates Infectious clones of Chikungunya virus (La Reunion isolate) for vector competence studies A single mutation in chikungunya virus affects vector specificity and epidemic potential Epistatic roles of E2 glycoprotein mutations in adaption of chikungunya virus to Aedes albopictus and Ae. aegypti mosquitoes Development and characterization of a double subgenomic chikungunya virus infectious clone to express heterologous genes in Aedes aegypti mosqutioes Novel chikungunya vaccine candidate with an IRES-based attenuation and host range alteration mechanism Attenuation of Chikungunya virus vaccine strain 181/clone 25 is determined by two amino acid substitutions in the E2 envelope glycoprotein Chimeric Chikungunya viruses are nonpathogenic in highly sensitive mouse models but efficiently induce a protective immune response DNA vaccine initiates replication of live attenuated chikungunya virus in vitro and elicits protective immune response in mice Development of an attenuated strain of chikungunya virus for use in vaccine production Neuroblastoma× spinal cord (NSC) hybrid cell lines resemble developing motor neurons This work was supported by a Johns Hopkins Catalyst Award (AKLL) and research grants from the Johns Hopkins University School of Medicine Sherrilyn and Ken Fisher Center for Environmental Infectious Disease (DEG and AKLL). This work is also in part supported by R01GM104135S1 (AKLL and RLM) and T32CA009110 (RLM) from the U.S. National Institutes of Health.