4 METHODS

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4.1  Site Directed Mutagenesis

In vitro site-directed mutagenesis was performed using QuikChangeTM site-directed mutagenesis kit from Stratagene. The reaction was based on PCR performed according to the manufacturer’s instructions.

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A typical PCR contained approximately 50 ng of dsDNA, 125 ng each of the two oligonucleotide primers, 1 µl of dNTP mix, 1 µl (2.5 U) of polymerase and 5 µl of 10X reaction buffer in a reaction volume of 50 µl. All the components except the oligonucleotide primers were provided in the kit. The reaction was performed using PfuTurbo DNA polymerase and two synthetic oligonucleotide primers comprising the desired mutation. Oligonucleotide primers (oligos) were designed to incorporate the desired mutation, and the PfuTurbo DNA polymerase replicates both the plasmid strands with high fidelity without displacing the mutant oligos. The thermal cycles consisted of an initial denaturation cycle of 95°C for 30 seconds, followed by 16 cycles of denaturation for 30 sec, annealing at 55°C for 1 minute and extension at 68°C for 15 min (2 min/kb of plasmid length). The oligos complementing the opposite strand of the plasmid DNA are extended during thermal cycling thus generating the entire copy of the mutant plasmid containing staggered nicks. Following the thermal cycling, the product was treated with Dpn I enzyme (target sequence 5’-Gm6ATC-3’). The nicked vector DNA containing the mutation was then transformed into XL1-Blue supercompetent cells. A volume of 1 µl of the product was used for transformation and the transformation mix was plated on an YT-ampicillin agar plates, incubated at 37°C for approximately 16 hrs. The recombinant colonies were screened by mini-prep, followed by RE analysis and further confirmed in some cases by sequencing.

Figure 4.1: Overview of the Quik-change Site-directed Mutagenesis experiment.

4.2 Oligonucleotide primer designing

Guidelines mentioned in the product manual of the QuikChangeTM site-directed mutagenesis kit from Stratagene were followed for designing of primers. In most cases, the primers were designed in such a manner so as to facilitate either incorporation of a new RE site or remove the existing RE site. This was achieved by creating silent mutations in or around the desired mutation region. Both the forward and reverse primers contained the desired mutant. The primers anneal to the same sequence on opposite strands of the plasmid (schematically shown in figure). The length of the primers was set to approximately 25 – 30 bases. Care was taken to have a minimum of 10 bases flanking the mutant site. GC content was maintained between 40 – 60 %. The melting temperature (T m) of the primers was calculated using the following formula

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T m = 81.5 +0.41(%GC) – 675/N- (% mismatch), where N is the primer length in bases, values of % GC and % mismatch are in whole numbers.

The oligonucleotide sequence was further analysed for any hairpin formation or dimmer formations using online software from “Net Primer” of Premierbiosoft or by using Jellyfish 1.5 from Biowire.

4.3 Extraction of plasmid DNA

4.3.1  Mini-Preparation

Mini-preparation was usually performed to screen the recombinants following the PCR based site-directed mutagenesis experiment.

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The plasmid extraction was done according to Sambrook et al. (1989). A single colony was grown overnight in 3 ml of YT medium containing ampicillin to a final concentration of 50 µg/ml. The culture was incubated overnight at 37°C with vigorous shaking at approximately 200 cycles/min.

An aliquot (1.5 ml) of the overnight culture was transferred into an Eppendorf tube, and centrifuged for 5 min at 21,000 x g. The pellet was resuspended in 200 µl of STET buffer containing 1 mg/ml lysozyme and incubated for 5 min at RT. The lysate was boiled for 45 sec in a water bath. The sample was centrifuged for 10 min at 21,000 x g, and the viscous pellet was removed with a tooth-pick. Eight microlitres of 5 % CTAB solution (prewarmed to 37°C) was added to the lysate followed by vortexing and centrifugation at 21,000 x g for 5 min. The supernatant was discarded, and the pellet was resuspended in 300 µl of 1.2 M sodium chloride. The DNA was precipitated using 2.5 volumes of ice-cold absolute alcohol (750 µl). The sample was centrifuged at 21,000 x g for 10 min, and supernatant was discarded. The pellet was washed for 10 min with 70 % ice-cold alcohol and air-dried. Finally, the pellet was resuspended in 20 µl of TE-buffer containing RNase A1 (20 µg/ml). The extracted plasmid DNA was further used for restriction analysis. The remaining bacterial suspension was either stored as glycerol stock (15 %) or used for Maxi-prep.

4.3.2  Maxi-preparation (Qiagen kit)

Maxi-prep was typically done according to the protocol of the manufacturer. One hundred millilitres of YT medium containing 50 µg/ml ampicillin was inoculated with either the bacterial glycerol stock or rest of the bacterial culture from the mini-prep. The culture was incubated over night at 37°C with vigorous shaking at approximately 200 cycles/min.

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The entire suspension was centrifuged at 6000 x g, 4°C for 10 min in a Beckmann JLA 16.250 rotor. The pellet was resuspended in 10 ml of resuspension buffer containing 100 µg/ml of RNase A. Further a volume of 10 ml of lysis buffer was added. The sample was gently mixed and left for 5 min at RT to allow bacterial cell lysis. A volume of 10 ml of ice-cold neutralisation buffer was added and kept on ice for another 20 min. The suspension was centrifuged at 20,000 x g, 4°C for 30 min. The resultant supernatant was passed through a previously equilibrated Qiagen Tip 500 column. Once the solution was filtered, the column was washed twice with 30 ml of wash buffer. Finally the DNA was eluted with 15 ml of elution buffer into a centrifuge tube. An additional 10.5 ml of isoproponal was added slowly to elute along the walls of the centrifuge tube. Immediately, the DNA precipitate could be visualised forming a white ring at the interface of the two solutions. The DNA was pelleted by ultra centrifugation for 30 min at 20,000 x g, and 4°C. The pellet was subsequently washed with 5 ml of 70 % ice-cold ethanol. Finally the pellet was air dried, and resuspended in appropriate amount of TE-buffer (pH 7.6). The purified DNA was stored at –20°C for further use.

4.4 Transformation

An aliquot (50µl) of frozen XL-1 Blue supercompetent cells (provided by manufacturer) were thawed on ice. The cells were mixed with 1 µl of PCR product and kept on ice for 30 min. The cells were then subjected to a heat shock at 42°C for 45 sec, and chilled on ice for 2 min. The cells were diluted 10 fold with prewarmed YT medium. The diluted suspension was incubated at 37°C for 1 hr to allow the expression of antibiotic resistance. Samples were pelleted and resuspended in 100 µl of fresh YT medium. The product was plated on an agar plate containing 50 µg/ml of sodium salt of ampicillin. The plates were incubated at 37°C for nearly 16 hrs for the appearance of colonies. The colonies were further screened for the presence of recombinant plasmid DNA with desired mutation.

4.4.1  Growth and Purification of vaccinia virus stocks

CV-1 cells were grown to nearly full confluence in two 15 cm petri dishes with 10 % FCS in DMEM. The cells were infected with a purified recombinant vaccinia virus (vTF7-3) at 0.05 – 0.1 pfu/cell in nearly 5 ml of serum-free DMEM per dish. The plates were incubated in an incubator at 37°C for 1 hr. At the end of infection time, the virus was removed from the plates and 20 ml of DMEM/5 % FCS was added to each dish. The cells were further incubated for another 2- 3 days at 37°C, by which time most or all the cells appear rounded, but remain attached to the dish. The purification of the virus was performed by scraping the cells into their medium with a rubber policeman. The medium from both petri dishes was transferred into a single 50 ml disposable sterile tube and centrifuged for 5 min at 200 g and at 4°C. The pellet was resuspended in 4 ml of 10 mM Tris Hcl, pH 9.0. The product was homogenized on ice in a 7 ml dounce using a tight pestle, and centrifuged for 5 min at 200 g at 4°C to remove nuclei. The supernatant was transferred into a fresh tube. The pellet was again resuspended with another 4 ml of 10 mM Tris-HCl (pH 9.0), and centrifuged for another 5 min at 200 g at 4°C. The supernatants from both centrifugations were mixed and recentrifuged at 650 g for 10 min at 4°C to remove any remaining debris. The supernatants were sonicated in a water bath for 5 min and centrifuged at 13,500 rpm (33,000 g)for 80 min in an ultra centrifuge. At the end of centrifugation, the pellet was resuspended in 2 ml of 1 mM Tris-Hcl, pH 9.0, aliquoted into 100 µl fractions and stored at –80°C. All the glassware and discarded solutions were treated with sodium hypo chlorite. The solutions were then discarded and glassware was further autoclaved.

4.5 Expression of HA mutants in CV-1 /COS-7 cells

4.5.1  Transient T7-RNA-polymerase vaccinia expression system

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CV-1 cells were grown as monolayers in DMEM supplemented with 5 % heat-inactivated FCS in an incubator at 37°C and 5 % CO2. Medium also contained 1 % penicillin and streptomycin. Usually FCS is contained in the medium only for the growth purpose and plain DMEM (without FCS and antibiotics) was used for the transfection experiments.

For expression purposes, CV-1 cells were grown near to confluence in 3.5 cm dishes. Cells were washed twice with 1 ml DMEM and infected with vTF7-3 virus at 10 pfu/cell in 500 µl of DMEM. The cells were incubated for 1 hr at 37°C with 5 % CO2. After optimising different plasmid DNA concentrations, finally 6 µg of plasmid DNA per 3.5 cm dish was used as standard for all the transfections. Transfections were performed using lipofectin (a cationic lipid liposome formulation used for transfection of DNA into tissue culture cells). Ten micro litres of lipofectin was diluted in 100 µl of DMEM medium and incubated at RT for 30 min along with 6 µg of plasmid DNA in 100 µl of DMEM medium. At the end of 30 min, the DNA was added drop-by-drop to the lipofectin and incubated for another 15 min. The virus inoculum was removed after 1 hr. The cells were washed twice with DMEM. A volume of 800 µl of DMEM was added to the cells and the DNA-lipofectin mix (200 µl /dish) was added to the medium. The cells were further incubated for another 4 hrs at 37°C with 5 % CO2. The DNA containing medium was replaced with plain DMEM and cells were incubated at 31°C with 5 % CO2 for 14 – 16 hrs.

4.5.2  Metabolic labelling with Trans S (cysteine & methionine)

CV-1 cells were grown nearer to confluence in 3.5 cm dishes. The cells were infected with vTF7-3 virus as mentioned before. The protocol was identical as mentioned earlier with the exception that lipofectin and DNA were diluted in MEM (deficient in methionine, cysteine and L-glutamine) instead of DMEM. The virus inoculum was removed, and the cells were washed twice with MEM. The cells were incubated for 4 hrs at 37°C with 5 % CO2 with MEM upon addition of lipofectin:DNA mixture. At the end of the transfection time, the DNA-lipofectin mixture was removed and substituted with MEM by the addition 3 µl of Trans [35S]-label (1000 Ci/mmol). In addition, L-Glutamine was added externally. Incubation was continued at 31°C with 5 % CO2 for 14-16 hrs.

4.5.3  Processing of surface expressed HA

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The cells were washed with pre-warmed (37°C) PBS and treated with TPCK-trypsin at 8 µg in 1 ml of PBS for 10 min at RT to cleave surface expressed HA0 to HA1 and HA2. Trypsin activity was inhibited by the addition of soyabean trypsin inhibitor (STI) to a final conc., of 50 µg /ml and incubated at RT of another 10 min. Finally, the cells were lysed in a cell lysis buffer of 800 µl containing 1 % NP-40 and protease inhibitors. The cell debris was removed by centrifugation at 12,000 rpm for 15 min.

4.6 Immunoprecipitation

An appropriate anti-HA antibody (mostly N2 antibody) was added to the clear lysate. The antigen- antibody complex was incubated at 4°C for either 1 hr or overnight on a shaker platform. At the end of the incubation, 35 µl of protein-A sepharose was added. The solution was further incubated for another 1 hr to allow the protein-A to bind the Fc portion of the antibodies. The immune complex (antigen:antibody: protein-A sepharose) was centrifuged at 12,000 rpm for 5 min. The immune complex was washed 3 times with 800 µl of RIPA buffer to remove unbound proteins. Sample buffer (reducing or non-reducing) was added to the pellet and boiled at 95°C for 5 min. The sample was centrifuged for 5 min and the supernatant was loaded onto a 12 % SDS-PAGE gel along with a pre-stained standard protein molecular weight marker.

4.7 Fluorography of the gels

The SDS-PAGE gel was treated with fixer solution for 30 min on a rotating platform. The gel was then washed twice with distilled water for 30 min each. Finally, the gel was treated with 1 M sodium salicylate for 30 min before it was dried on a gel-dryer for 90 min at 85°C. The dried gel was exposed to a X-ray film in a cassette. The fluorogram was developed after over-night incubation of the cassette at –80°C. Quantification of fluorogram was carried out with an Epson GT-9000 scanner and ScanPack software.

4.8 Glycosidase assay

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The glycosylation pattern was determined by using endoglycosidases - Endoglycosidase H (Endo H) and peptide-N-glycosidsae F (PNGase F). The HA protein for this assay was expressed as previously mentioned by using 35S metabolic labelling. The cells expressing HA protein were trypsin processed if needed. The processed cells were lysed, and digested with either Endo H or PNGase F following the manufacturers instructions. The digested product was routinely processed for immunoprecipitation and visualised by fluorography after SDS-PAGE in a 12 % polyacrylamide gel.

4.9 Cross-Linking Experiment

CV-1 cells were routinely infected, transfected and metabolically labelled, lysed and centrifuged as described earlier. The surface expressed HA was not trypsin processed. The cells were lysed in a lysis buffer without amines in a volume of 500 µl. The lysate was aliquoted into two Eppendorf tubes with 200 µl each. To one aliquot, 4 µl of DMSO was added which served as a control. To the other aliquot, 4 µl of cross-linking reagent DSP (40 mM in DMSO) to a final conc., of 0.3 mM was added. Both the samples were incubated at 15°C for 15 min. The reaction was stopped by addition of 4 µl of 1 M ammonium chloride and immunoprecipitation was carried out using N2 antibody. The samples were analysed on a 6 % SDS-PAGE gel using non-reducing sample buffer. The gel was further processed for visible bands on a fluorogram.

4.10 Conformational change assay (Proteinase-K assay)

The wild type and mutant forms of HA were analysed for variation in their conformations at a gradient of reducing pH conditions. HA expressed in CV-1 cells were metabolically labelled as mentioned above. After treatment with TPCK-trypsin and trypsin inhibitor, cells were incubated at 37°C for 6 min in fusion buffer adjusted to desired pH (7.0, 6.0, 5.8, 5.6, 5.4, 5.2 and 5.0). The cells were then re-neutralised with pH 7.0 fusion buffer and lysed with 800 µl of lysis buffer. The lysate from centrifugation was digested with 0.2 mg/ml of Proteinase-K and 2 mM CaCl2 at 37°C for 30- 45 min. The digestion was stopped by addition of 1 mM PMSF and a protease inhibitor cocktail. The samples were immunoprecipitated with N2 antibody, and subsequently the protein bands were analysed using fluorography.

4.11 Densitometric analysis

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The fluorograms were scanned using Epson scanner (GT-9000) attached to a Personal Computer. The scanned pictures were analysed using ScanPack version 2.0 software. The quantity of protein seen on the fluorogram was expressed as a percentage taking the sample treated with pH 7.0 buffer as 100 % for that particular mutant. The graphs were plotted using MS-Office Excel software using standard error as statistical tool to know the sampling fluctuations.

4.12 Fusion assays

4.12.1  Preparation of HA-expressing cells for fusion assay

CV-1 cells were grown on sterile glass sildes of 20 x 20 mm placed inside 3.5 cm dishes using DMEM with 10 % FCS. HA protein was expressed in CV-1 cells for nearly 14-16 hrs as mentioned earlier with out any metabolic labelling. The cells were processed with TPCK-trypsin and additionally with neuraminidase (0.2 mg/ml), followed by trypsin inhibitor. Further the cells were treated with pre-adjusted low pH fusion buffer and re-neutralised. As a control, CV-1 cells infected with vaccinia virus and treated according to the transfection protocol, but without DNA, were used.

4.12.2  Preparation, labelling of RBC for fusion assay

Human RBC were prepared and labelled as described by Kozerski et al. (2000). Human RBC was washed for three times with PBS (centrifuged at 2,000 x g for 10 min), and RBC was resuspended in PBS (50 % hematocrit). Twenty microliters R18-solution (octadecylrhodamine B chloride; 2 mM in ethanol) was added to 5 ml RBC suspension in PBS (heamtocrit 4 %) under gentle vortexing. After addition of 10 ml of PBS and incubation of the suspension for 30 min at RT in the dark, 25 ml of DMEM with 5 % FCS were added to the suspension to absorb unbound label. After further incubation for 20 min at RT in the dark, the RBC suspension was washed in 40 ml PBS. The RBC pellet was resuspended in 2 ml PBS, after addition of the calcein-AM solution (50 µg freshly dissolved in 50 µl DMSO), cells were incubated for 45 min at 37°C in the dark. Calcein-AM is known to enter the cell cytoplasm where it is cleaved by unspecific esterases of the RBC. The resulting fluorescent calcein becomes trapped in the cytoplasm because it cannot permeate the membrane. Subsequently, the suspension was centrifuged and washed in 20 ml PBS. After resuspension of the pellet in 10 ml PBS, cells were incubated for another 20 min at 37°C in the dark to allow cleavage of intracellular calcein-AM. The suspension of R18 and calcein-AM labelled RBCs was washed five times in 30 ml PBS, rsuspended in 5 ml PBS++, was kept at 4°C until binding to the HA expressing CV-1 cells.

4.12.3  Fusion assay and fluorescence microscopy

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For binding, 1 ml of labelled RBC with a hematocrit of 0.4 % was added to the monolayer of HA expressing cells and incubated for 15 min at RT in the dark with occassinal gentle shaking. Unbound RBCs were removed by 4-5 washes with PBS++. The medium was replaced by MES-saline adjusted to indicated pH and incubated at 37°C for 5 min, followed by neutral pH buffer for a brief 2 min at 37°C. Finally the slide is carefully lifted from the dish and placed inverted on a 24 x 60 mm cover slip with a drop of PBS++. The corners of the slide were fixed with a clear transparent nail polish. The slides were observed for fusion under a fluorescence microscope. Fusion was monitored at RT by transfer of fluorescent dyes from labelled RBCs to HA expressing cells. Label redistribution was observed under the fluorescence microscope (Axiovert 100 with a 40x Achroplan objective [Zeiss]). R18 fluoroscence was visualised using a green filter (excitation: 510-560 nm and emission: 590 nm). Similarly calcein fluorescence was observed with a blue filter (excitation: 450-490 nm & emission: 520 nm). Images were acquired with a CCD camera Coolsnap fx (Photometrics, AZ, USA) and analysed using Metamorph 6.1 software (Universal Imaging, PA, USA).


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