Analysis of Group IV Viral SSHHPS Using In Vitro and In Silico Methods

We've shown that group IV viral genomes encode short stretches of host protein sequences. These sequences can be found within the viral protease cleavage sites. They're being used for the targeted destruction of host proteins, typically proteins involved in generating the immune responses.

A major advantage of using protease assays is that it removes the complexity of a cell and shows whether or not a viral protease can cleave a particular sequence. So when we analyzed Zika SSHHPS sequences, we found that the protease could cut sequences in proteins involved in generating immune responses and that some of these proteins also had roles in brain and eye development. When performing this technique for the first time, one should remember that if cleavage is not observed, that it may be due to a variety of factors such as the activity of the protease.

Demonstrating the procedure will be Jaimee Compton, a technician from my laboratory. Start by opening Protein BLAST and put the 20 amino acids surrounding the scissile bond and the viral polyprotein and select non-redundant protein sequences then type in the host genome to be searched. If needed, select PHI-BLAST and type in a pattern sequence where square brackets indicate that either amino acid within the brackets can be at the substitute position, then hit BLAST.

Rank order the BLAST hits based on the number of consecutive identical or tolerated residues that match a cleavage site sequence. From the list, select the proteins containing six or more identical or similar residues for analysis in the protease assays. Construct a plasmid encoding the cyan fluorescent protein, up to 25 amino acids of the cleavage sequence, and the yellow fluorescent protein.

Next, prepare the CFP and YFP substrates by inoculating four four liter flasks with 25 milliliters of an overnight culture. Shake the cultures at 37 degrees Celsius and monitor growth hourly by UV-Vis spectroscopy at 600 nanometers. When the bacteria reach an absorbance of about one, induce protein expression by adding 0.5 milliliters of one molar IPTG to each flask, then lower the temperature of the shaking incubator to 17 degrees Celsius and allow the expression to continue overnight for 17 to 20 hours.

On the next day, pellet the bacteria by centrifugation at 7000 times g for 10 minutes at 4 degrees Celsius. Remove and discard the liquid media then store the pellets at minus 80 degrees Celsius or proceed with cell lysis. To lyse the cells, prepare 100 milliliters of lysis buffer according to manuscript directions, resuspend the pellets in the buffer, and transfer 25 to 25 milliliters of the suspension to 50 milliliter disposable conical tubes.

Place the tubes in a plastic beaker with ice water then insert the sonicator tip into the tube so that the tip is about one centimeter from the bottom of the tube and sonicate the lysate 10 to 20 times until it becomes fluid and liquified. After sonication, transfer the lysate to high speed centrifuge tubes and centrifuge it at 20, 500 x g for 30 minutes at 4 degrees Celsius. Transfer the supernatant to a clean bottle and discard the pellets.

Load the lysate onto the nickel column then wash the column with two column volumes of buffer A followed by five column volumes of 20%buffer B.Absorbance at 280 nanometers will increase during the wash as contaminants elute from the column. Continue washing until A280 returns to baseline values. Then elute the protein with two to three column volumes of 100%buffer B and collect 10 milliliter fractions, making sure to measure the A280 of each fraction.

Prepare eight reactions mixes according to manuscript directions and pipette 45 microliters of each mix into the first three wells of each row of a black half-area 96-well plate. Set up the plate reader for simultaneous detection of florescence at two wavelengths with a fixed photomultiplier tube setting. Program the read time to 20 to 30 minutes with one read per minute and select the wells to be read.

Insert the plate into the machine and start the read, monitoring the emission ratios over time. Run an endpoint read of the plate containing the uncut substrate. Remove the plate and pipette five microliters of enzyme into each well.

One can save the first column as an uncut control. Then read the plate again for 20 to 30 minutes, making sure to set the plate reader to output absolute values. Once the read is complete, seal the plate with film to prevent evaporation and leave it overnight at room temperature to allow the enzyme to completely cut the substrate.

After 24 hours, remove the film and perform an endpoint read of the plate. Average the emission ratios and record them as cut. Confirm the cleavage of the substrate using SDS-PAGE.

Load a molecular weight marker into the first or last lane and load five microliters of each reaction mixture into a lane of the gel, starting with the uncut reaction. Attach the electrodes of the gel tank to the power supply and run the products at 110 volts for 60 minutes. Remove the gel from the cassette using a cracking tool and submerge it in five to 10 milliliters of staining solution.

After one to 24 hours, remove the excess stain and submerge the gel in water. Then take a picture of the gel using a gel imager. To prepare the protein structure, load the protein PDB file into MOE.

Click Select and Solvent, then delete the solvent. Open the Structure pPreparation panel from the Compute top menu bar and automatically correct all structural items by clicking on Correct. Protonate the structure by clicking Protonate 3D.

Add partial charges to the protein by opening the Partial Charges panel and selecting AMBER 99 and Adjust Hydrogens and Lone Pairs as Required. Then save the structure file. To build the structure for the substrate peptides and TRIM14, open the Protein Builder panel, enter the substrate sequence, and select Auto Repack.

Next, set Geometry as Extended and click on Build. Finally minimize the structure and save it as a PDB file. Dock the substrate peptides to VEEV-nsP2 using PyRx AudoDock 4.2 software.

Load the protein, right-click the name, and select Make Macromolecule to prepare the PDBQT docking file. Then load the substrate molecule and select Make Ligand to prepare the ligand docking file. Start the AutoDock wizard on the docking panel at the bottom and select the prepared ligand and protein files.

Define the protein-binding pocket by manually adjusting the grid dimension, then run AuotGrid. Next, run AutoDock and select the Lamarckian genetic algorithm method. Click on Docking Parameters and set the number of GA runs to 50 then click on Forward to start the docking run.

When AutoDock is complete, open the Analyze Results panel and inspect all predicted binding poses. Select the best model with the lowest predicted binding energy and reasonable binding interactions between the cis-477 and substrate on the cleavage site then save it as a PDB file for further MD simulations. After reading the binding poses with AutoDock, it is important to perform post-docking analysis to identify the plausible binding model for refinement with MD simulations.

This protocol was used to find short stretches of homologous host-pathogen protein sequences or SSHHPS in the Zika virus ns2B/3 protease. Four host protein targets were identified. FOXGS, SFRP1, a Gsalpha subunit from a retinal cDNA library, and the NT5M mitochondrial 5'3'nucleotidase.

Sequence alignments of the SSHHPS allowed species-specific differences in the cleavage site sequences. The SFRP1 sequence was identical in humans and chickens which is noteworthy because the Zika virus can induce mortality and microcephaly in chicken embryos. The continuous assay was used to measure steady state kinetic parameters and inhibition constants for the viral polyprotein sequences and the discontinuous assay was used to obtain qualitative cleavage information, such as cleavage of a particular sequence or the inhibition of the protease by various compounds.

A model of the Venezuelan equine encephalitis nsP1-nsP2 junction was made using in silico methods. For the nsP2 protease, lengthening the substrate sequence and reducing the ionic strength of the buffer led to a significant reduction in the Michaelis constant of cleavage of a Semliki Forest virus sequence. When attempting this procedure, it's important to run the controls.

Substrates containing the viral protease cleavage site sequences should be tested before proceeding with SSHHPS sequence analysis. If cleavage of a host protein is observed, follow up experiments should be carried out to confirm that that host protein affects viral replication. This can be done by overexpressing or silencing the protein, and then examining the effects on viral replication.

Group IV contains a large number of new and emerging pathogens. The SSHHPS sequences link specific host proteins and pathways with the viral proteases in a very predictable manner.