This work was supported by Defense Threat Reduction Agency (DTRA) project numbers CB-SEED-SEED09-2-0061 and CBCall4-CBM-05-2-0019, and in part by the Intramural/Extramural research program of the NCATS, NIH (XH) and Naval Research Laboratory base funds.

The opinions expressed here are those of the authors and do not represent those of the U. S. Navy, U. S. Army, U. S. Department of Defense, or the U. S. government.

Sequence-specific destruction of a protein or a nucleic acid guided by a foreign sequence is only seen in a few cases in biology. The mechanisms shown in Figure 1 are defensive mechanisms that protect a host from a virus, or a virus from a host.
Using bioinformatic methods we can identify the targets that are destroyed by these systems. In our analyses of SSHHP sequences, we discovered that many of them could be found in proteins needed to generate innate immune responses. Some had obvious roles such as MAVS and TRIF (TIR-domain-containing adapter-inducing interferon-&#946;), while others were related to immunity though more complex mechanisms (e.g., Histone H3, SFRP1, FOXG1)8,9. The target information stored in the SSHHP sequence has the potential to identify pathways that have antiviral effects against these viruses. Antiviral responses in vivo are often virus-specific26,43. For example, subsets of TRIM proteins have antiviral effects on different viruses43,44,45, some are viral restriction factors (e.g., HIV and TRIM5&#945;). The specificity of TRIM proteins (~70 have been identified) still is being examined44,45. The information within SSHHPS may contribute to our understanding of how these viruses evade the innate immune responses. Other patterns and correlations may be uncovered as more SSHHPS are examined.
Species-specific differences were apparent in our analyses (Figure 2, Figure 10). These viruses are known to affect some species more than others. Information about host range, host susceptibility, and host defenses may be present within SSHHPS. For example, equine, the most susceptible species to equine encephalitis viruses, lacked the region of human TRIM14 that was transiently cut by the VEEV nsP2 protease. Humans rarely die from VEEV infections but can be infected24. The human TRIM14 protein carried an nsP2 protease cleavage sequence6. The presence of the cleavage site suggest that humans have a defense mechanism against these viruses. Birds have been thought to be potential reservoirs of these viruses46. The corresponding SSHHP sequence in the TRIM14 protein from chickens differed from the sequences found in humans and other species. Subtle differences like these may make a target host protein uncleavable or more readily cleaved. Aguirre et al.16 showed that an uncleavable mutated STRING protein induced higher levels of IFN after Dengue virus infection and that mice naturally carry a version of STING that is not cut by the Dengue ns2B3 protease. The murine STING protein was not cut by the ZIKV protease47. In our SSHHPS analysis, we also observed differences in the ZIKV protease cleavage site sequences when we compared the human proteins with those of rodents6 (Figure 10D). Reproducing the species-specific proteolytic cleavages of host proteins may be important in animal models used for Group IV viruses. The inhibition of host protein cleavage also has implications with regards to the development of Group IV protease inhibitors. In our previous publication, we showed that we could inhibit TRIM14 cleavage by the VEEV nsP2 protease using CA074 methyl ester6. This result suggests that small molecule inhibitors of these proteases may be able to modulate the innate immune responses that are capable of suppressing the infection6,31.
Genetic variation within a species also has the potential to produce differences in proteolytic cleavage. Subtle differences in codon usage could affect ribosome pausing48. Since some Group IV viral proteases are embedded in the ER membrane, differences in these pauses could affect cleavage of a target if cleavage occurs co-translationally. Some of the cleavage sites that we identified were in predicted signal peptide sequences (e.g., SFRP1) while others were internal.
SSHHPS analysis can produce information that differs from other methods of host protein analyses. SSHHPS analysis was inexpensive and easy to employ. The use of a bacterial expression system allowed testing of short segments (~25 amino acids) of mammalian sequences without the use of mammalian cell culture. We found that the CFP-YFP substrates were able to tolerate all of the tested human protein sequences; however, yields varied. In similar assays, substrates containing human protein sequences as long as 63 amino acids were successfully expressed, purified, and utilized for kinetic analyses and inhibitor screening49,50,51. Since only small amounts of the substrate are needed for the discontinuous assay, a large number of targets can be explored. One advantage of the system is that the CFP/YFP substrates can be used for SDS-PAGE analyses and for more elaborate kinetic analyses (i.e., IC50, Ki, Km, Vmax). For drug discovery, inhibitory compounds can produce artifacts in fluorescent assays. Thus, the discontinuous assay in combination with a continuous assay allows one to confirm cleavage or inhibition of cleavage. The samples for the discontinuous SDS-PAGE assay can be taken directly out of the 96-well plates. CFP/YFP substrates have been used for compound library screening52. However, additional analyses are required to determine if a substrate is suitable for high throughput screening such as the calculation of a Z-factor53.
One challenge in designing a substrate is identifying the region around the scissile bond that is bound and recognized by the protease. In the examples shown here, we began with 12 residue sequences that were centered around the scissile bond. After analyzing sequence alignments of the cleavage sites homology to the residues N-terminal of the scissile bond was found for the VEEV protease, whereas for the ZIKV protease homology to several of the C-terminal residues was found. An in silico model of the docked substrate can be used to design site-directed mutagenesis experiments that probe the binding sites of the substrate. Since the substrate and enzyme sequences are on plasmids, either can be mutated to test the in silico models or subsite tolerances. This can be advantageous if a crystal structure of the bound substrate(s) is not available.
SSHHPS analysis may also yield new information about the mechanisms by which virus-induced phenotypes are produced by viral enzymes. One of the ZIKV targets, SFRP1, is part of the Wnt signaling pathway and has roles in both brain and eye development and in immune responses36,37,54,55,56,57. We found that the other protein sequences that could be cut by the ZIKV ns2B/ns3 protease were also in proteins involved in brain and eye development; abnormalities in both have been observed in congenital Zika syndrome and are thought to be part of the virus-induced phenotype58.
The predictability of host-pathogen interactions could be exploited for a variety of applications: target-specific oncolytic viral therapies; de-risking live virus vaccines; refinement, prediction or selection of animal models; prediction of host-range or susceptibility; prediction of zoonotic events; and prediction of host-defenses. Since the methods described are sequence-based, they may be of value to incorporate into software in the future.

Evidence of horizontal gene transfer from virus to host, or host to virus, can be found in a variety of genomes1,2,3,4. Examples of viral endogenization are the CRISPR spacer sequences found in bacterial host genomes4. Recently, we have found evidence of host protein sequences embedded in the nonstructural polyproteins of (+)ssRNA Group IV viruses. These sequences within the coding regions of the viral genome can be propagated generationally. The short stretches of homologous host-pathogen protein sequences (SSHHPS) are found in the virus and host5,6. SSHHPS are the conserved cleavage site motif sequences recognized by viral proteases that have homology to specific host proteins. These sequences direct the destruction of specific host proteins.
In our previous publication6, we compiled a list of all of the host proteins that were targeted by viral proteases and found that the list of targets was non-random (Table 1). Two trends were apparent. First, the majority of the viral proteases that cut host proteins belonged to Group IV viruses (24 of 25 cases involved Group IV viral proteases), and one protease belonged to the (+)ssRNA Group VI retroviruses (HIV, human immunodeficiency virus)7. Second, the host protein targets being cut by the viral proteases were generally involved in generating the innate immune responses suggesting that the cleavages were intended to antagonize the host&#39;s immune responses. Half of the host proteins targeted by the viral proteases were known components of signaling cascades that generate interferon (IFN) and proinflammatory cytokines (Table 1). Others were involved in host cell transcription8,9,10 or translation11. Interestingly, Shmakov et al.4 have shown that many CRISPR protospacer sequences correspond to genes involved in plasmid conjugation or replication4.
Group IV includes, among others, Flaviviridae, Picornaviridae, Coronaviridae, Calciviridae, and Togaviridae. Several new and emerging pathogens belong to Group IV such as the Zika virus (ZIKV), West Nile (WNV), Chikungunya (CHIKV), severe acute respiratory syndrome virus (SARS) and Middle East respiratory syndrome virus (MERS). The (+)ssRNA genome is essentially a piece of mRNA. To produce the enzymes necessary for genome replication, the (+)ssRNA genome first must be translated. In alphaviruses and other Group IV viruses, the enzymes necessary for replication are produced in a single polyprotein (i.e., nsP1234 for VEEV). The nonstructural polyprotein (nsP) is proteolytically processed (nsP1234 nsP1, nsP2, nsP3, nsP4) by the nsP2 protease to produce active enzymes12 (Figure 1). Cleavage of the polyprotein by the nsP2 protease is essential for viral replication; this has been demonstrated by deletion and site-directed mutagenesis of the active site cysteine of the nsP2 protease13,14. Notably, the translation of viral proteins precedes genome replication events. For example, nsP4 contains the RNA-dependent RNA polymerase needed to replicate the (+)ssRNA genome. Genome replication can produce dsRNA intermediates; these intermediates can trigger the host&#39;s innate immune responses. Thus, these viruses may cleave host innate immune response proteins early in infection in order to suppress their effects15,16,17.
Silencing can occur at the level of DNA, RNA, and protein. What is common to each of the silencing mechanisms shown in Figure 1 is that short foreign DNA, RNA, or protein sequences are used to guide the destruction of specific targets to antagonize their function. The silencing mechanisms are analogous to &#34;search and delete&#34; programs that have been written in three different languages. The short cleavage site sequence is analogous to a &#34;keyword&#34;. Each program has an enzyme that recognizes the match between the short sequence (the &#34;keyword&#34;) and a word in the &#34;file&#34; that is to be deleted. Once a match is found, the enzyme cuts (&#34;deletes&#34;) the larger target sequence. The three mechanisms shown in Figure 1 are used to defend the host from viruses, or to defend a virus from a host&#39;s immune system.
Viral proteases recognize short cleavage site motif sequences between ~2-11 amino acids; in nucleotides, this would correspond to 6-33 bases. For comparison, CRISPR spacer sequences are ~26-72 nucleotides and RNAi are ~20-22 nucleotides18,19. While these sequences are relatively short, they can be recognized specifically. Given the higher diversity of amino acids, the probability of a random cleavage event is relatively low for a viral protease recognizing protein sequences of 6-8 amino acids or longer. The prediction of SSHHPS in host proteins will largely depend upon the specificity of the viral protease being examined. If the protease has strict sequence specificity requirements the chance of finding a cleavage site sequence is 1/206 = 1 in 64 million or 1/208 = 1 in 25.6 billion; however, most proteases have variable subsite tolerances (e.g., R or K may be tolerated at the S1 site). Consequently, there is no requirement for sequence identity between the sequences found in the host versus the virus. For viral proteases that have looser sequence requirements (such as those belonging to Picornaviridae) the probability of finding a cleavage site in a host protein may be higher. Many of the entries in Table 1 are from the Picornaviridae family.
Schechter &#38; Berger notation20 is commonly used to describe the residues in a protease substrate and the subsites to which they bind, we utilize this notation throughout. The residues in the substrate that are N-terminal of the scissile bond are denoted as P3-P2-P1 while those that are C-terminal are denoted as P1&#39;-P2&#39;-P3&#39;. The corresponding subsites in the protease that bind these amino acid residues are S3-S2-S1 and S1&#39;-S2&#39;-S3&#39;, respectively.
To determine which host proteins are being targeted, we can identify SSHHPS in the viral polyprotein cleavage sites and search for the host proteins that contain them. Herein, we outline procedures for identifying SSHHPS using known viral protease cleavage site sequences. The bioinformatic methods, protease assays, and in silico methods described are intended to be used in conjunction with cell-based assays.
Sequence alignments of the host proteins targeted by viral proteases have revealed species-specific differences within these short cleavage site sequences. For example, the Venezuelan equine encephalitis virus (VEEV) nsP2 protease was found to cut human TRIM14, a tripartite motif (TRIM) protein6. Some TRIM proteins are viral restriction factors (e.g., TRIM5&#945;21), most are thought to be ubiquitin E3 ligases. TRIM14 lacks a RING (really interesting new gene) domain and is not thought to be an E3 ligase22. TRIM14 has been proposed to be an adaptor in the mitochondrial antiviral signalosome (MAVS)22, but may have other antiviral functions23. Alignment of TRIM14 sequences from various species shows that equine lack the cleavage site and harbor a truncated version of TRIM14 that is missing the C-terminal PRY/SPRY domain. This domain contains a polyubiquitination site (Figure 2). In equine, these viruses are highly lethal (~20-80% mortality) whereas in humans only ~1% die from VEEV infections24. Cleavage of the PRY/SPRY domain may transiently short circuit the MAVS signaling cascade. This cascade can be triggered by dsRNA and leads to the production of interferon and pro-inflammatory cytokines. Thus, the presence of the SSHHPS may be useful for predicting which species have defense systems against specific Group IV viruses.
In Group IV viruses, IFN antagonism mechanisms are thought to be multiply redundant25. Host protein cleavage may be transient during infection and concentrations may recover over time. We found in cells that TRIM14 cleavage products could be detected very early after transfection (6 h) with a plasmid encoding the protease (cytomegalovirus promoter). However, at longer periods, the cleavage products were not detected. In virus-infected cells, the kinetics were different and cleavage products could be detected between 6-48 h6. Others have reported the appearance of host protein cleavage products as early as 3-6 h post infection9,11.
Proteolytic activity in cells is often difficult to catch; the cleavage products can vary in their solubility, concentration, stability, and lifetime. In cell-based assays, it cannot be assumed that cleavage products will accumulate in a cell or that the band intensities of cut and uncut protein will show compensatory increases and decreases as the cut protein may be degraded very quickly and may not be detectable in a Western blot at an expected molecular weight (MW) (e.g., the region containing the epitope could be cleaved by other host proteases or could be ubiquitinated). If the substrate of the viral protease is an innate immune response protein, its concentration may vary during infection. For example, some innate immune response proteins are present prior to viral infection and are induced further by interferon26. The concentration of the target protein may therefore fluctuate during infection and comparison of uninfected vs. infected cell lysates may be difficult to interpret. Additionally, all cells may not be uniformly transfected or infected. In vitro protease assays using purified proteins from E. coli on the other hand have fewer variables for which to control and such assays can be done using SDS-PAGE rather than immunoblots. Contaminating proteases can be inhibited in the early steps of the protein purification of the CFP/YFP substrate, and mutated viral proteases can be purified and tested as controls to determine if the cleavage is due to the viral protease or a contaminating bacterial protease.
One limitation of in vitro protease assays is that they lack the complexity of a mammalian cell. For an enzyme to cut its substrate, the two must be co-localized. Group IV viral proteases differ in structure and localization. For example, the ZIKV protease is embedded in the endoplasmic reticulum (ER) membrane and faces the cytosol, whereas the VEEV nsP2 protease is a soluble protein in the cytoplasm and nucleus27. Some of the cleavage site sequences found in the ZIKV SSHHPS analysis were in signal peptides suggesting that cleavage might occur co-translationally for some targets. Thus, the location of the protease and the substrate in the cell also needs to be considered in these analyses.
Cell-based assays can be valuable for establishing a role for the identified host protein(s) in infection. Methods that aim to halt viral protease cleavage of host proteins such as the addition of a protease inhibitor6 or a mutation in the host target16 can be used to examine their effects on viral replication. Overexpression of the targeted protein also may affect viral replication28. Plaque assays or other methods can be used to quantify viral replication.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>250 mL Erlenmeyer Flask</td>
			<td>VWR</td>
			<td>89000-362</td>
			<td></td>
		</tr>
		<tr>
			<td>2-mercaptoethanol</td>
			<td>Acros Organics (Fisher)</td>
			<td>125472500</td>
			<td>Danger: Acutely Toxic. Open bottle in hood to avoid inhaling the fumes.</td>
		</tr>
		<tr>
			<td>4L Pyrex wide-mouth graduated Erlenmeyer flask with screw-cap</td>
			<td>Millipore Sigma</td>
			<td>CLS49954L-1EA</td>
			<td></td>
		</tr>
		<tr>
			<td>AKTA Prime Plus</td>
			<td>GE Healthcare</td>
			<td>17-0729-01</td>
			<td></td>
		</tr>
		<tr>
			<td>AKTA XK 16/20 Column</td>
			<td>GE Healthcare</td>
			<td>28988937</td>
			<td></td>
		</tr>
		<tr>
			<td>Amicon Ultra-0.5 Centrifugal Filter Unit</td>
			<td>Millipore Sigma</td>
			<td>UFC501096</td>
			<td></td>
		</tr>
		<tr>
			<td>Amicon Ultra-15 Centrifugal Filter Unit</td>
			<td>Millipore Sigma</td>
			<td>UFC901096</td>
			<td></td>
		</tr>
		<tr>
			<td>Amicon Ultra-4 Centrifugal Filter Unit</td>
			<td>Millipore Sigma</td>
			<td>UFC801024</td>
			<td></td>
		</tr>
		<tr>
			<td>Ampicillin</td>
			<td>Sigma</td>
			<td>A0166</td>
			<td>Danger: Allergic reactions (skin or breathing).</td>
		</tr>
		<tr>
			<td>Chelating Sepharose Fast Flow</td>
			<td>GE Healthcare</td>
			<td>17-0575-02</td>
			<td>Once the resin is equilibrated with 0.2 M Nickel Sulfate it is refered to as a Nickel Column in the text. Column will have a green color after washing with water. The column will have a blue color after equilibrating with buffer.</td>
		</tr>
		<tr>
			<td>Chloramphenicol</td>
			<td>RPI</td>
			<td>C61000</td>
			<td>Danger: May cause cancer.</td>
		</tr>
		<tr>
			<td>Corning 50 mL centrifuge tubes</td>
			<td>Corning</td>
			<td>430828</td>
			<td>Suggestion: Polypropylene tubes are less likely to crack during sonication than Polyethylene tubes</td>
		</tr>
		<tr>
			<td>Corning 96 Well Half-Area Microplate, Non-Binding Surface</td>
			<td>Corning</td>
			<td>3993</td>
			<td></td>
		</tr>
		<tr>
			<td>Dialysis Tubing Clips</td>
			<td>Fisher Scientific</td>
			<td>PI68011</td>
			<td></td>
		</tr>
		<tr>
			<td>Disposable PD-10 Desalting Column</td>
			<td>GE Healthcare</td>
			<td>17-0851-01</td>
			<td></td>
		</tr>
		<tr>
			<td>DNAse</td>
			<td>Sigma</td>
			<td>DN25-1G</td>
			<td></td>
		</tr>
		<tr>
			<td>DTT (DL-Dithiothreitol)</td>
			<td>RPI</td>
			<td>D11000-50.0</td>
			<td>Warning: Acute Oral Toxicity; skin and eye irritation</td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>Fisher Scientific</td>
			<td>S311-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Fisherbrand Petri Dishes with Clear Lid</td>
			<td>Fisher Scientific</td>
			<td>FB0875712</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>Acros Organics (Fisher)</td>
			<td>15892-0010</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Millipore Sigma</td>
			<td>H4034-1KG</td>
			<td></td>
		</tr>
		<tr>
			<td>Imidazole</td>
			<td>Acros Organics</td>
			<td>301870010</td>
			<td>Danger: Toxic, Irritant</td>
		</tr>
		<tr>
			<td>IPTG (Isopropyl &#946;-D-thiogalactopyranoside)</td>
			<td>Calbiochem (Millipore Sigma)</td>
			<td>420291</td>
			<td>Do not breathe dust. Avoid contact with eyes and skin.</td>
		</tr>
		<tr>
			<td>Laemmli Sample Buffer</td>
			<td>BIO-RAD</td>
			<td>1610737</td>
			<td></td>
		</tr>
		<tr>
			<td>Luria Bertani Agar</td>
			<td>Fluka (Millipore Sigma)</td>
			<td>L3027-1KG</td>
			<td>Suggestion: Autoclave with magnetic stirrer in the liquid, and stir while cooling. Wait to add antibiotic until you can hold your hands on the bottle without pain for 30 seconds.</td>
		</tr>
		<tr>
			<td>Luria Bertani Media</td>
			<td>Fisher Bioreagents</td>
			<td>BP1426-2</td>
			<td></td>
		</tr>
		<tr>
			<td>Lysozyme</td>
			<td>Sigma</td>
			<td>L4919-5g</td>
			<td></td>
		</tr>
		<tr>
			<td>Mini-PROTEAN Tetra Vertical Electrophoresis CellGel Box</td>
			<td>BIO-RAD</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Nalgene Oak Ridge High-Speed PPCO Centrifuge Tubes</td>
			<td>Nalgene (Thermo Scientific)</td>
			<td>3119-0050</td>
			<td></td>
		</tr>
		<tr>
			<td>Nanodrop</td>
			<td>Thermo Fisher</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>New Brunswick Innova 42R Shaker Incubator</td>
			<td>Eppendorf</td>
			<td>M1335</td>
			<td></td>
		</tr>
		<tr>
			<td>Nickel Sulfate Hexahydrate (Crystalline/Certified ACS), Fisher Chemical</td>
			<td>Fisher Scientific</td>
			<td>N73-500</td>
			<td>Danger: Harmful if swallowed or inhaled, skin and eye irritation</td>
		</tr>
		<tr>
			<td>One Shot BL21(DE3) Chemically Competent E. coli</td>
			<td>Invitrogen (Thermo Fisher)</td>
			<td>C600003</td>
			<td>May be harmful if inhaled or swallowed. May cause skin and eye irritation with susceptible people.</td>
		</tr>
		<tr>
			<td>One Shot BL21(DE3) pLysS Chemically Competent E. coli</td>
			<td>Invitrogen (Thermo Fisher)</td>
			<td>C606003</td>
			<td>May be harmful if inhaled or swallowed. May cause skin and eye irritation with susceptible people.</td>
		</tr>
		<tr>
			<td>pet15b plasmid DNA</td>
			<td>Novagen (Millipore Sigma)</td>
			<td>69661</td>
			<td>GenScript Inc. was used for commerical DNA synthesis. The pet15b plasmid was used for the CFP/YFP substrates.</td>
		</tr>
		<tr>
			<td>pet32b</td>
			<td>Novagen (Millipore Sigma)</td>
			<td>69016-3</td>
			<td>The pet32b plasmid was used for the cysteine protease construct.</td>
		</tr>
		<tr>
			<td>Pierce Protease Inhibitor Mini Tablets, EDTA-free</td>
			<td>Thermo Fisher</td>
			<td>A32955</td>
			<td>Warning: Skin corrosion/irriation; eye damage</td>
		</tr>
		<tr>
			<td>Plate Reader</td>
			<td>Molecular Devices</td>
			<td>Model M5</td>
			<td></td>
		</tr>
		<tr>
			<td>Precision Plus Protein All Blue Prestained Protein Standard</td>
			<td>BIO-RAD</td>
			<td>161-0373</td>
			<td></td>
		</tr>
		<tr>
			<td>Protein Extraction Reagent</td>
			<td>Novagen (Millipore Sigma)</td>
			<td>70584-4</td>
			<td>BugBuster or Bper (Catalog # 78248, ThermoFisher)</td>
		</tr>
		<tr>
			<td>Q-Sepharose Fast Flow</td>
			<td>G.E. Healthcare</td>
			<td>17-0510-01</td>
			<td>Anion exchange resin</td>
		</tr>
		<tr>
			<td>RunBlue (12%) 17-well PAGE gels</td>
			<td>Expedeon</td>
			<td>BCG01227</td>
			<td>Any 12% pre-cast polyacrylamide gel can be used</td>
		</tr>
		<tr>
			<td>RunBlue 20x SDS Running Buffer</td>
			<td>Expedeon</td>
			<td>NXB50500</td>
			<td>Dilute 50 mL with 950 mL deionized water to obtain 1x</td>
		</tr>
		<tr>
			<td>RunBlue Instant Blue Gel Stain</td>
			<td>Expedeon</td>
			<td>ISB1L</td>
			<td>Do not dilute, use as directed</td>
		</tr>
		<tr>
			<td>Sodium Chloride</td>
			<td>Fisher Chemical</td>
			<td>S271-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Sonifier Cell Disrupter 450 Sonicator</td>
			<td>Branson Ultrasonics (VWR)</td>
			<td>Model No. 101-063-346R</td>
			<td>Sonicator was used on level 5</td>
		</tr>
		<tr>
			<td>Spectra/Por 6-8 kD MWCO</td>
			<td>Spectrum Labs</td>
			<td>132645T</td>
			<td>Dialysis Tubing</td>
		</tr>
		<tr>
			<td>SP-Sepharose Fast Flow</td>
			<td>G.E. Healthcare</td>
			<td>17-0729-01</td>
			<td>Cation exchange resin</td>
		</tr>
		<tr>
			<td>Thrombin from bovine plasma</td>
			<td>Sigma</td>
			<td>T6634-500UN</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris Base</td>
			<td>Fisher Scientific</td>
			<td>BP152-500</td>
			<td>Caution: Eye/Skin Irritant</td>
		</tr>
	</tbody>
</table>

analysis of group iv viral sshhps using in vitro and in silico methods

Alphaviral enzymes are synthesized in a single polypeptide. The nonstructural polyprotein (nsP) is processed by its nsP2 cysteine protease to produce active enzymes essential for viral replication. Viral proteases are highly specific and recognize conserved cleavage site motif sequences (~6-8 amino acids). In several Group IV viruses, the nsP protease(s) cleavage site motif sequences can be found in specific host proteins involved in generating the innate immune responses and, in some cases, the targeted proteins appear to be linked to the virus-induced phenotype. These viruses utilize short stretches of homologous host-pathogen protein sequences (SSHHPS) for targeted destruction of host proteins. To identify SSHHPS the viral protease cleavage site motif sequences can be inputted into BLAST and the host genome(s) can be searched. Cleavage initially can be tested using the purified nsP viral protease and fluorescence resonance energy transfer (FRET) substrates made in E. coli. The FRET substrates contain cyan and yellow fluorescent protein and the cleavage site sequence (CFP-sequence-YFP). This protease assay can be used continuously in a plate reader or discontinuously in SDS-PAGE gels. Models of the bound peptide substrates can be generated in silico to guide substrate selection and mutagenesis studies. CFP/YFP substrates have also been utilized to identify protease inhibitors. These in vitro and in silico methods can be used in combination with cell-based assays to determine if the targeted host protein affects viral replication.

SSHHPS analysis of the ZIKV ns2B/3 protease identified 4 host protein targets: FOXG1, SFRP1, a Gs alpha subunit from a retinal cDNA library, and the NT5M mitochondrial 5&#39;,3&#39;-nucleotidase (Figure 10)6. Notably, no other method predicted these proteins as potential targets of the ZIKV protease. Mutations in the FOXG1 gene have been linked to a congenital syndrome characterized by impaired development and structural brain abnormalities such as microcephaly. SFRP1 is a secreted frizzled-related protein (SFRP); these soluble receptors can competitively bind Wnt ligands to antagonize and inhibit Wnt signaling. The Wnt signaling pathway is involved in the regulation of the IFN response during Flavivirus infection36. The cleavage of SFRP1 would be expected to enhance flavivirus replication. SFRP1 is also involved in Th17-cell differentiation37. Sequence alignments of the SSHHPS showed species-specific differences in the cleavage site sequences (Figure 10D). The cleavage site sequence in SFRP1 was identical in humans and chickens; ZIKV can induce mortality and microcephaly in chicken embryos38. In rodents, the highly conserved P1 residue (K/R)R&#8595;G is substituted by a glycine (RGG). Immunocompetent strains of mice are generally resistant to ZIKV infection and disease39.
Steady state kinetic parameters and inhibition constants can be measured for the viral polyprotein sequences and for the host protein sequences using the continuous assay in a plate reader31,40,41 (Figure 11A). For qualitative cleavage information, such as cleavage of a particular sequence or the inhibition of the protease by various compounds, the discontinuous assay can be used (Figure 11B).
Optimization of the number of residues in between CFP and YFP may be required. A substrate-bound model can be made using the in silico methods. A representative docked model of the nsP1/nsP2 junction is shown in Figure 9. For the VEEV nsP2 protease, cleavage of a 12-amino acid Semliki Forest Virus (SFV) sequence had been reported (Km = 0.58 mM)33. Lengthening the substrate sequence to 19, 22, and 25 residues and reducing the ionic strength of the buffer led to a significant reduction in Km. Examination of the VEEV nsP2 crystal structure and crystal packing also showed that a portion of one of the junctions was packed against the protease domain and was helical. Thus, the longer VEEV substrates may bind better due to the recognition of a secondary structural motif.
For TRIM14, we obtained a Km = 21 &#181;M6,33. The Km for the substrate carrying the host protein sequence was comparable to the Km values of the substrates containing the viral polyprotein cleavage site sequences (Km(V12) = 12 &#181;M and Km(V34) = 21 &#181;M). The cleavage site sequences at the nsP1/nsP2, nsP2/nsP3, and nsP3/nsP4 junctions were cut with different efficiencies. In the cell, this is thought to allow for sequential cleavage of the polyprotein42.
Caution should be taken in interpreting negative results. If no cleavage occurs, the cleavage site may be too short or the purified protease may be inactive. For substrates that are cut, additional experiments are needed to confirm cleavage of the full length protein or cleavage in virus-infected cells. Appropriate follow-on experiments should be chosen. The effects of overexpression or silencing of the target protein on viral replication also can be tested.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/60421/60421fig1a.jpg" /> 
Figure 1: Three mechanisms of silencing. Silencing can occur at the level of DNA, RNA, or protein. These &#34;search and delete&#34; algorithms each use a &#34;keyword&#34; to direct the cleavage of a file containing the word. This figure has been modified from Morazzani et al.32 and the references therein. <a href="https://www.jove.com/files/ftp_upload/60421/60421fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/60421/60421fig2a.jpg" /> 
Figure 2: Species-specific differences in cleavage site sequences. The C-terminal PRY/SPRY domains of TRIM14 homologues are shown in the alignment. The PRY/SPRY domain can be identified by the conserved motifs highlighted in gray. Human TRIM14 is cut at QEAGA&#8595;G by the VEEV nsP2 cysteine protease. The SSHHP sequence is shown in color. The residue in green is the P1&#39; residue; in blue is the P4 residue, and in red are other conserved residues within the cleavage site motif sequence. Equine harbor a truncated version of TRIM14 lacking the PRY/SPRY domain. The lysine highlighted in cyan is poly-ubiquitinated and is important for the assembly of the MAVS signalosome. The C-terminal PRY/SPRY domain may be transiently cut by the nsP2 protease to impair the host&#39;s antiviral response intracellularly during an acute viral infection. In equine, this domain is always absent. This suggests that the PRY/SPRY domain of TRIM14 may have a protective function against VEEV infections. This figure has been reproduced from Morazanni et al.6 <a href="https://www.jove.com/files/ftp_upload/60421/60421fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/60421/60421fig3a.jpg" /> 
Figure 3: SSHHPS identification using BLAST. The cleavage site motif sequence at the VEEV nsP1/nsP2 junction is aligned with the SSHHP sequence in the host protein TRIM14. The residue colored in green is the P1&#39; residue; in blue is the P4 residue and in red are other conserved residues of the cleavage site motif sequence. Most alignments contained homology to regions outside of the conserved cleavage site motif or did not include the P1/P1&#39; scissile bond residues. TRIM14 showed a match to 6 residues in sequential order that included P1 and P1&#39;. <a href="https://www.jove.com/files/ftp_upload/60421/60421fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/60421/60421fig4a.jpg" /> 
Figure 4: Protein and DNA sequences of the CFP-V12-YFP substrate for the VEEV nsP2 cysteine protease. The NdeI (CATATG) and XhoI (CTCGAG) restriction sites are shown in capital letters. In red is the cleavage site sequence from the viral polyprotein that is in between nsP1 and nsP2. The residue in green is the P1&#39; residue and in blue is the P4 residue of the cleavage site. <a href="https://www.jove.com/files/ftp_upload/60421/60421fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/60421/60421fig5a.jpg" /> 
Figure 5: Protein sequence of the Trx-VEEV-nsP2 cysteine protease construct. Thioredoxin (Trx) is shown in yellow. The thrombin cleavage site and His-tag are shown in cyan. The Cys-His dyad are labeled in red. <a href="https://www.jove.com/files/ftp_upload/60421/60421fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/60421/60421fig6a.jpg" /> 
Figure 6: Peptide structures in MOE. <a href="https://www.jove.com/files/ftp_upload/60421/60421fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/60421/60421fig7a.jpg" /> 
Figure 7: Docking of substrate peptide using PyRx/AutoDock. <a href="https://www.jove.com/files/ftp_upload/60421/60421fig7large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 8" class="xfigimg" src="/files/ftp_upload/60421/60421fig8a.jpg" /> 
Figure 8: Jobs running on the Biowulf cluster. <a href="https://www.jove.com/files/ftp_upload/60421/60421fig8large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 9" class="xfigimg" src="/files/ftp_upload/60421/60421fig9a.jpg" /> 
Figure 9: Model of the VEEV P12 substrate containing the cleavage site sequence at the nsP1/nsP2 junction. The Cys-477/His-546 catalytic dyad is shown in blue. Figure was made using Pymol (https://pymol.org). <a href="https://www.jove.com/files/ftp_upload/60421/60421fig9large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 10" class="xfigimg" src="/files/ftp_upload/60421/60421fig10a.jpg" /> 
Figure 10: SSHHPS Analysis of the Zika virus ns2B/ns3 protease. (A) Predicted host protein targets of the ZIKV ns2B/ns3 protease. Residues in red match a single cleavage site sequence. Residues in green are tolerated at the subsite and match residues at other cleavage sites. SFRP1 had the highest number of identical residues in consecutive order. (B) CFP-substrate-YFP proteins (~50-60 kDa) were expressed and purified containing the predicted SSHHP sequence from each host protein (human). The ZIKV protease cut human FOXG1, SFRP1, NT5M and a Gsalpha subunit isolated from a retinal cDNA library. The cleavage products are approximately 28-30 kDa. The substrate sequences are available in Morazzani et al.6 (C) While the ns2B/ns3 protease cut SFRP1, it did not cut its homologues (SFRP2 and SFRP4). (D) Alignment of the cleavage sites from different animal species may be useful in selecting an animal model for a Group IV virus. Note that the conserved R&#8595;G sequence differs between humans and rodents in SFRP1. Figure reproduced from Morazzani et al.6 <a href="https://www.jove.com/files/ftp_upload/60421/60421fig10large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 11" class="xfigimg" src="/files/ftp_upload/60421/60421fig11a.jpg" /> 
Figure 11: Steady state kinetic analysis using the continuous and discontinuous assays. (A) The kinetic data shown in Table 5 was plotted in GraFit. The inset shows the Lineweaver-Burk plot. (B) SDS-PAGE gel showing the cleavage products of the CFP-V12-YFP substrate. In lane 1 is the &#34;UNCUT&#34; substrate (48 kDa). In lane 2 is the &#34;CUT&#34; substrate (31 kDa and 27 kDa). In lanes 3-9 different compounds were included to test their inhibitory activity. Lane 4 contains the E64d covalent inhibitor. These reactions were run overnight for ~17 h at room temperature. Boiling of the samples was required to achieve the sharp banding pattern. The nsP2 protease is visible (56 kDa) in the reactions containing enzyme, but not in lane 1. Lane 1 is the &#34;no enzyme&#34; control. <a href="https://www.jove.com/files/ftp_upload/60421/60421fig11large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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

We present a general protocol for identifying short stretches of homologous host-pathogen protein sequences (SSHHPS) embedded in the viral polyprotein. SSHHPS are recognized by viral proteases and direct the targeted destruction of specific host proteins by several Group IV viruses.

Alphaviral enzymes are synthesized in a single polypeptide. The nonstructural polyprotein (nsP) is processed by its nsP2 cysteine protease to produce ...

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.

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

Abstract