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>

Watch this Scientific Journal Video about Analysis of Group IV Viral SSHHPS Using In Vitro and In Silico Methods at JoVE.com

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 ...

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

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Immunology and Infection

25.8K Views.  National Institutes of Health. 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.

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

Genotypic Inference of HIV-1 Tropism Using Population-based Sequencing of V3

Background: Prior to receiving a drug from CCR5-antagonist class in HIV therapy, a patient must undergo an HIV tropism test to confirm that his or her viral population uses the CCR5 coreceptor for cellular entry, and not an alternative coreceptor. One approach to tropism testing is to examine the sequence of the V3 region of the HIV envelope, which interacts with the coreceptor.
Methods: Viral RNA is extracted from blood plasma. The V3 region is amplified in triplicate with nested reverse transcriptase-PCR. The amplifications are then sequenced and analyzed using the software, RE_Call. Sequences are then submitted to a bioinformatic algorithm such as geno2pheno to infer viral tropism from the V3 region. Sequences are inferred to be non-R5 if their geno2pheno false positive rate falls below 5.75%. If any one of the three sequences from a sample is inferred to be non-R5, the patient is unlikely to respond to a CCR5-antagonist.

HIV tropism can be inferred from the V3 region of the viral envelope. V3 is PCR amplified in triplicate using nested RT-PCR, sequenced, and interpreted using bioinformatic software. Samples with with 1 or more sequence(s) with low g2P scores are classified as non-R5 virus.

Background: Prior to receiving a drug from CCR5-antagonist class in HIV therapy, a patient must undergo an HIV tropism test to confirm that his or her ...

Establishing a Liquid-covered Culture of Polarized Human Airway Epithelial Calu-3 Cells to Study Host Cell Response to Respiratory Pathogens In vitro

The apical and basolateral surfaces of airway epithelial cells demonstrate directional responses to pathogen exposure in vivo. Thus, ideal in vitro models for examining cellular responses to respiratory pathogens polarize, forming apical and basolateral surfaces. One such model is differentiated normal human bronchial epithelial cells (NHBE). However, this system requires lung tissue samples, expertise isolating and culturing epithelial cells from tissue, and time to generate an air-liquid interface culture.
Calu-3 cells, derived from a human bronchial adenocarcinoma, are an alternative model for examining the response of proximal airway epithelial cells to respiratory insult1, pharmacological compounds2-6, and bacterial7-9 and viral pathogens, including influenza virus, rhinovirus and severe acute respiratory syndrome - associated coronavirus10-14. Recently, we demonstrated that Calu-3 cells are susceptible to respiratory syncytial virus (RSV) infection in a manner consistent with NHBE15,16 . Here, we detail the establishment of a polarized, liquid-covered culture (LCC) of Calu-3 cells, focusing on the technical details of growing and culturing Calu-3 cells, maintaining cells that have been cultured into LCC, and we present the method for performing respiratory virus infection of polarized Calu-3 cells.
To consistently obtain polarized Calu-3 LCC, Calu-3 cells must be carefully subcultured before culturing in Transwell inserts. Calu-3 monolayer cultures should remain below 90% confluence, should be subcultured fewer than 10 times from frozen stock, and should regularly be supplied with fresh medium. Once cultured in Transwells, Calu-3 LCC must be handled with care. Irregular media changes and mechanical or physical disruption of the cell layers or plates negatively impact polarization for several hours or days. Polarization is monitored by evaluating trans-epithelial electrical resistance (TEER) and is verified by evaluating the passive equilibration of sodium fluorescein between the apical and basolateral compartments17,18 . Once TEER plateaus at or above 1,000 &Omega;&times;cm2, Calu-3 LCC are ready to use to examine cellular responses to respiratory pathogens.

Establishing a Liquid-covered Culture of Polarized Human Airway Epithelial Calu-3 Cells to Study Host Cell Response to Respiratory Pathogens In vitro

The findings and conclusions in this report are those of the authors and do not necessarily represent the views of the Centers for Disease Control and Prevention.

The  apical and basolateral surfaces of airway epithelial cells demonstrate  directional responses to pathogen exposure in  vivo. Thus, ideal in vitro ...

Quantitative In vitro Assay to Measure Neutrophil Adhesion to Activated Primary Human Microvascular Endothelial Cells under Static Conditions

The vascular endothelium plays an integral part in the inflammatory response. During the acute phase of inflammation, endothelial cells (ECs) are activated by host mediators or directly by conserved microbial components or host-derived danger molecules. Activated ECs express cytokines, chemokines and adhesion molecules that mobilize, activate and retain leukocytes at the site of infection or injury. Neutrophils are the first leukocytes to arrive, and adhere to the endothelium through a variety of adhesion molecules present on the surfaces of both cells. The main functions of neutrophils are to directly eliminate microbial threats, promote the recruitment of other leukocytes through the release of additional factors, and initiate wound repair. Therefore, their recruitment and attachment to the endothelium is a critical step in the initiation of the inflammatory response. In this report, we describe an in vitro neutrophil adhesion assay using calcein AM-labeled primary human neutrophils to quantitate the extent of microvascular endothelial cell activation under static conditions. This method has the additional advantage that the same samples quantitated by fluorescence spectrophotometry can also be visualized directly using fluorescence microscopy for a more qualitative assessment of neutrophil binding.

Quantitative In vitro Assay to Measure Neutrophil Adhesion to Activated Primary Human Microvascular Endothelial Cells under Static Conditions

Neutrophil adherence to the activated endothelium at sites of infection is an integral component of the host's inflammatory response. Described in this report is a neutrophil binding assay that allows for the in vitro quantitation of primary human neutrophil binding to endothelial cells activated by inflammatory mediators under static conditions.

The vascular  endothelium plays an integral part in the inflammatory response. During the  acute phase of inflammation, endothelial cells (ECs) are ...

Total Protein Extraction and 2-D Gel Electrophoresis Methods for Burkholderia Species

The investigation of the intracellular protein levels of bacterial species is of importance to understanding the pathogenic mechanisms of diseases caused by these organisms. Here we describe a procedure for protein extraction from Burkholderia species based on mechanical lysis using glass beads in the presence of ethylenediamine tetraacetic acid and phenylmethylsulfonyl fluoride in phosphate buffered saline. This method can be used for different Burkholderia species, for different growth conditions, and it is likely suitable for the use in proteomic studies of other bacteria. Following protein extraction, a two-dimensional (2-D) gel electrophoresis proteomic technique is described to study global changes in the proteomes of these organisms. This method consists of the separation of proteins according to their isoelectric point by isoelectric focusing in the first dimension, followed by separation on the basis of molecular weight by acrylamide gel electrophoresis in the second dimension. Visualization of separated proteins is carried out by silver staining.

Total Protein Extraction and 2-D Gel Electrophoresis Methods for Burkholderia Species

Members of the Burkholderia genus are pathogens of clinical importance. We describe a method for total bacterial protein extraction, using mechanical disruption, and 2-D gel electrophoresis for subsequent proteomic analysis.

The investigation of  the intracellular protein levels of bacterial species is of importance to  understanding the pathogenic mechanisms of diseases ...

Neutrophil Isolation and Analysis to Determine their Role in Lymphoma Cell Sensitivity to Therapeutic Agents

Neutrophils are the most abundant (40% to 75%) type of white blood cells and among the first inflammatory cells to migrate towards the site of inflammation. They are key players in the innate immune system and play major roles in cancer biology. Neutrophils have been proposed as key mediators of malignant transformation, tumor progression, angiogenesis and in the modulation of the antitumor immunity; through their release of soluble factors or their interaction with tumor cells. To characterize the specific functions of neutrophils, a fast and reliable method is coveted for in vitro isolation of neutrophils from human blood. Here, a density gradient separation method is demonstrated to isolate neutrophils as well as mononuclear cells from the blood. The procedure consists of layering the density gradient solution such as Ficoll carefully above the diluted blood obtained from patients diagnosed with chronic lymphocytic leukemia (CLL), followed by centrifugation, isolation of mononuclear layer, separation of neutrophils from RBCsby dextran then lysis of residual erythrocytes. This method has been shown to isolate neutrophils &#8805; 90 % pure. To mimic the tumor microenvironment, 3-dimensional (3D) experiments were performed using basement membrane matrix such as Matrigel. Given the short half-life of neutrophils in vitro, 3D experiments with fresh human neutrophils cannot be performed. For this reason promyelocytic HL60 cells are differentiated along the granulocytic pathway using the differentiation inducers dimethyl sulfoxide (DMSO) and retinoic acid (RA). The aim of our experiments is to study the role of neutrophils on the sensitivity of lymphoma cells to anti-lymphoma agents. However these methods can be generalized to study the interactions of neutrophils or neutrophil-like cells with a large range of cell types in different situations.

Neutrophils are the most abundant type of white blood cells. The isolation of neutrophils from human blood by density gradient separation method and the differentiation of human promyelocytic (HL60) cells along the granulocytic pathway are described here; to test their role on sensitivity of lymphoma cells to anti-lymphoma agents.

Neutrophils are the most abundant (40% to 75%) type of white blood cells and among the first inflammatory cells to migrate towards the site of ...

Analysis of Lymphocyte Extravasation Using an In Vitro Model of the Human Blood-brain Barrier

Lymphocyte extravasation into the central nervous system (CNS) is critical for immune surveillance. Disease-related alterations of lymphocyte extravasation might result in pathophysiological changes in the CNS. Thus, investigation of lymphocyte migration into the CNS is important to understand inflammatory CNS diseases and to develop new therapy approaches. Here we present an in vitro model of the human blood-brain barrier to study lymphocyte extravasation. Human brain microvascular endothelial cells (HBMEC) are confluently grown on a porous polyethylene terephthalate transwell insert to mimic the endothelium of the blood-brain barrier. Barrier function is validated by zonula occludens immunohistochemistry, transendothelial electrical resistance (TEER) measurements as well as analysis of evans blue permeation. This model allows investigation of the diapedesis of rare lymphocyte subsets such as CD56brightCD16dim/- NK cells. Furthermore, the effects of other cells, cytokines and chemokines, disease-related alterations, and distinct treatment regimens on the migratory capacity of lymphocytes can be studied. Finally, the impact of inflammatory stimuli as well as different treatment regimens on the endothelial barrier can be analyzed.

Analysis of Lymphocyte Extravasation Using an In Vitro Model of the Human Blood-brain Barrier

Here, we describe a human blood-brain barrier model enabling to investigate lymphocyte transmigration into the central nervous system in vitro.

Lymphocyte extravasation into the central nervous system (CNS) is critical for immune surveillance. Disease-related alterations of lymphocyte ...

In Vitro Methods for Comparing Target Binding and CDC Induction Between Therapeutic Antibodies: Applications in Biosimilarity Analysis

Therapeutic monoclonal antibodies (mAbs) are relevant to the treatment of different pathologies, including cancers. The development of biosimilar mAbs by pharmaceutical companies is a market opportunity, but it is also a strategy to increase drug accessibility and reduce therapy-associated costs. The protocols detailed here describe the evaluation of target binding and CDC induction by rituximab in Daudi cells. These two functions require different structural regions of the antibody and are relevant to the clinical effect induced by rituximab. The protocols allow the side-to-side comparison of a reference rituximab and a marketed rituximab biosimilar. The evaluated products showed differences both in target binding and CDC induction, suggesting that there are underlying physicochemical differences and highlighting the need to analyze the impact of those differences in the clinical setting. The methods reported here constitute simple and inexpensive in vitro models for the evaluation of the activity of rituximab biosimilars. Thus, they can be useful during biosimilar development, as well as for quality control in biosimilar production. Furthermore, the presented methods can be extrapolated to other therapeutic mAbs.

In Vitro Methods for Comparing Target Binding and CDC Induction Between Therapeutic Antibodies: Applications in Biosimilarity Analysis

This protocol describes the in vitro comparison of two key functional characteristics of rituximab: target binding and complement-dependent cytotoxicity (CDC) induction. The methods were employed for a side-to-side comparison between reference rituximab and a rituximab biosimilar. These assays can be employed during biosimilar development or as a quality control in their production.

Therapeutic monoclonal antibodies (mAbs) are relevant to the treatment of different pathologies, including cancers. The development of biosimilar mAbs by ...

Alternative In Vitro Methods for the Determination of Viral Capsid Structural Integrity

Human norovirus exacts considerable public health and economic losses worldwide. Emerging in vitro cultivation advances are not yet applicable for routine detection of the virus. The current detection and quantification techniques, which rely primarily on nucleic acid amplification, do not discriminate infectious from non-infectious viral particles. The purpose of this article is to present specific details on recent advances in techniques used together in order to acquire further information on the infectivity status of viral particles. One technique involves assessing binding of a norovirus ssDNA aptamer to capsids. Aptamers have the advantage of being easily synthesized and modified, and are inexpensive and stable. Another technique, dynamic light scattering (DLS), has the advantage of observing capsid behavior in solution. Electron microscopy allows for visualization of the structural integrity of the viral capsids. Although promising, there are some drawbacks to each technique, such as non-specific aptamer binding to positively-charged molecules from sample matrices, requirement of purified capsid for DLS, and poor sensitivity for electron microscopy. Nonetheless, when these techniques are used in combination, the body of data produced provides more comprehensive information on norovirus capsid integrity that can be used to infer infectivity, information which is essential for accurate evaluation of inactivation methods or interpretation of virus detection. This article provides protocols for using these methods to discriminate infectious human norovirus particles.

Alternative In Vitro Methods for the Determination of Viral Capsid Structural Integrity

Routine detection methods utilizing viral genome amplification are limited by their inability to discriminate infectious from non-infectious particles. The purpose of this article is to provide detailed protocols for alternative methods to aid in discrimination of infectious norovirus particles using aptamer binding, dynamic light scattering, and transmission electron microscopy.

Human norovirus exacts considerable public health and economic losses worldwide. Emerging in vitro cultivation advances are not yet applicable for routine ...

Detection of Low Copy Number Integrated Viral DNA Formed by In Vitro Hepatitis B Infection

Hepatitis B Virus (HBV) is a common blood-borne pathogen causing liver cancer and liver cirrhosis resulting from chronic infection. The virus generally replicates through an episomal DNA intermediate; however, integration of HBV DNA fragments into the host genome has been observed, even though this form is not necessary for viral replication. The exact purpose, timing, and mechanism(s) by which HBV DNA integration occurs is not yet clear, but recent data show that it occurs very early after infection. Here, the in vitro generation and detection of HBV DNA integrations are described in detail. Our protocol specifically amplifies single copies of virus-cell DNA integrations and allows both absolute quantification and single-base pair resolution of the junction sequence. This technique has been applied to various HBV-susceptible cell types (including primary human hepatocytes), to various HBV mutants, and in conjunction with various drug exposures. We foresee this technique becoming a key assay to determine the underlying molecular mechanisms of this clinically relevant phenomenon.

Detection of Low Copy Number Integrated Viral DNA Formed by In Vitro Hepatitis B Infection

We describe here the in vitro generation of HBV DNA via a Hepatitis B virus infection system and the highly sensitive detection of its (1&#8211;2 copies) integration using inverse nested PCR.

Hepatitis B Virus (HBV) is a common blood-borne pathogen causing liver cancer and liver cirrhosis resulting from chronic infection. The virus generally ...

Establishment of Viral Infection and Analysis of Host-Virus Interaction in Drosophila Melanogaster

Virus spreading is a major cause of epidemic diseases. Thus, understanding the interaction between the virus and the host is very important to extend our knowledge of prevention and treatment of viral infection. The fruit fly Drosophila melanogaster has proven to be one of the most efficient and productive model organisms to screen for antiviral factors and investigate virus-host interaction, due to powerful genetic tools and highly conserved innate immune signaling pathways. The procedure described here demonstrates a nano-injection method to establish viral infection and induce systemic antiviral responses in adult flies. The precise control of the viral injection dose in this method enables high experimental reproducibility. Protocols described in this study include the preparation of flies and the virus, the injection method, survival rate analysis, the virus load measurement, and an antiviral pathway assessment. The influence effects of viral infection by the flies' background were mentioned here. This infection method is easy to perform and quantitatively repeatable; it can be applied to screen for host/viral factors involved in virus-host interaction and to dissect the crosstalk between innate immune signaling and other biological pathways in response to viral infection.

Establishment of Viral Infection and Analysis of Host-Virus Interaction in Drosophila Melanogaster

This protocol describes how to establish viral infection in vivo in Drosophila melanogaster using the nano-injection method and basic techniques to analyze virus-host interaction.

Virus spreading is a major cause of epidemic diseases. Thus, understanding the interaction between the virus and the host is very important to extend our ...