Name: analysis of group iv viral sshhps using in vitro and in silico methods
Uploaded: 2019-12-21T12:00:00.000+00:00
Duration: 10 min 40 s
Description: Watch this Scientific Journal Video about Analysis of Group IV Viral SSHHPS Using In Vitro and In Silico Methods at JoVE.com

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><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>4 L 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 &amp;beta;-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 &lt;em&gt;E. coli&lt;/em&gt;</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 &lt;em&gt;E. coli&lt;/em&gt;</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

Research

JoVE Journal

Immunology and Infection

25.9K 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

Genome-wide RNAi Screening to Identify Host Factors That Modulate Oncolytic Virus Therapy

High-throughput genome-wide RNAi (RNA interference) screening technology has been widely used for discovering host factors that impact&#160;virus replication. Here we present the application of this technology to uncovering host targets that specifically modulate the replication of Maraba virus, an oncolytic rhabdovirus, and vaccinia virus with the goal of enhancing therapy. While the protocol has been tested for use with oncolytic Maraba virus and oncolytic vaccinia virus, this approach is applicable to other oncolytic viruses and can also be utilized for identifying host targets that modulate virus replication in mammalian cells in general. This protocol describes the development and validation of an assay for high-throughput RNAi screening in mammalian cells, the key considerations and preparation steps important for conducting a primary high-throughput RNAi screen, and a step-by-step guide for conducting a primary high-throughput RNAi screen; in addition, it broadly outlines the methods for conducting secondary screen validation and tertiary validation studies. The benefit of high-throughput RNAi screening is that it allows one to catalogue, in an extensive and unbiased fashion, host factors that modulate any aspect of virus replication for which one can develop an in vitro assay such as infectivity, burst size, and cytotoxicity. It has the power to uncover biotherapeutic targets unforeseen based on current knowledge.

Here we describe a protocol for employing high-throughput RNAi screening to uncover host targets that can be manipulated to enhance oncolytic virus therapy, specifically rhabodvirus and vaccinia virus therapy, but it can be readily adapted to other oncolytic virus platforms or for discovering host genes that modulate virus replication generally.

High-throughput genome-wide RNAi (RNA interference) screening technology has been widely used for discovering host factors that impact&#160;virus ...

Cancer Research

Dissecting Host-virus Interaction in Lytic Replication of a Model Herpesvirus

In response to viral infection, a host develops various defensive responses, such as activating innate immune signaling pathways that lead to antiviral cytokine production1,2. In order to colonize the host, viruses are obligate to evade host antiviral responses and manipulate signaling pathways. Unraveling the host-virus interaction will shed light on the development of novel therapeutic strategies against viral infection.
Murine &gamma;HV68 is closely related to human oncogenic Kaposi's sarcoma-associated herpesvirus and Epsten-Barr virus3,4. &gamma;HV68 infection in laboratory mice provides a tractable small animal model to examine the entire course of host responses and viral infection in vivo, which are not available for human herpesviruses. In this protocol, we present a panel of methods for phenotypic characterization and molecular dissection of host signaling components in &gamma;HV68 lytic replication both in vivo and ex vivo. The availability of genetically modified mouse strains permits the interrogation of the roles of host signaling pathways during &gamma;HV68 acute infection in vivo. Additionally, mouse embryonic fibroblasts (MEFs) isolated from these deficient mouse strains can be used to further dissect roles of these molecules during &gamma;HV68 lytic replication ex vivo.
Using virological and molecular biology assays, we can pinpoint the molecular mechanism of host-virus interactions and identify host and viral genes essential for viral lytic replication. Finally, a bacterial artificial chromosome (BAC) system facilitates the introduction of mutations into the viral factor(s) that specifically interrupt the host-virus interaction. Recombinant &gamma;HV68 carrying these mutations can be used to recapitulate the phenotypes of &gamma;HV68 lytic replication in MEFs deficient in key host signaling components. This protocol offers an excellent strategy to interrogate host-pathogen interaction at multiple levels of intervention in vivo and ex vivo.
Recently, we have discovered that &gamma;HV68 usurps an innate immune signaling pathway to promote viral lytic replication5. Specifically, &gamma;HV68 de novo infection activates the immune kinase IKK&beta; and activated IKK&beta; phosphorylates the master viral transcription factor, replication and transactivator (RTA), to promote viral transcriptional activation. In doing so, &gamma;HV68 efficiently couples its transcriptional activation to host innate immune activation, thereby facilitating viral transcription and lytic replication. This study provides an excellent example that can be applied to other viruses to interrogate host-virus interaction.

We describe a protocol to identify key roles of host signaling molecules in lytic replication of a model herpesvirus, gamma herpesvirus 68 (&gamma;HV68). Utilizing genetically modified mouse strains and embryonic fibroblasts for &gamma;HV68 lytic replication, the protocol permits both phenotypic characterization and molecular interrogation of virus-host interactions in viral lytic replication.

In response to viral infection, a host develops various defensive responses, such as activating innate immune signaling pathways that lead to antiviral ...

Simple and Robust in vivo and in vitro Approach for Studying Virus Assembly

In viruses with positive-sense RNA genomes pathogenic to humans, animals and plants, progeny encapsidation into mature and stable virions is a cardinal phase during establishment of infection in a given host. Consequently, study of encapsidation deciphers the information regarding the know-how of the mechanism regulating virus assembly to form infectious virions. Such information is vital in formulating novel methods of curbing virus spread and disease control. Virus encapsidation can be studied in vivo and in vitro. Genome encapsidation in vivo is a highly regulated selective process involving macromolecular interactions and subcellular compartmentalization. Therefore, study leading to dissect events encompassing virus encapsidation in vivo would provide basic knowledge to understand how viruses proliferate and assemble. Recently in vitro encapsidation has been exploited for the research in the area of biomedical imaging and therapeutic applications. Non-enveloped plant viruses stand far ahead in the venture of in vitro encapsidation of the negatively charged foreign material. Brome mosaic virus (BMV), a non-enveloped multicomponent RNA virus pathogenic to plants, has been used as a model system for studying genome packaging in vivo and in vitro. For encapsidation assays in Nicotiana benthamiana plants, Agrobacterium -mediated transient expression, refer to as agroinfiltration, is an efficient and robust technique for the synchronized delivery and expression of multiple components to the same cell. In this approach, a suspension of Agrobacterium tumefaciens cells carrying binary plasmid vectors carrying cDNAs of desiredviral mRNAs is infiltrated into the intercellular space withina leaf using nothing more sophisticated than a 1 ml disposable syringe (without needle). This process results in the transfer of DNA insert into plant cells; the T-DNA insert remains transiently in the nucleus and is then transcribed by the host polymerase II, leading to the transient expression. The resulting mRNA transcript (capped and polyadenylated) is then exported to the cytoplasm for translation. After approximately 24 to 48 hours of incubation,sections of infiltrated leaves can be sampled for microscopyor biochemical analyses. Agroinfiltration permits large numbers (hundreds to thousands) of cells to be transfected simultaneously. For in vitro encapsidation, purified virions of BMV are dissociated into capsid protein by dialyzing against dissociation buffer containing calcium chloride followed by removal of RNA and un-dissociated virions by centrifugation. Genome depleted capsid protein subunits are then reassembled with desired viral genome components or non-viral components such as indocyanine dye.

Simple and Robust in vivo and in vitro Approach for Studying Virus Assembly

A simple, efficient and robust way to synchronize the delivery of multiple viral components to plant cells via Agrobacterium-mediated transient expression is described. This approach is amenable for studying replication, encapsidation followed by in vitro reassembly of non-viral components into genome depleted optical viral ghosts suitable for biomedical applications.

In viruses with positive-sense RNA genomes pathogenic to humans, animals and plants, progeny encapsidation into mature and stable virions is a cardinal ...

Isolation and Genome Analysis of Single Virions using 'Single Virus Genomics'

Whole genome amplification and sequencing of single microbial cells enables genomic characterization without the need of cultivation 1-3. Viruses, which are ubiquitous and the most numerous entities on our planet 4 and important in all environments 5, have yet to be revealed via similar approaches. Here we describe an approach for isolating and characterizing the genomes of single virions called 'Single Virus Genomics' (SVG). SVG utilizes flow cytometry to isolate individual viruses and whole genome amplification to obtain high molecular weight genomic DNA (gDNA) that can be used in subsequent sequencing reactions.

Single Virus Genomics (SVG) is a method to isolate and amplify the genomes of single virons. Viral suspensions of a mixed assemblage are sorted using flow cytometry onto a microscope slide with discrete wells containing agarose, thereby capturing the virion and reducing genome shearing during downstream processing. Whole genome amplification is achieved using multiple displacement amplification (MDA) resulting in genomic material that is suitable for sequencing.

Whole genome amplification and sequencing of single microbial cells enables genomic characterization without the need of cultivation 1-3. Viruses, which ...

High-throughput Screening for Broad-spectrum Chemical Inhibitors of RNA Viruses

RNA viruses are responsible for major human diseases such as flu, bronchitis, dengue, Hepatitis C or measles. They also represent an emerging threat because of increased worldwide exchanges and human populations penetrating more and more natural ecosystems. A good example of such an emerging situation is chikungunya virus epidemics of 2005-2006 in the Indian Ocean. Recent progresses in our understanding of cellular pathways controlling viral replication suggest that compounds targeting host cell functions, rather than the virus itself, could inhibit a large panel of RNA viruses. Some broad-spectrum antiviral compounds have been identified with host target-oriented assays. However, measuring the inhibition of viral replication in cell cultures using reduction of cytopathic effects as a readout still represents a paramount screening strategy. Such functional screens have been greatly improved by the development of recombinant viruses expressing reporter enzymes capable of bioluminescence such as luciferase. In the present report, we detail a high-throughput screening pipeline, which combines recombinant measles and chikungunya viruses with cellular viability assays, to identify compounds with a broad-spectrum antiviral profile.

In vitro assays to measure virus replication have been greatly improved by the development of recombinant RNA viruses expressing luciferase or other enzymes capable of bioluminescence. Here we detail a high-throughput screening pipeline that combines such recombinant strains of measles and chikungunya viruses to isolate broad-spectrum antivirals from chemical libraries.

RNA viruses are responsible for major human diseases such as flu, bronchitis, dengue, Hepatitis C or measles. They also represent an emerging threat ...

Early Viral Entry Assays for the Identification and Evaluation of Antiviral Compounds

Cell-based systems are useful for discovering antiviral agents. Dissecting the viral life cycle, particularly the early entry stages, allows a mechanistic approach to identify and evaluate antiviral agents that target specific steps of the viral entry. In this report, the methods of examining viral inactivation, viral attachment, and viral entry/fusion as antiviral assays for such purposes are described, using hepatitis C virus as a model. These assays should be useful for discovering novel antagonists/inhibitors to early viral entry and help expand the scope of candidate antiviral agents for further drug development.

Here, we present a protocol that examines specific steps of the viral entry to identify and evaluate novel antiviral agents.

Cell-based systems are useful for discovering antiviral agents. Dissecting the viral life cycle, particularly the early entry stages, allows a mechanistic ...

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 ...

Arbovirus Infections As Screening Tools for the Identification of Viral Immunomodulators and Host Antiviral Factors

RNA interference- and genome editing-based screening platforms have been widely used to identify host cell factors that restrict virus replication. However, these screens are typically conducted in cells that are naturally permissive to the viral pathogen under study. Therefore, the robust replication of viruses in control conditions may limit the dynamic range of these screens. Furthermore, these screens may be unable to easily identify cellular defense pathways that restrict virus replication if the virus is well-adapted to the host and capable of countering antiviral defenses. In this article, we describe a new paradigm for exploring virus-host interactions through the use of screens that center on naturally abortive infections by arboviruses such as vesicular stomatitis virus (VSV). Despite the ability of VSV to replicate in a wide range of dipteran insect and mammalian hosts, VSV undergoes a post-entry, abortive infection in a variety of cell lines derived from lepidopteran insects, such as the gypsy moth (Lymantria dispar). However, these abortive VSV infections can be &#34;rescued&#34; when host cell antiviral defenses are compromised. We describe how VSV strains encoding convenient reporter genes and restrictive L. dispar cell lines can be paired to set-up screens to identify host factors involved in arbovirus restriction. Furthermore, we also show the utility of these screening tools in the identification of virally encoded factors that rescue VSV replication during coinfection or through ectopic expression, including those encoded by mammalian viruses. The natural restriction of VSV replication in L. dispar cells provides a high signal-to-noise ratio when screening for the conditions that promote VSV rescue, thus enabling the use of simplistic luminescence- and fluorescence-based assays to monitor the changes in VSV replication. These methodologies are valuable for understanding the interplay between host antiviral responses and viral immune evasion factors.

Here, we present the protocols to identify 1) virus-encoded immunomodulators that promote arbovirus replication and 2) eukaryotic host factors that restrict arbovirus replication. These fluorescence- and luminescence-based methods allow researchers to rapidly obtain quantitative readouts of arbovirus replication in simplistic assays with low signal-to-noise ratios.

RNA interference- and genome editing-based screening platforms have been widely used to identify host cell factors that restrict virus replication. ...

Use of Viral Entry Assays and Molecular Docking Analysis for the Identification of Antiviral Candidates against Coxsackievirus A16

Antiviral assays that mechanistically examine viral entry are pertinent to discern at which step the evaluated agents are most effective, and allow for the identification of candidate viral entry inhibitors. Here, we present the experimental approaches for the identification of small molecules capable of blocking infection by the non-enveloped coxsackievirus A16 (CVA16) through targeting the virus particles or specific steps in early viral entry. Assays include the time-of-drug-addition analysis, flow cytometry-based viral binding assay, and viral inactivation assay. We also present a molecular docking protocol utilizing virus capsid proteins to predict potential residues targeted by the antiviral compounds. These assays should help in the identification of candidate antiviral agents that act on viral entry. Future directions can explore these possible inhibitors for further drug development.

The goal of the protocol is to illustrate the different assays relating to viral entry that can be used to identify candidate viral entry inhibitors.

Antiviral assays that mechanistically examine viral entry are pertinent to discern at which step the evaluated agents are most effective, and allow for ...

A Viral Attachment Assay for Screening of Antiviral Compounds

Source: Tai, C. et al., <a href="https://app.jove.com/v/53124">Early Viral Entry Assays for the Identification and Evaluation of Antiviral Compounds.</a> J. Vis. Exp.&#160;(2015)
This video demonstrates a viral attachment assay for screening antiviral compounds. Using genetically modified hepatitis C virus and luciferase, it assesses inhibitory effects on viral attachment, as indicated by lower light intensity in co-treated wells compared to wells treated with the virus alone.

NOTE: Ensure that all procedures involving cell culture and virus infection are conducted in certified biosafety hoods that are appropriate for the ...

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

Summary

Explore More Videos

Chapters in this video

Genome-wide RNAi Screening to Identify Host Factors That Modulate Oncolytic Virus Therapy

Dissecting Host-virus Interaction in Lytic Replication of a Model Herpesvirus

Simple and Robust in vivo and in vitro Approach for Studying Virus Assembly

Isolation and Genome Analysis of Single Virions using 'Single Virus Genomics'

High-throughput Screening for Broad-spectrum Chemical Inhibitors of RNA Viruses

Early Viral Entry Assays for the Identification and Evaluation of Antiviral Compounds

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

Arbovirus Infections As Screening Tools for the Identification of Viral Immunomodulators and Host Antiviral Factors

Use of Viral Entry Assays and Molecular Docking Analysis for the Identification of Antiviral Candidates against Coxsackievirus A16

A Viral Attachment Assay for Screening of Antiviral Compounds