Journal of Visualized Experiments www.jove.com Copyright © 2019 Journal of Visualized Experiments December 2019 | 154 | e60421 | Page 1 of 15 Video Article Analysis of Group IV Viral SSHHPS Using In Vitro and In Silico Methods Xin Hu 1 , Jaimee R. Compton 2 , Patricia M. Legler 2 1 National Center for Advancing Translational Sciences, National Institutes of Health 2 United States Naval Research Laboratory Correspondence to: Patricia M. Legler at patricia.legler@nrl.navy.mil URL: https://www.jove.com/video/60421 DOI: doi:10.3791/60421 Keywords: Immunology and Infection, Issue 154, enzyme, assay, FRET, nonstructural protein, alphavirus, Group IV, protease, SSHHPS, gel assay, in vitro, docking Date Published: 12/21/2019 Citation: Hu, X., Compton, J.R., Legler, P.M. Analysis of Group IV Viral SSHHPS Using In Vitro and In Silico Methods. J. Vis. Exp. (154), e60421, doi:10.3791/60421 (2019). Abstract 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. Video Link The video component of this article can be found at https://www.jove.com/video/60421/ Introduction Evidence of horizontal gene transfer from virus to host, or host to virus, can be found in a variety of genomes 1,2,3,4 . Examples of viral endogenization are the CRISPR spacer sequences found in bacterial host genomes 4 . 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 host 5,6 . 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 publication 6 , 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'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 transcription 8,9,10 or translation 11 . Interestingly, Shmakov et al. 4 have shown that many CRISPR protospacer sequences correspond to genes involved in plasmid conjugation or replication 4 . 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 enzymes 12 (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 protease 13,14 . 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