Characterization of the cysteine protease domain of Semliki Forest virus replicase protein nsP2 by in vitro mutagenesis.
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ABSTRACT:
SUBMITTER: Golubtsov A
PROVIDER: S-EPMC7118720 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Semliki Forest virus (SFV) is a mosquito-transmitted pathogen of small rodents, and infection of adult mice with SFV4, a neurovirulent strain of SFV, leads to lethal encephalitis in a few days, whereas mice infected with the avirulent A7(74) strain remain asymptomatic. In adult neurons, A7(74) is unable to form virions and hence does not reach a critical threshold of neuronal damage. To elucidate the molecular mechanisms of neurovirulence, we have cloned and sequenced the entire 11,758-nucleotide genome of A7(74) and compared it to the highly neurovirulent SFV4 virus. We found several sequence differences and sought to localize determinants conferring the neuropathogenicity by using a panel of chimeras between SFV4 and a cloned recombinant, rA774. We first localized virulence determinants in the nonstructural region by showing that rA774 structural genes combined with the SFV4 nonstructural genome produced a highly virulent virus, while a reciprocal recombinant was asymptomatic. In addition to several amino acid mutations in the nonstructural region, the nsp3 gene of rA774 displayed an opal termination codon and an in-frame 21-nucleotide deletion close to the nsp4 junction. Replacement in rA774 of the entire nsp3 gene with that of SFV4 reconstituted the virulent phenotype, whereas an arginine at the opal position significantly increased virulence, leading to clinical symptoms in mice. Completion of the nsp3 deletion in rA774 did not increase virulence. We conclude that the opal codon and amino acid mutations other than the deleted residues are mainly responsible for the attenuation of A7(74) and that the attenuating determinants reside entirely in the nonstructural region.
| S-EPMC111978 | biostudies-literature
Project description:Chikungunya virus (CHIKV) is a mosquito-borne alphavirus that causes epidemic fever, rash and polyarthralgia in Africa and Asia. Although it is known since the 1950s, new epidemiological and clinical features reported during the recent outbreak in the Indian Ocean can be regarded as the emergence of a new disease. Numerous severe forms of the infection have been described that put emphasis on the lack of efficient antiviral therapy. Among the virus-encoded enzymes, nsP2 constitutes an attractive target for the development of antiviral drugs. It is a multifunctional protein of approximately 90 kDa with a helicase motif in the N-terminal portion of the protein while the papain-like protease activity resides in the C-terminal portion. The nsP2 proteinase is an essential enzyme whose proteolytic activity is critical for virus replication. In this work, a recombinant CHIKV nsP2pro and a C-terminally truncated variant were expressed in Escherichia coli and purified by metal-chelate chromatography. The enzymatic properties of the proteinase were then determined using specific synthetic fluorogenic substrates. This study constitutes the first characterization of a recombinant CHIKV nsP2 cysteine protease, which may be useful for future drug screening.
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Project description:Chikungunya virus (CHIKV) infection is one of the major public health concerns, leading thousands of cases every year in rural as well as urban regions of several countries worldwide, few to mention are India, Philippines, Indonesia, and also in American countries. The structural and non-structural proteins of CHIKV are structurally and functionally similar to other alphaviruses such as Sindbis virus, Venezuelan Equine Encephalitis virus. The precursor protein of non-structural proteins is cleaved by proteolytic activity of non-structural protein (nsp2). This multifunctional nsp2 carry out nucleoside-triphosphatase (NTPase) and RNA helicase activity at its N-terminal and protease activity at C-terminal that makes it primarily a drug target to inhibit CHIKV replication. Until the current date, no suitable treatment for chikungunya infection is available. The introduction of a new drug into the market is a lengthy process, therefore, drug repurposing is now familiar approach that cut off the time and cost of drug discovery. In this study, we have implemented this approach with Food and Drug Administration (FDA) approved drugs and known cysteine protease inhibitors against CHIKV nsp2 protease using structure-based drug discovery. Our extensive docking and molecular dynamics simulations studies leads to two best interacting compounds, Ribostamycin sulfate and E-64, with utmost stable complexes at active site of nsp2 protease. Therefore, these compounds could be suitable for inhibiting CHIKV protease activity, and ultimately the viral replication.
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Project description:A common motif found in invertebrate serine proteases involved in immunity and development is the clip domain, proposed to regulate catalytic activity or protein-protein interactions within proteolytic cascades. Snake functions in a cascade that patterns the Drosophila embryo, and provides an accessible model for exploring the structural requirements for clip domain function. We tested Snake zymogens bearing charged-to-alanine mutations in the clip domain for their ability to rescue embryos lacking endogenous Snake and for their interactions by S2 cell co-transfection with upstream Gastrulation Defective and downstream Easter in the protease cascade. Of 13 single and multiple substitutions, one double mutant in a predicted protruding region exhibited a severe defect in embryonic rescue but showed only minimal defects in the co-transfection assay. We discuss implications of these and other results for potential biological roles of the Snake clip domain and for use of the in vitro assay in predicting protease behavior.
| S-EPMC2518291 | biostudies-literature
Project description:The N terminus of the replicase nonstructural protein 2 (nsp2) of porcine reproductive and respiratory syndrome virus (PRRSV) contains a putative cysteine protease domain (PL2). Previously, we demonstrated that deletion of either the PL2 core domain (amino acids [aa] 47 to 180) or the immediate downstream region (aa 181 to 323) is lethal to the virus. In this study, the PL2 domain was found to encode an active enzyme that mediates efficient processing of nsp2-3 in CHO cells. The PL2 protease possessed both trans- and cis-cleavage activities, which were distinguished by individual point mutations in the protease domain. The minimal size required to maintain these two enzymatic activities included nsp2 aa 47 to 240 (Tyr(47) to Cys(240)) and aa 47 to 323 (Tyr(47) to Leu(323)), respectively. Introduction of targeted amino acid mutations in the protease domain confirmed the importance of the putative Cys(55)- His(124) catalytic motif for nsp2/3 proteolysis in vitro, as were three additional conserved cysteine residues (Cys(111), Cys(142), and Cys(147)). The conserved aspartic acids (e.g., Asp(89)) were essential for the PL2 protease trans-cleavage activity. Reverse genetics revealed that the PL2 trans-cleavage activity played an important role in the PRRSV replication cycle in that mutations that impaired the PL2 protease trans function, but not the cis activity, were detrimental to viral viability. Lastly, the potential nsp2/3 cleavage site was probed. Mutations with the largest impact on in vitro cleavage were at or near the G(1196)|G(1197) dipeptide.
| S-EPMC2738230 | biostudies-literature
Project description:Using photoactivatable ribonucleoside crosslinking and biotinylated Cp retrieval, multiple binding sites on viral gRNA where identified.
2020-08-17 | GSE156313 | GEO
Project description:Chikungunya virus is the pathogenic alphavirus that causes chikungunya fever in humans. In the last decade millions of cases have been reported around the world from Africa to Asia to the Americas. The alphavirus nsP2 protein is multifunctional and is considered to be pivotal to viral replication, as the nsP2 protease activity is critical for proteolytic processing of the viral polyprotein during replication. Classically the alphavirus nsP2 protease is thought to be papain-like with the enzyme reaction proceeding through a cysteine/histidine catalytic dyad. We performed structure-function studies on the chikungunya nsP2 protease and show that the enzyme is not papain-like. Characterization of the catalytic dyad cysteine residue enabled us to identify a nearby serine that is catalytically interchangeable with the dyad cysteine residue. The enzyme retains activity upon alanine replacement of either residue but a replacement of both cysteine and serine residues results in no detectable activity. Protein dynamics appears to allow the use of either the cysteine or the serine residue in catalysis. This switchable dyad residue has not been previously reported for alphavirus nsP2 proteases and would have a major impact on the nsP2 protease as an anti-viral target.
| S-EPMC4657084 | biostudies-literature
Project description:Transmembrane (TM) helix-helix interactions are important for virus budding and fusion. We have developed a simulation strategy that reveals the main features of the helical packing between the TM domains of the two glycoproteins E1 and E2 of the alpha-virus Semliki Forest virus and that can be extrapolated to sketch TM helical packing in other alpha-viruses. Molecular dynamics simulations were performed in wild-type and mutant peptides, both isolated and forming E1/E2 complexes. The simulations revealed that the isolated wild-type E1 peptide formed a more flexible helix than the rest of peptides and that the wild-type E1/E2 complex consists of two helices that intimately pack their N-terminals. The residues located at the interhelical interface displayed the typical motif of the left-handed coiled-coils. These were small and medium residues as Gly, Ala, Ser, and Leu, which also had the possibility to form interhelical Calpha-H...O hydrogen bonds. Results from the mutant complexes suggested that correct packing is a compromise between these residues at both E1 and E2 interhelical interfaces. This compromise allowed prediction of E1-E2 contact residues in the TM spanning domain of other alphaviruses even though the sequence identity of E2 peptides is low in this domain.
| S-EPMC1303669 | biostudies-literature
Project description:Alphavirus nsP2 proteins are multifunctional and essential for viral replication. The protease role of nsP2 is critical for virus replication as only the virus protease activity is used for processing of the viral non-structural polypeptide. Chikungunya virus is an emerging disease problem that is becoming a world-wide health issue. We have generated purified recombinant chikungunya virus nsP2 proteins, both full length and a truncated protease domain from the C-terminus of the nsP2 protein. Enzyme characterization shows that the protease domain alone has different properties compared with the full length nsP2 protease. We also show chikungunya nsP2 protease possesses different substrate specificity to the canonical alphavirus nsP2 polyprotein cleavage specificity. Moreover, the chikungunya nsP2 also appears to differ from other alphavirus nsP2 in its distinctive ability to recognize small peptide substrates.
| S-EPMC4445351 | biostudies-literature
Project description:The nsp2 replicase protein of porcine reproductive and respiratory syndrome virus (PRRSV) was recently demonstrated to be processed from its precursor by the PL2 protease at or near the G(1196)|G(1197) dipeptide in transfected CHO cells. Here the proteolytic cleavage of PRRSV nsp2 was further investigated in virally infected MARC-145 cells by using two recombinant PRRSVs expressing epitope-tagged nsp2. The data revealed that PRRSV nsp2 exists as different isoforms, termed nsp2a, nsp2b, nsp2c, nsp2d, nsp2e, and nsp2f, during PRRSV infection. Moreover, on the basis of deletion mutagenesis and antibody probing, these nsp2 species appeared to share the same N terminus but to differ in their C termini. The largest protein, nsp2a, corresponded to the nsp2 product identified in transfected CHO cells. nsp2b and nsp2c were processed within or near the transmembrane (TM) region, presumably at or near the conserved sites G(981)|G(982) and G(828)|G(829)|G(830), respectively. The C termini for nsp2d, -e, and -f were mapped within the nsp2 middle hypervariable region, but no conserved cleavage sites could be definitively predicted. The larger nsp2 species emerged almost simultaneously in the early stage of PRRSV infection. Pulse-chase analysis revealed that all six nsp2 species were relatively stable and had low turnover rates. Deletion mutagenesis revealed that the smaller nsp2 species (e.g., nsp2d, nsp2e, and nsp2f) were not essential for viral replication in cell culture. Lastly, we identified a cellular chaperone, named heat shock 70-kDa protein 5 (HSPA5), that was strongly associated with nsp2, which may have important implications for PRRSV replication. Overall, these findings indicate that PRRSV nsp2 is increasingly emerging as a multifunctional protein and may have a profound impact on PRRSV replication and viral pathogenesis.
| S-EPMC2937792 | biostudies-literature