A fluorescence-activatable reporter of flavivirus NS2B-NS3 protease activity enables live imaging of infection in single cells and viral plaques.

The genus Flavivirus in the family Flaviviridae comprises many medically important viruses, such as dengue virus (DENV), Zika virus (ZIKV), and yellow fever virus. The quest for therapeutic targets to combat flavivirus infections requires a better understanding of the kinetics of virus-host interactions during infections with native viral strains. However, this is precluded by limitations of current cell-based systems for monitoring flavivirus infection in living cells. In the present study, we report the construction of fluorescence-activatable sensors to detect the activities of flavivirus NS2B-NS3 serine proteases in living cells. The system consists of GFP-based reporters that become fluorescent upon cleavage by recombinant DENV-2/ZIKV proteases in vitro A version of this sensor containing the flavivirus internal NS3 cleavage site linker reported the highest fluorescence activation in stably transduced mammalian cells upon DENV-2/ZIKV infection. Moreover, the onset of fluorescence correlated with viral protease activity. A far-red version of this flavivirus sensor had the best signal-to-noise ratio in a fluorescent Dulbecco's plaque assay, leading to the construction of a multireporter platform combining the flavivirus sensor with reporter dyes for detection of chromatin condensation and cell death, enabling studies of viral plaque formation with single-cell resolution. Finally, the application of this platform enabled the study of cell-population kinetics of infection and cell death by DENV-2, ZIKV, and yellow fever virus. We anticipate that future studies of viral infection kinetics with this reporter system will enable basic investigations of virus-host interactions and facilitate future applications in antiviral drug research to manage flavivirus infections.

A Nanopore Approach for Analysis of Caspase-7 Activity in Cell Lysates.

Caspases are an important protease family that coordinate inflammation and programmed cell death. Two closely related caspases, caspase-3 and caspase-7, exhibit largely overlapping substrate specificities. Assessing their proteolytic activities individually has therefore proven extremely challenging. Here, we constructed an outer membrane protein G (OmpG) nanopore with a caspase substrate sequence DEVDG grafted into one of the OmpG loops. Cleavage of the substrate sequence in the nanopore by caspase-7 generated a characteristic signal in the current recording of the OmpG nanopore that allowed the determination of the activity of caspase-7 in Escherichia coli cell lysates. Our approach may provide a framework for the activity-based profiling of proteases that share highly similar substrate specificity spectrums.

Cysteine Disulfide Traps Reveal Distinct Conformational Ensembles in Dengue Virus NS2B-NS3 Protease.

The dengue virus protease (NS2B-NS3pro) plays a critical role in the dengue viral life cycle, making it an attractive drug target for dengue-related pathologies, including dengue hemorrhagic fever. A number of studies indicate that NS2B-NS3pro undergoes a transition between two widely different conformational states: an "open" (inactive) conformation and a "closed" (active) conformation. For the past several years, the equilibrium between these states and the resting conformation of NS2B-NS3pro have been debated, although a strong consensus is emerging. To investigate the importance of such conformational states, we developed versions of NS2B-NS3pro that allow us to trap the enzyme in various distinct conformations. Our data from these variants suggest that the enzymatic activity appears to be dependent on the movement of NS2B and may rely on the flexibility of the protease core. Locking the enzyme into the "closed" conformation dramatically increased activity, strongly suggesting that the "closed" conformation is the active conformation. The observed resting state of the enzyme depends largely on the construct used to express the NS2B-NS3pro complex. In an "unlinked" construct, in which the NS2B and NS3 regions exist as independent, co-expressed polypeptides, the enzyme rests predominantly in a "closed", active conformation. In contrast, in a "linked" construct, in which NS2B and NS3 are attached by a nine-amino acid linker, NS2B-NS3pro adopts a more relaxed, alternative conformation. Nevertheless, even the unlinked construct samples both the "closed" and other alternative conformations. Given our findings, and the more realistic resemblance of NS2B-NS3pro to the native enzyme, these data strongly suggest that studies should focus on the "unlinked" constructs moving forward. Additionally, the results from these studies provide a more detailed understanding of the various poses of the dengue virus NS2B-NS3 protease and should help guide future drug discovery efforts aimed at this enzyme.

The Unique Cofactor Region of Zika Virus NS2B-NS3 Protease Facilitates Cleavage of Key Host Proteins.

Zika virus is an emerging mosquito-borne pathogen capable of severely damaging developing fetuses as well as causing neurological abnormalities in adults. The molecular details of how Zika virus causes pathologies that are unique among the flavivirus family remain poorly understood and have contributed to the lack of Zika antiviral therapies. To elucidate how Zika virus protease (ZVP) affects host cellular pathways and consequent pathologies, we used unbiased N-terminomics to identify 31 human proteins cleaved by the NS2B-NS3 protease. In particular, autophagy-related protein 16-1 (ATG16L1) and eukaryotic translation initiation factor 4 gamma 1 (eIF4G1) are dramatically depleted during Zika virus infection. ATG16L1 and eIF4G1 mediate type-II interferon production and host-cell translation, respectively, likely aiding immune system evasion and driving the Zika life cycle. Intriguingly, the NS2B cofactor region from Zika virus protease is essential for recognition of host cell substrates. Replacing the NS2B region in another flavivirus protease enabled recognition of novel Zika-specific substrates by hybrid proteases, suggesting that the cofactor is the principal determinant in ZVP substrate selection.

Tumor-Associated Mutations in Caspase-6 Negatively Impact Catalytic Efficiency.

Unregulated, particularly suppressed programmed cell death is one of the distinguishing features of many cancer cells. The cysteine protease caspase-6, one of the executioners of apoptotic cell death, plays a crucial role in regulation of apoptosis. Several somatic mutations in the CASP6 gene in tumor tissues have been reported. This work explores the effect of CASP6 tumor-associated mutations on the catalytic efficiency and structure of caspase-6. In general, these mutations showed decreased overall rates of catalytic turnover. Mutations within 8 Å of the substrate-binding pocket of caspase-6 were found to be the most catalytically deactivating. Notably, the R259H substitution decreased activity by 457-fold. This substitution disrupts the cation-π stacking interaction between Arg-259 and Trp-227, which is indispensable for proper assembly of the substrate-binding loops in caspase-6. Sequence conservation analysis at the homologous position across the caspase family suggests a role for this cation-π stacking in the catalytic function of caspases generally. These data suggest that caspase-6 deactivating mutations may contribute to multifactorial carcinogenic transformations.

Reprogramming Caspase-7 Specificity by Regio-Specific Mutations and Selection Provides Alternate Solutions for Substrate Recognition.

The ability to routinely engineer protease specificity can allow us to better understand and modulate their biology for expanded therapeutic and industrial applications. Here, we report a new approach based on a caged green fluorescent protein (CA-GFP) reporter that allows for flow-cytometry-based selection in bacteria or other cell types enabling selection of intracellular protease specificity, regardless of the compositional complexity of the protease. Here, we apply this approach to introduce the specificity of caspase-6 into caspase-7, an intracellular cysteine protease important in cellular remodeling and cell death. We found that substitution of substrate-contacting residues from caspase-6 into caspase-7 was ineffective, yielding an inactive enzyme, whereas saturation mutagenesis at these positions and selection by directed evolution produced active caspases. The process produced a number of nonobvious mutations that enabled conversion of the caspase-7 specificity to match caspase-6. The structures of the evolved-specificity caspase-7 (esCasp-7) revealed alternate binding modes for the substrate, including reorganization of an active site loop. Profiling the entire human proteome of esCasp-7 by N-terminomics demonstrated that the global specificity toward natural protein substrates is remarkably similar to that of caspase-6. Because the esCasp-7 maintained the core of caspase-7, we were able to identify a caspase-6 substrate, lamin C, that we predict relies on an exosite for substrate recognition. These reprogrammed proteases may be the first tool built with the express intent of distinguishing exosite dependent or independent substrates. This approach to specificity reprogramming should also be generalizable across a wide range of proteases.