DUAL FUNCTION OF ZIKA VIRUS NS2B-NS3 PROTEASE

Zika virus (ZIKV) serine protease, indispensable for viral polyprotein processing and replication, is composed of an NS2B polypeptide that associates with a proteolytic N terminal fragment of NS3 polypeptide (NS3pro) to form NS2B-NS3pro. The larger C-terminal fragment of NS3 polypeptide contains helicase activity. In the present study, we discovered that ZIKV NS2BNS3pro efficiently binds single-stranded (ss) RNA (Kd ~0.3 uM), suggesting that the protease may have a novel function. We tested an array of NS2B-NS3pro modifications and found that NS2B NS3pro constructs that adopt the recently discovered super-open conformation could not bind ssRNA. Likewise, stabilization of NS2B-NS3pro in the closed (proteolytically active) conformation by substrate-like inhibitors abolished ssRNA binding. Therefore, we suggest that ssRNA binding occurs when ZIKV protease adopts the open conformation, which could be modeled using dengue NS2B-NS3pro in the open conformation. ssRNA binding competes with ZIKV NS2B-NS3pro protease activity, likely by shifting the complex into the open conformation. Modeling of ZIKV NS3 helicase activity based on homologous crystal structures suggests that the open conformation of NS3pro domains provides a positively charged surface contiguous with the NS3 helicase domain. Such a positively charged surface is well poised to bind ssRNA, providing an explanation for the previously observed requirement of NS3pro for RNA processivity by viral helicase. Our structure-function analyses suggest that binding of ssRNA by the protease domain of NS3 is likely to be a universal feature of Flaviviridae, given the high level of homology between NS3 protease-helicase proteins in this family.

Targeting DDX3X Helicase Activity with BA103 Shows Promising Therapeutic Effects in Preclinical Glioblastoma Models

DDX3X is an ATP-dependent RNA helicase that has recently attracted interest for its involvement in viral replication and oncogenic progression. Starting from hit compounds previously identified by our group, we have designed and synthesized a new series of DDX3X inhibitors that effectively blocked its helicase activity. These new compounds were able to inhibit the proliferation of cell lines from different cancer types, also in DDX3X low-expressing cancer cell lines. According to the absorption, distribution, metabolism, elimination properties, and antitumoral activity, compound BA103 was chosen to be further investigated in glioblastoma models. BA103 determined a significant reduction in the proliferation and migration of U87 and U251 cells, downregulating the oncogenic protein β-catenin. An in vivo evaluation demonstrated that BA103 was able to reach the brain and reduce the tumor growth in xenograft and orthotopic models without evident side effects. This study represents the first demonstration that DDX3X-targeted small molecules are feasible and promising drugs also in glioblastoma.

Variable inhibition of unwinding rates of DNA catalyzed by the SARS-Cov-2 (COV19) helicase nsp13 by structurally distinct single DNA lesions.

The SARS 2 (Covid 19) helicase nsp13 plays a critically important role in the replication of the Corona virus by unwinding double-stranded RNA (and DNA) with a 5 prime to 3 prime strand polarity. Here we explored the impact of single, structurally defined covalent DNA lesions on the helicase activity of nsp13 in aqueous solutions, The objectives were to derive mechanistic insights into the relationships between the structures of DNA lesions, the DNA distortions that they engender, and the inhibition of helicase activity. The lesions included two bulky stereoisomeric N2-guanine adducts derived from the reactions of benzo[a]pyrene diol epoxide with DNA. The trans-adduct assumes a minor groove conformation, while the cis-product adopts a base-displaced intercalated conformation. The non-bulky DNA lesions included the intra-strand cross-linked thymine dimers, the cis-syn-cyclobutane pyrimidine dimer, and the pyrimidine (6–4) pyrimidone photoproduct. All four lesions strongly inhibit the helicase activity of nsp13, The UV photolesions feature a 2 - 5-fold smaller inhibition of the nsp13 unwinding activity than the bulky DNA adducts, and the kinetics of these two pairs of DNA lesions are also different. The connections between the structural features of these four DNA lesions and their impact on nsp13 unwinding efficiencies are discussed.

RNA helicase DDX6 in P-bodies is essential for the assembly of stress granules

Two prominent cytoplasmic RNA granules, ubiquitous RNA-processing bodies (PB) and inducible stress granules (SG), regulate storage of translationally arrested mRNAs and are intimately related. In this study, we found the dependence of SG formation on PB in the cells under arsenite (ARS) stress, but not the other way around. GW182, 4E-T and DDX6 essential for PB formation differentially affect SG formation in the cells under ARS stress, with DDX6 being the most prominent. The cells with DDX6 deficiency display irregular shape of SG which could be rescued by ectopic wt DDX6, but not its helicase mutant E247A DDX6, which induces SG in the cells without stress, indicating that DDX6 helicase activity is essential for PB, but suppressive for SG. DDX6's dual roles are independent of DDX6 interactors EDC3, CNOT1, and PAT1B. This study provides a conceptual advance of how DDX6 involves in the biogenesis of PB and SG.

Large-scale ratcheting in a bacterial DEAH/RHA-type RNA helicase that modulates antibiotics susceptibility

Many bacteria harbor RNA-dependent nucleoside-triphosphatases of the DEAH/RHA family, whose molecular mechanisms and cellular functions are poorly understood. Here, we show that the Escherichia coli DEAH/RHA protein, HrpA, is an ATP-dependent 3 to 5′ RNA helicase and that the RNA helicase activity of HrpA influences bacterial survival under antibiotics treatment. Limited proteolysis, crystal structure analysis, and functional assays showed that HrpA contains an N-terminal DEAH/RHA helicase cassette preceded by a unique N-terminal domain and followed by a large C-terminal region that modulates the helicase activity. Structures of an expanded HrpA helicase cassette in the apo and RNA-bound states in combination with cross-linking/mass spectrometry revealed ratchet-like domain movements upon RNA engagement, much more pronounced than hitherto observed in related eukaryotic DEAH/RHA enzymes. Structure-based functional analyses delineated transient interdomain contact sites that support substrate loading and unwinding, suggesting that similar conformational changes support RNA translocation. Consistently, modeling studies showed that analogous dynamic intramolecular contacts are not possible in the related but helicase-inactive RNA-dependent nucleoside-triphosphatase, HrpB. Our results indicate that HrpA may be an interesting target to interfere with bacterial tolerance toward certain antibiotics and suggest possible interfering strategies.

The intra-S phase checkpoint directly regulates replication elongation to preserve the integrity of stalled replisomes

DNA replication is dramatically slowed down under replication stress. The regulation of replication speed is a conserved response in eukaryotes and, in fission yeast, requires the checkpoint kinases Rad3ATR and Cds1Chk2. However, the underlying mechanism of this checkpoint regulation remains unresolved. Here, we report that the Rad3ATR-Cds1Chk2 checkpoint directly targets the Cdc45-MCM-GINS (CMG) replicative helicase under replication stress. When replication forks stall, the Cds1Chk2 kinase directly phosphorylates Cdc45 on the S275, S322, and S397 residues, which significantly reduces CMG helicase activity. Furthermore, in cds1Chk2-mutated cells, the CMG helicase and DNA polymerases are physically separated, potentially disrupting replisomes and collapsing replication forks. This study demonstrates that the intra-S phase checkpoint directly regulates replication elongation, reduces CMG helicase processivity, prevents CMG helicase delinking from DNA polymerases, and therefore helps preserve the integrity of stalled replisomes and replication forks.