Author Correction: A naturally occurring antiviral ribonucleotide encoded by the human genome.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Publisher Correction: A naturally occurring antiviral ribonucleotide encoded by the human genome.

Change history: In the HTML version of this Letter, Extended Data Fig. 4 incorrectly corresponded to Fig. 4 (the PDF version of the figure was correct). This has been corrected online.

A naturally occurring antiviral ribonucleotide encoded by the human genome.

Viral infections continue to represent major challenges to public health, and an enhanced mechanistic understanding of the processes that contribute to viral life cycles is necessary for the development of new therapeutic strategies 1 . Viperin, a member of the radical S-adenosyl-L-methionine (SAM) superfamily of enzymes, is an interferon-inducible protein implicated in the inhibition of replication of a broad range of RNA and DNA viruses, including dengue virus, West Nile virus, hepatitis C virus, influenza A virus, rabies virus 2 and HIV3,4. Viperin has been suggested to elicit these broad antiviral activities through interactions with a large number of functionally unrelated host and viral proteins3,4. Here we demonstrate that viperin catalyses the conversion of cytidine triphosphate (CTP) to 3'-deoxy-3',4'-didehydro-CTP (ddhCTP), a previously undescribed biologically relevant molecule, via a SAM-dependent radical mechanism. We show that mammalian cells expressing viperin and macrophages stimulated with IFNα produce substantial quantities of ddhCTP. We also establish that ddhCTP acts as a chain terminator for the RNA-dependent RNA polymerases from multiple members of the Flavivirus genus, and show that ddhCTP directly inhibits replication of Zika virus in vivo. These findings suggest a partially unifying mechanism for the broad antiviral effects of viperin that is based on the intrinsic enzymatic properties of the protein and involves the generation of a naturally occurring replication-chain terminator encoded by mammalian genomes.

S100A4 regulates macrophage invasion by distinct myosin-dependent and myosin-independent mechanisms.

S100A4, a member of the S100 family of Ca2+-binding proteins, is a key regulator of cell migration and invasion. Our previous studies showed that bone marrow-derived macrophages from S100A4-/- mice exhibit defects in directional motility and chemotaxis in vitro and reduced recruitment to sites of inflammation in vivo. We now show that the loss of S100A4 produces two mechanistically distinct phenotypes with regard to macrophage invasion: a defect in matrix degradation, due to a disruption of podosome rosettes caused by myosin-IIA overassembly, and a myosin-independent increase in microtubule acetylation, which increases podosome rosette stability and is sufficient to inhibit macrophage invasion. Our studies point to S100A4 as a critical regulator of matrix degradation, whose actions converge on the dynamics and degradative functions of podosome rosettes.

Structure of the S100A4/myosin-IIA complex.

BACKGROUND: S100A4, a member of the S100 family of Ca2+-binding proteins, modulates the motility of both non-transformed and cancer cells by regulating the localization and stability of cellular protrusions. Biochemical studies have demonstrated that S100A4 binds to the C-terminal end of the myosin-IIA heavy chain coiled-coil and disassembles myosin-IIA filaments; however, the mechanism by which S100A4 mediates myosin-IIA depolymerization is not well understood. RESULTS: We determined the X-ray crystal structure of the S100A4Δ8C/MIIA(1908-1923) peptide complex, which showed an asymmetric binding mode for the myosin-IIA peptide across the S100A4 dimer interface. This asymmetric binding mode was confirmed in NMR studies using a spin-labeled myosin-IIA peptide. In addition, our NMR data indicate that S100A4Δ8C binds the MIIA(1908-1923) peptide in an orientation very similar to that observed for wild-type S100A4. Studies of complex formation using a longer, dimeric myosin-IIA construct demonstrated that S100A4 binding dissociates the two myosin-IIA polypeptide chains to form a complex composed of one S100A4 dimer and a single myosin-IIA polypeptide chain. This interaction is mediated, in part, by the instability of the region of the myosin-IIA coiled-coil encompassing the S100A4 binding site. CONCLUSION: The structure of the S100A4/MIIA(1908-1923) peptide complex has revealed the overall architecture of this assembly and the detailed atomic interactions that mediate S100A4 binding to the myosin-IIA heavy chain. These structural studies support the idea that residues 1908-1923 of the myosin-IIA chain heavy represent a core sequence for the S100A4/myosin-IIA complex. In addition, biophysical studies suggest that structural fluctuations within the myosin-IIA coiled-coil may facilitate S100A4 docking onto a single myosin-IIA polypeptide chain.

The heavy chain has its day: regulation of myosin-II assembly.

Nonmuscle myosin-II is an actin-based motor that converts chemical energy into force and movement, and thus functions as a key regulator of the eukaryotic cytoskeleton. Although it is established that phosphorylation on the regulatory light chain increases the actin-activated MgATPase activity of the motor and promotes myosin-II filament assembly, studies have begun to characterize alternative mechanisms that regulate filament assembly and disassembly. These investigations have revealed that all three nonmuscle myosin-II isoforms are subject to additional regulatory controls, which impact diverse cellular processes. In this review, we discuss current knowledge on mechanisms that regulate the oligomerization state of nonmuscle myosin-II filaments by targeting the myosin heavy chain.

Two functional S100A4 monomers are necessary for regulating nonmuscle myosin-IIA and HCT116 cell invasion.

S100A4, a member of the Ca(2+)-activated S100 protein family, regulates the motility and invasiveness of cancer cells. Moreover, high S100A4 expression levels correlate with poor patient survival in several cancers. Although biochemical, biophysical, and structural data indicate that S100A4 is a noncovalent dimer, it is unknown if two functional S100A4 monomers are required for the productive recognition of protein targets and the promotion of cell invasion. To address this question, we created covalently linked S100A4 dimers using a glycine rich flexible linker. The single-chain S100A4 (sc-S100A4) proteins exhibited wild-type affinities for calcium and nonmuscle myosin-IIA, retained the ability to regulate nonmuscle myosin-IIA assembly, and promoted tumor cell invasion when expressed in S100A4-deficient colon carcinoma cells. Mutation of the two calcium-binding EF-hands in one monomer, while leaving the other monomer intact, caused a 30-60-fold reduction in binding affinity for nonmuscle myosin-IIA concomitant with a weakened ability to regulate the monomer-polymer equilibrium of nonmuscle myosin-IIA. Moreover, sc-S100A4 proteins with one monomer deficient in calcium responsiveness did not support S100A4-mediated colon carcinoma cell invasion. Cross-linking and titration data indicate that the S100A4 dimer binds a single myosin-IIA target peptide. These data are consistent with a model in which a single peptide forms interactions in the vicinity of the canonical target binding cleft of each monomer in such a manner that both target binding sites are required for the efficient interaction with myosin-IIA.

Cysteine 81 is critical for the interaction of S100A4 and myosin-IIA.

Overexpression of S100A4, a member of the S100 family of Ca(2+)-binding proteins, is associated with a number of human pathologies, including fibrosis, inflammatory disorders, and metastatic disease. The identification of small molecules that disrupt S100A4/target interactions provides a mechanism for inhibiting S100A4-mediated cellular activities and their associated pathologies. Using an anisotropy assay that monitors the Ca(2+)-dependent binding of myosin-IIA to S100A4, NSC 95397 was identified as an inhibitor that disrupts the S100A4/myosin-IIA interaction and inhibits S100A4-mediated depolymerization of myosin-IIA filaments. Mass spectrometry demonstrated that NSC 95397 forms covalent adducts with Cys81 and Cys86, which are located in the canonical target binding cleft. Mutagenesis studies showed that covalent modification of just Cys81 is sufficient to inhibit S100A4 function with respect to myosin-IIA binding and depolymerization. Remarkably, substitution of Cys81 with serine or alanine significantly impaired the ability of S100A4 to promote myosin-IIA filament disassembly. As reversible covalent cysteine modifications have been observed for several S100 proteins, we propose that modification of Cys81 may provide an additional regulatory mechanism for mediating the binding of S100A4 to myosin-IIA.

S100A4 regulates macrophage chemotaxis.

S100A4, a member of the S100 family of Ca(2+)-binding proteins, is directly involved in tumor metastasis. In addition to its expression in tumor cells, S100A4 is expressed in normal cells and tissues, including fibroblasts and cells of the immune system. To examine the contribution of S100A4 to normal physiology, we established S100A4-deficient mice by gene targeting. Homozygous S100A4(-/-) mice are fertile, grow normally and exhibit no overt abnormalities; however, the loss of S100A4 results in impaired recruitment of macrophages to sites of inflammation in vivo. Consistent with these observations, primary bone marrow macrophages (BMMs) derived from S100A4(-/-) mice display defects in chemotactic motility in vitro. S100A4(-/-) BMMs form unstable protrusions, overassemble myosin-IIA, and exhibit altered colony-stimulating factor-1 receptor signaling. These studies establish S100A4 as a regulator of physiological macrophage motility and demonstrate that S100A4 mediates macrophage recruitment and chemotaxis in vivo.

Phenothiazines inhibit S100A4 function by inducing protein oligomerization.

S100A4, a member of the S100 family of Ca(2+)-binding proteins, regulates carcinoma cell motility via interactions with myosin-IIA. Numerous studies indicate that S100A4 is not simply a marker for metastatic disease, but rather has a direct role in metastatic progression. These observations suggest that S100A4 is an excellent target for therapeutic intervention. Using a unique biosensor-based assay, trifluoperazine (TFP) was identified as an inhibitor that disrupts the S100A4/myosin-IIA interaction. To examine the interaction of S100A4 with TFP, we determined the 2.3 A crystal structure of human Ca(2+)-S100A4 bound to TFP. Two TFP molecules bind within the hydrophobic target binding pocket of Ca(2+)-S100A4 with no significant conformational changes observed in the protein upon complex formation. NMR chemical shift perturbations are consistent with the crystal structure and demonstrate that TFP binds to the target binding cleft of S100A4 in solution. Remarkably, TFP binding results in the assembly of five Ca(2+)-S100A4/TFP dimers into a tightly packed pentameric ring. Within each pentamer most of the contacts between S100A4 dimers occurs through the TFP moieties. The Ca(2+)-S100A4/prochlorperazine (PCP) complex exhibits a similar pentameric assembly. Equilibrium sedimentation and cross-linking studies demonstrate the cooperative formation of a similarly sized S100A4/TFP oligomer in solution. Assays examining the ability of TFP to block S100A4-mediated disassembly of myosin-IIA filaments demonstrate that significant inhibition of S100A4 function occurs only at TFP concentrations that promote S100A4 oligomerization. Together these studies support a unique mode of inhibition in which phenothiazines disrupt the S100A4/myosin-IIA interaction by sequestering S100A4 via small molecule-induced oligomerization.

Multiple regulatory steps control mammalian nonmuscle myosin II assembly in live cells.

To better understand the mechanism controlling nonmuscle myosin II (NM-II) assembly in mammalian cells, mutant NM-IIA constructs were created to allow tests in live cells of two widely studied models for filament assembly control. A GFP-NM-IIA construct lacking the RLC binding domain (DeltaIQ2) destabilizes the 10S sequestered monomer state and results in a severe defect in recycling monomers during spreading, and from the posterior to the leading edge during polarized migration. A GFP-NM-IIA construct lacking the nonhelical tailpiece (Deltatailpiece) is competent for leading edge assembly, but overassembles, suggesting defects in disassembly from lamellae subsequent to initial recruitment. The Deltatailpiece phenotype was recapitulated by a GFP-NM-IIA construct carrying a mutation in a mapped tailpiece phosphorylation site (S1943A), validating the importance of the tailpiece and tailpiece phosphorylation in normal lamellar myosin II assembly control. These results demonstrate that both the 6S/10S conformational change and the tailpiece contribute to the localization and assembly of myosin II in mammalian cells. This work furthermore offers cellular insights that help explain platelet and leukocyte defects associated with R1933-stop alleles of patients afflicted with human MYH9-related disorder.

TRPM7 regulates myosin IIA filament stability and protein localization by heavy chain phosphorylation.

Deregulation of myosin II-based contractility contributes to the pathogenesis of human diseases, such as cancer, which underscores the necessity for tight spatial and temporal control of myosin II activity. Recently, we demonstrated that activation of the mammalian alpha-kinase TRPM7 inhibits myosin II-based contractility in a Ca(2+)- and kinase-dependent manner. However, the molecular mechanism is poorly defined. Here, we demonstrate that TRPM7 phosphorylates the COOH-termini of both mouse and human myosin IIA heavy chains--the COOH-terminus being a region that is critical for filament stability. Phosphorylated residues were mapped to Thr1800, Ser1803 and Ser1808. Mutation of these residues to alanine and that to aspartic acid lead to an increase and a decrease, respectively, in myosin IIA incorporation into the actomyosin cytoskeleton and accordingly affect subcellular localization. In conclusion, our data demonstrate that TRPM7 regulates myosin IIA filament stability and localization by phosphorylating a short stretch of amino acids within the alpha-helical tail of the myosin IIA heavy chain.

Visual snapshots of intracellular kinase activity at the onset of mitosis.

Visual snapshots of intracellular kinase activity can be acquired with exquisite temporal control by using a light-activatable (caged) sensor, thereby providing a means to interrogate enzymatic activity at any point during the cell-division cycle. Robust protein kinase activity transpires just prior to, but not immediately after, nuclear envelope breakdown (NEB). Furthermore, kinase activity is required for the progression from prophase into metaphase. Finally, the application of selective protein kinase C (PKC) inhibitors, in combination with the caged sensor, correlates the action of the PKC beta isoform with subsequent NEB.

Myosin-IIA heavy-chain phosphorylation regulates the motility of MDA-MB-231 carcinoma cells.

In mammalian nonmuscle cells, the mechanisms controlling the localized formation of myosin-II filaments are not well defined. To investigate the mechanisms mediating filament assembly and disassembly during generalized motility and chemotaxis, we examined the EGF-dependent phosphorylation of the myosin-IIA heavy chain in human breast cancer cells. EGF stimulation of MDA-MB-231 cells resulted in transient increases in both the assembly and phosphorylation of the myosin-IIA heavy chains. In EGF-stimulated cells, the myosin-IIA heavy chain is phosphorylated on the casein kinase 2 site (S1943). Cells expressing green fluorescent protein-myosin-IIA heavy-chain S1943E and S1943D mutants displayed increased migration into a wound and enhanced EGF-stimulated lamellipod extension compared with cells expressing wild-type myosin-IIA. In contrast, cells expressing the S1943A mutant exhibited reduced migration and lamellipod extension. These observations support a direct role for myosin-IIA heavy-chain phosphorylation in mediating motility and chemotaxis.

Regulation of myosin-IIA assembly and Mts1 binding by heavy chain phosphorylation.

Previous studies suggested that heavy chain phosphorylation regulates non-muscle myosin-II assembly in an isoform-specific manner, affecting the assembly of myosin-IIB, but not myosin-IIA. We re-examined the effects of heavy chain phosphorylation on myosin-IIA filament formation and also examined mts1 binding. We demonstrated that heavy chain phosphorylation by either protein kinase C (PKC) or casein kinase 2 (CK2) inhibits the assembly of myosin-IIA into filaments. PKC phosphorylation had no affect on mts1 binding, but CK2 phosphorylation decreased the affinity of mts1 for the myosin-IIA rod by approximately 6.5-fold. Mts1 destabilized PKC-phosphorylated myosin-IIA filaments and inhibited the assembly of myosin-IIA monomers with maximal inhibition of assembly and promotion of disassembly occurring at a molar ratio of one mts1 dimer per myosin-IIA rod. At this molar ratio, mts1 only weakly disassembled CK2-phosphorylated myosin-IIA filaments and weakly inhibited the assembly of CK2-phosphorylated myosin-IIA monomers. These observations demonstrate that CK2 phosphorylation of the myosin-IIA heavy chain protects against mts1-induced filament disassembly and inhibition of assembly, and suggest that heavy chain phosphorylation provides an additional level of regulation for the mts1-myosin-IIA interaction.

The long myosin light chain kinase is differentially phosphorylated during interphase and mitosis.

We have shown previously that the activity of the long myosin light chain kinase (MLCK) is cell cycle regulated with a decrease in specific activity during mitosis that can be restored following treatment with alkaline phosphatase. To better understand the role and significance of phosphorylation in regulating MLCK function during mitosis, we examined the phosphorylation state of in vivo derived MLCK. Phosphoamino acid analysis and phosphopeptide mapping demonstrate that the long MLCK is differentially phosphorylated on serine residues during interphase and mitosis with the majority of the phosphorylation sites located within the N-terminal IgG domain. Biochemical assays show that Aurora B binds and phosphorylates the IgG domain of the long MLCK. In addition, phosphopeptide maps of the endogenous full-length MLCK from mitotic cells and in vitro phosphorylated IgG domain demonstrate that Aurora B phosphorylates the same sites as those observed in vivo. Altogether, these studies suggest that the long MLCK may be a cellular target for Aurora B during mitosis.

Cell type-specific regulation of RhoA activity during cytokinesis.

Rho family GTPases play pivotal roles in cytokinesis. By using probes based on the principle of fluorescence resonance energy transfer (FRET), we have shown that in HeLa cells RhoA activity increases with the progression of cytokinesis. Here we show that in Rat1A cells RhoA activity remained suppressed during most of the cytokinesis. Consistent with this observation, the expression of C3 toxin inhibited cytokinesis in HeLa cells but not in Rat1A cells. Furthermore, the expression of a dominant negative mutant of Ect2, a Rho GEF, or Y-27632, an inhibitor of the Rho-dependent kinase ROCK, inhibited cytokinesis in HeLa cells but not in Rat1A cells. In contrast to the activity of RhoA, the activity of Rac1 was suppressed during cytokinesis and started increasing at the plasma membrane of polar sides before the abscission of the daughter cells in both HeLa and Rat1A cells. This type of Rac1 suppression was shown to be essential for cytokinesis because a constitutively active mutant of Rac1 induced a multinucleated phenotype in both HeLa and Rat1A cells. Moreover, the involvement of MgcRacGAP/CYK-4 in this suppression of Rac1 during cytokinesis was shown by the use of a dominant negative mutant. Because ML-7, an inhibitor of myosin light chain kinase, delayed the cytokinesis of Rat1A cells and because Pak, a Rac1 effector, is known to suppress myosin light chain kinase, the suppression of the Rac1-Pak pathway by MgcRacGAP may play a pivotal role in the cytokinesis of Rat1A cells.

The N-terminus of the long MLCK induces a disruption in normal spindle morphology and metaphase arrest.

We have shown previously that only the long myosin light chain kinase (MLCK), which is the predominant MLCK isoform expressed in nonmuscle cells, localizes to the cleavage furrow. To further examine the in vivo localization of the long MLCK in HeLa cells and the mechanisms responsible for kinase targeting during the cell cycle, we examined the distribution of the endogenous kinase and constructed green fluorescent protein (GFP) fusions of long HeLa MLCK truncations. A GFP fusion containing the N-terminal IgG domain and the five DXR motifs localized to stress fibers during interphase and the cleavage furrow during mitosis. Although individual fusions of the five DXRs and IgG domain both independently localized to stress fibers, only the five DXRs demonstrated a cortical localization in mitotic cells. Thus, robust targeting of the long MLCK to the cleavage furrow required the five DXRs and additional sequences from the IgG domain. Expression of the IgG domain alone or with five DXRs increased the number of multinucleate cells tenfold, whereas expression of the five DXRs or GFP had no effect. Furthermore, expression of the IgG domain alone or with five DXRs disrupted normal spindle morphology during mitosis. Extended astral microtubules and increased bundling of kinetochore microtubules, and spindle pole fragmentation were detected in mitotic cells. These microtubule defects were associated with abnormalities in metaphase chromosome alignment and a subsequent metaphase arrest caused by activation of the spindle assembly checkpoint at the kinetochores of mono-oriented chromosomes. Together, these results suggest that MLCK has an unexpected regulatory function during mitosis.