LIM domain binding (Ldb) proteins are important regulators of LIM homeodomain and LIM-only proteins that specify cell fate in many different tissues. An essential feature of these proteins is the ability to self-associate, but there have been no studies that characterise the nature of this self-association. We have used deletion mutagenesis with yeast two-hybrid analysis to define the minimal self-association domains of Ldb1 and Ldb2 as residues 14-200 and 21-197, respectively. We then used a range of different biophysical methods, including sedimentation equilibrium and small-angle X-ray scattering to show that Ldb1(14-200) forms a trimer and Ldb2(21-197) undergoes a monomer-tetramer-octamer equilibrium, where the association in each case is of moderate affinity ( approximately 10(5) M(-1)). These modes of association represent a clear physical difference between these two proteins that otherwise appear to have very similar properties. The levels of association are more complex than previously assumed and emphasise roles of avidity and DNA looping in transcriptional regulation by Ldb1/LIM protein complexes. The abilities of Ldb1 and Ldb2 to form trimers and higher oligomers, respectively, should be considered in models of transcriptional regulation by Ldb1-containing complexes in a wide range of biological processes.

The LIM domain-containing proteins Pinch1/2 regulate integrin activation and cell-extracellular matrix interaction and adhesion. Here, we report that deleting Pinch1 in limb mesenchymal stem cells (MSCs) and Pinch2 globally (double knockout; dKO) in mice causes severe chondrodysplasia, while single mutant mice do not display marked defects. Pinch deletion decreases chondrocyte proliferation, accelerates cell differentiation and disrupts column formation. Pinch loss drastically reduces Smad2/3 protein expression in proliferative zone (PZ) chondrocytes and increases Runx2 and Col10a1 expression in both PZ and hypertrophic zone (HZ) chondrocytes. Pinch loss increases sclerostin and Rankl expression in HZ chondrocytes, reduces bone formation, and increases bone resorption, leading to low bone mass. In vitro studies revealed that Pinch1 and Smad2/3 colocalize in the nuclei of chondrocytes. Through its C-terminal region, Pinch1 interacts with Smad2/3 proteins. Pinch loss increases Smad2/3 ubiquitination and degradation in primary bone marrow stromal cells (BMSCs). Pinch loss reduces TGF-β-induced Smad2/3 phosphorylation and nuclear localization in primary BMSCs. Interestingly, compared to those from single mutant mice, BMSCs from dKO mice express dramatically lower protein levels of β-catenin and Yap1/Taz and display reduced osteogenic but increased adipogenic differentiation capacity. Finally, ablating Pinch1 in chondrocytes and Pinch2 globally causes severe osteopenia with subtle limb shortening. Collectively, our findings demonstrate critical roles for Pinch1/2 and a functional redundancy of both factors in the control of chondrogenesis and bone mass through distinct mechanisms.

Drosophila melanogaster has been used as a model organism for study on development and pathophysiology of the heart. LIM domain proteins act as adaptors or scaffolds to promote the assembly of multimeric protein complexes. We found a total of 75 proteins encoded by 36 genes have LIM domain in Drosophila melanogaster by the tools of SMART, FLY-FISH, and FlyExpress, and around 41.7% proteins with LIM domain locate in lymph glands, muscles system, and circulatory system. Furthermore, we summarized functions of different LIM domain proteins in the development and physiology of fly heart and hematopoietic systems. It would be attractive to determine whether it exists a probable "LIM code" for the cycle of different cell fates in cardiac and hematopoietic tissues. Next, we aspired to propose a new research direction that the LIM domain proteins may play an important role in fly cardiac and hematopoietic morphogenesis.

POT1 is the single stranded telomeric overhang binding protein, and is part of the shelterin complex, a group of six proteins essential for proper telomere function. The reduction or abrogation of POT1 DNA binding activity in mammalian cells results in telomere elongation, or activation of the ATR DNA damage response at telomeres. Therefore, overhang binding represents the functionally relevant activity of POT1. To better understand the roles of POT1, we sought to isolate proteins that interact with the DNA binding domain of the protein. A yeast two-hybrid screen was implemented using a C-terminal truncation termed POT1DeltaC, retaining the DNA binding domain. This screen yielded a partial cDNA corresponding to TRIP6, a member of the LIM domain protein family. TRIP6 could co-immunoprecipitate with POT1, TRF2 and TIN2 in human cells, arguing for association with the whole shelterin complex, and was detected at telomeres by ChIP. TRIP6 depletion by siRNA led to the induction of telomere dysfunction induced foci (TIFs), indicating a role in telomere protection. A closely related LIM domain protein, LPP, was also found at telomeres and was also important for repressing the DNA damage response. We propose that TRIP6 and LPP are both required for telomere protection.

A full-length cDNA of the filarial nematode Acanthocheilonema viteae was isolated from a cDNA library of female worms, using a partial cDNA of the OvL3-1 gene of Onchocerca volvulus as a probe. The AvL3-1 cDNA contained an open reading frame which encoded for a protein with a theoretical molecular weight of 64 kDa. The deduced protein contained a predicted signal sequence, a short repetitive motive of unknown function, and three LIM domains. The structure of the LIM domains was identical to those of zyxin, a cytoskeleton-associated protein of chicken fibroblasts, suggesting that AvL3-1 has a similar role in filarial nematodes. The sequence information was used to isolate the homologous cDNA of O. volvulus by PCR from a cDNA library of female O. volvulus, which showed an overall identity of 76.9% to AvL3-1 on the protein level. AvL3-1 was expressed in Escherichia coli and the affinity-purified fusion free protein was used to immunized jirds (Meriones unguiculatus). Immunization together with the adjuvant STP or with Freund's adjuvant induced IgG and IgM antibody responses, but no significant protection against a challenge infection with L3 of A. viteae, compared to appropriate control groups.

As core components of the microRNA-induced silencing complex (miRISC), Argonaute (AGO) proteins interact with TNRC6 proteins, recruiting other effectors of translational repression/mRNA destabilization. Here, we show that LIMD1 coordinates the assembly of an AGO-TNRC6 containing miRISC complex by binding both proteins simultaneously at distinct interfaces. Phosphorylation of AGO2 at Ser 387 by Akt3 induces LIMD1 binding, which in turn enables AGO2 to interact with TNRC6A and downstream effector DDX6. Conservation of this serine in AGO1 and 4 indicates this mechanism may be a fundamental requirement for AGO function and miRISC assembly. Upon CRISPR-Cas9-mediated knockout of LIMD1, AGO2 miRNA-silencing function is lost and miRNA silencing becomes dependent on a complex formed by AGO3 and the LIMD1 family member WTIP. The switch to AGO3 utilization occurs due to the presence of a glutamic acid residue (E390) on the interaction interface, which allows AGO3 to bind to LIMD1, AJUBA, and WTIP irrespective of Akt signaling.

Tumour radiotherapy resistance involves the cell cycle pathway. CDC25 phosphatases are key cell cycle regulators. However, how CDC25 activity is precisely controlled remains largely unknown. Here, we show that LIM domain-containing proteins, such as FHL1, increase inhibitory CDC25 phosphorylation by forming a complex with CHK2 and CDC25, and sequester CDC25 in the cytoplasm by forming another complex with 14-3-3 and CDC25, resulting in increased radioresistance in cancer cells. FHL1 expression, induced by ionizing irradiation in a SP1- and MLL1-dependent manner, positively correlates with radioresistance in cancer patients. We identify a cell-penetrating 11 amino-acid motif within LIM domains (eLIM) that is sufficient for binding CHK2 and CDC25, reducing the CHK2-CDC25 and CDC25-14-3-3 interaction and enhancing CDC25 activity and cancer radiosensitivity accompanied by mitotic catastrophe and apoptosis. Our results provide novel insight into molecular mechanisms underlying CDC25 activity regulation. LIM protein inhibition or use of eLIM may be new strategies for improving tumour radiosensitivity.

Carcinoma cells can lose their epithelial cell characteristics and dedifferentiate into a fibroblast-like cell during progression of a neoplasm. Aberrant expression of oligomeric transcriptional complexes contributes to progression of carcinomas. Although individual transcription factors initiating progression remain unknown, LIM-only protein (LMO) and LIM-domain binding protein (LDB) negatively regulate breast carcinoma cell differentiation. In this study, we investigated the expression of LMO4 and LDB in squamous cell carcinomas of the oral cavity. LMO4 mRNA was amplified in four of six carcinoma tissues and eight of 12 carcinoma cell lines, and LDB1 in three carcinoma tissues and 11 cell lines examined. Immunoprecipitation studies revealed that LMO4 and LDB1 interact with each other in the nuclear milieu of the carcinoma cells indicating the presence of an LMO4-LDB1-mediated transcription complex. Both LMO4 and LDB1 proteins were preferentially localised in the nuclei of carcinoma cells at the invasive front and the immunoreactivity was increased in less-differentiated carcinoma tissues (P<0.01). Carcinoma cells metastasised to the cervical lymph nodes with increased immunoreactivity compared to the primary site of neoplasm (P<0.05). These data suggest that the LMO4-LDB1 complexes may be involved in carcinoma progression possibly through dedifferentiation of squamous carcinoma cells of the oral cavity.

The crucial involvement of CLIM/NLI/Ldb cofactors for the exertion of the biological activity of LIM homeodomain transcription factors (LIM-HD) has been demonstrated. In this paper we show that CLIM cofactors are widely expressed during zebrafish development with high protein levels in specific neuronal cell types where LIM-HD proteins of the Isl class are synthesized. The overexpression of a dominant-negative CLIM molecule (DN-CLIM) that contains the LIM interaction domain (LID) during early developmental stages of zebrafish embryos results in an impairment of eye and midbrain-hindbrain boundary (MHB) development and disturbances in the formation of the anterior midline. On a cellular level we show that the outgrowth of peripheral but not central axons from Rohon Beard (RB) and trigeminal sensory neurons is inhibited by DN-CLIM overexpression. We demonstrate a further critical role of CLIM cofactors for axonal outgrowth of motor neurons. Additionally, DN-CLIM overexpression causes an increase of Isl-protein expression levels in specific neuronal cell types, likely due to a protection of the DN-CLIM/LIM-HD complex from proteasomal degradation. Our results demonstrate multiple roles of the CLIM cofactor family for the development of entire organs, axonal outgrowth of specific neurons and protein expression levels.

Members of the cysteine-rich protein (CRP) family are LIM domain proteins that have been implicated in muscle differentiation. One strategy for defining the mechanism by which CRPs potentiate myogenesis is to characterize the repertoire of CRP binding partners. In order to identify proteins that interact with CRP1, a prominent protein in fibroblasts and smooth muscle cells, we subjected an avian smooth muscle extract to affinity chromatography on a CRP1 column. A 100-kD protein bound to the CRP1 column and could be eluted with a high salt buffer; Western immunoblot analysis confirmed that the 100-kD protein is alpha-actinin. We have shown that the CRP1-alpha-actinin interaction is direct, specific, and saturable in both solution and solid-phase binding assays. The Kd for the CRP1-alpha-actinin interaction is 1.8 +/- 0.3 microM. The results of the in vitro protein binding studies are supported by double-label indirect immunofluorescence experiments that demonstrate a colocalization of CRP1 and alpha-actinin along the actin stress fibers of CEF and smooth muscle cells. Moreover, we have shown that alpha-actinin coimmunoprecipitates with CRP1 from a detergent extract of smooth muscle cells. By in vitro domain mapping studies, we have determined that CRP1 associates with the 27-kD actin-binding domain of alpha-actinin. In reciprocal mapping studies, we showed that alpha-actinin interacts with CRP1-LIM1, a deletion fragment that contains the NH2-terminal 107 amino acids (aa) of CRP1. To determine whether the alpha-actinin binding domain of CRP1 would localize to the actin cytoskeleton in living cells, expression constructs encoding epitope-tagged full-length CRP1, CRP1-LIM1(aa 1-107), or CRP1-LIM2 (aa 108-192) were microinjected into cells. By indirect immunofluorescence, we have determined that full-length CRP1 and CRP1-LIM1 localize along the actin stress fibers whereas CRP1-LIM2 fails to associate with the cytoskeleton. Collectively these data demonstrate that the NH2-terminal part of CRP1 that contains the alpha-actinin-binding site is sufficient to localize CRP1 to the actin cytoskeleton. The association of CRP1 with alpha-actinin may be critical for its role in muscle differentiation.

RLIM acts as a negative regulator of LIM-Homeodomain proteins either by recruiting Sin3A/Histone Deacetylase (HDAC) co-repressor complex or through degradation of CLIM coactivator, thus playing an important role in embryonic development. Recent studies by different research groups have shown that RLIM acts as an X-encoded, dose-dependent inducer of X chromosome inactivation in mouse embryonic stem cells. However, until now, very little is known about the expression regulation of RLIM gene, and we tried to study the transcriptional regulation of RLIM gene. In the present study, we identified RLIM as a novel target of p53 and demonstrated that p53 repressed both mRNA and protein levels of RLIM. Expression of wild type p53, but not p53 mutants, led to repression of the RLIM promoter activity. We further identified four putative Sp1 elements (S1 to S4) on the RLIM promoter that are essential for p53-mediated repression of RLIM. Although p53 does not directly bind to the RLIM promoter, it physically interacts with and prevents the binding of Sp1 to the RLIM promoter. Thus, RLIM is a novel target of p53, and p53 exerts its inhibitory effect on RLIM expression by interfering with Sp1-mediated transcriptional activation on RLIM. Our results provided data to enlarge the knowledge of transcriptional regulation of RLIM and suggested a new pathway by which physiological and pathological activators of p53 may affect development.

Several LIM domain proteins regulate transcription. They are thought to act through their LIM protein-protein interaction domains as adaptors for the recruitment of transcriptional co-regulators. An intriguing example is nTRIP6, the nuclear isoform of the focal adhesion protein TRIP6. nTRIP6 interacts with AP-1 and enhances its transcriptional activity. nTRIP6 is also essential for the transrepression of AP-1 by the glucocorticoid receptor (GR), by mediating GR tethering to promoter-bound AP-1. Here we report on the molecular mechanism by which nTRIP6 exerts these effects. Both the LIM domains and the pre-LIM region of nTRIP6 are necessary for its co-activator function for AP-1. Discrete domains within the pre-LIM region mediate the dimerization of nTRIP6 at the promoter, which enables the recruitment of the Mediator complex subunits THRAP3 and Med1. This recruitment is blocked by GR, through a competition between GR and THRAP3 for the interaction with the LIM domains of nTRIP6. Thus, nTRIP6 both positively and negatively regulates transcription by orchestrating the recruitment of the Mediator complex to AP-1-regulated promoters.

Cholestasis occurs in different clinical circumstances and leads to severe hepatic disorders. The four-and-a-half LIM-domain protein 2 (FHL2) is a scaffolding protein that modulates multiple signal transduction pathways in a tissue- and cell context-specific manner. In this study, we aimed to gain insight into the function of FHL2 in cholestatic liver injury. FHL2 expression was significantly increased in the bile duct ligation (BDL) model in mice. In Fhl2-deficient (Fhl2-ko) mice, BDL caused a more severe portal and parenchymal inflammation, extended portal fibrosis, higher serum transaminase levels, and higher pro-inflammatory and pro-fibrogenic gene expression compared to wild type (wt) mice. FHL2 depletion in HepG2 cells with siRNA resulted in a higher expression of the bile acid transporter Na+-taurocholate cotransporting polypeptide (NTCP) gene. Furthermore, FHL2-depleted HepG2 cells showed higher expression of markers for oxidative stress, lower B-cell lymphoma 2 (Bcl2) expression, and higher Bcl2-associated X protein (BAX) expression after stimulation with deoxycholic acid (DCA). In hepatic stellate cells (HSCs), FHL2 depletion caused an increased expression of TGF-β and several pro-fibrogenic matrix metalloproteinases. In summary, our study shows that deficiency in FHL2 aggravates cholestatic liver injury and suggests FHL2-mediated effects on bile acid metabolisms and HSCs as potential mechanisms for pronounced hepatocellular injury and fibrosis.

The two LIM domain-containing proteins from plants (LIMs) typically exhibit a dual cytoplasmic-nuclear distribution, suggesting that, in addition to their previously described roles in actin cytoskeleton organization, they participate in nuclear processes. Using a south-western blot-based screen aimed at identifying factors that bind to plant histone gene promoters, we isolated a positive clone containing the tobacco LIM protein WLIM2 (NtWLIM2) cDNA. Using both green fluorescent protein (GFP) fusion- and immunology-based strategies, we provide clear evidence that NtWLIM2 localizes to the actin cytoskeleton, the nucleus, and the nucleolus. Interestingly, the disruption of the actin cytoskeleton by latrunculin B significantly increases NtWLIM2 nuclear fraction, pinpointing a possible novel cytoskeletal-nuclear crosstalk. Biochemical and electron microscopy experiments reveal the ability of NtWLIM2 to directly bind to actin filaments and to crosslink the latter into thick actin bundles. Electrophoretic mobility shift assays show that NtWLIM2 specifically binds to the conserved octameric cis-elements (Oct) of the Arabidopsis histone H4A748 gene promoter and that this binding largely relies on both LIM domains. Importantly, reporter-based experiments conducted in Arabidopsis and tobacco protoplasts confirm the ability of NtWLIM2 to bind to and activate the H4A748 gene promoter in live cells. Expression studies indicate the constitutive presence of NtWLIM2 mRNA and NtWLIM2 protein during tobacco BY-2 cell proliferation and cell cycle progression, suggesting a role of NtWLIM2 in the activation of basal histone gene expression. Interestingly, both live cell and in vitro data support NtWLIM2 di/oligomerization. We propose that NtWLIM2 functions as an actin-stabilizing protein, which, upon cytoskeleton remodeling, shuttles to the nucleus in order to modify gene expression.

Cardiomyopathies are a severe and chronic cardiovascular burden worldwide, affecting a large cohort in the general population. Cysteine and glycine-rich protein 3 (CSRP3) is one of key proteins implicated in dominant dilated cardiomyopathy (DCM) and hypertrophic cardiomyopathy (HCM). In this study, we device a rapid in silico screening protocol that creates a mutational landscape map for all possible allowed and disallowed substitutions in the protein of interest. This map provides the structural and functional insights on the stability of LIM domains of CSRP3. Further, the sequence analysis delineates the eukaryotic CSRP3 protein orthologs which complements the mutational map, but provide limited information of amino acid exchanges. Next, we also evaluated the effect of HCM/DCM mutations on these domains. One of highly destabilising mutations-L44P (also disease causing) and a neutral mutation-L44M were further subjected to molecular dynamics (MD) simulations. The results establish that L44P substitution affects the LIM domain structure by altering secondary structure and due to loss of hydrophobic interaction with Phenylananine 35. The present study provides a useful perspective to our understanding of the role of mutations in the CSRP3 LIM domains and their evolution. This study provides a novel computational screening method for quick identification of key mutation sites for specific protein structures that can reduce the burden on experimental research.

Genomic defects with large effect size can help elucidate unknown pathologic architecture of mental disorders. We previously reported on a patient with schizophrenia and a balanced translocation between chromosomes 4 and 13 and found that the breakpoint within chromosome 4 is located near the LDB2 gene. We show here that Ldb2 knockout (KO) mice displayed multiple deficits relevant to mental disorders. In particular, Ldb2 KO mice exhibited deficits in the fear-conditioning paradigm. Analysis of the amygdala suggested that dysregulation of synaptic activities controlled by the immediate early gene Arc is involved in the phenotypes. We show that LDB2 forms protein complexes with known transcription factors. Consistently, ChIP-seq analyses indicated that LDB2 binds to > 10,000 genomic sites in human neurospheres. We found that many of those sites, including the promoter region of ARC, are occupied by EGR transcription factors. Our previous study showed an association of the EGR family genes with schizophrenia. Collectively, the findings suggest that dysregulation in the gene expression controlled by the LDB2-EGR axis underlies a pathogenesis of subset of mental disorders.

Predictive approaches such as virtual screening have been used in drug discovery with the objective of reducing developmental time and costs. Current machine learning and network-based approaches have issues related to generalization, usability, or model interpretability, especially due to the complexity of target proteins' structure/function, and bias in system training datasets. Here, we propose a new method "DRUIDom" (DRUg Interacting Domain prediction) to identify bio-interactions between drug candidate compounds and targets by utilizing the domain modularity of proteins, to overcome problems associated with current approaches. DRUIDom is composed of two methodological steps. First, ligands/compounds are statistically mapped to structural domains of their target proteins, with the aim of identifying their interactions. As such, other proteins containing the same mapped domain or domain pair become new candidate targets for the corresponding compounds. Next, a million-scale dataset of small molecule compounds, including those mapped to domains in the previous step, are clustered based on their molecular similarities, and their domain associations are propagated to other compounds within the same clusters. Experimentally verified bioactivity data points, obtained from public databases, are meticulously filtered to construct datasets of active/interacting and inactive/non-interacting drug/compound-target pairs (~2.9M data points), and used as training data for calculating parameters of compound-domain mappings, which led to 27,032 high-confidence associations between 250 domains and 8,165 compounds, and a finalized output of ~5 million new compound-protein interactions. DRUIDom is experimentally validated by syntheses and bioactivity analyses of compounds predicted to target LIM-kinase proteins, which play critical roles in the regulation of cell motility, cell cycle progression, and differentiation through actin filament dynamics. We showed that LIMK-inhibitor-2 and its derivatives significantly block the cancer cell migration through inhibition of LIMK phosphorylation and the downstream protein cofilin. One of the derivative compounds (LIMKi-2d) was identified as a promising candidate due to its action on resistant Mahlavu liver cancer cells. The results demonstrated that DRUIDom can be exploited to identify drug candidate compounds for intended targets and to predict new target proteins based on the defined compound-domain relationships. Datasets, results, and the source code of DRUIDom are fully-available at: https://github.com/cansyl/DRUIDom.

PINCH is a five LIM domain protein involved in the regulation of integrin-mediated cell adhesion. It has been shown that PINCH interacts with integrin-linked kinase and Nck2. Here we describe a new isoform of PINCH, which we call PINCH2. Therefore, we rename PINCH to PINCH1. PINCH2 has an overall similarity of 92% to PINCH1 and contains five LIM domains like PINCH1. While protein and gene structure of the PINCH homologues are very similar and well conserved during evolution, we observed differential expression pattern of the mRNAs. Based on northern hybridization of mouse embryo RNA, PINCH1 is already detectable at E8.5. It is highly expressed during later stages of development and in all adult mouse tissues analyzed, with the highest levels in heart, lung, bladder, skin, and uterus. In contrast, significant PINCH2 expression starts at E14.5. In adult mice it is widely expressed, similar to PINCH1, but absent from spleen and thymus. In situ hybridization confirmed the Northern data and showed differential expression of PINCH1 and PINCH2 in embryonic intestine. Finally, we demonstrate that PINCH2 localizes to focal adhesions in NIH 3T3 cells and to Z-disks in primary rat cardiomyocytes.

During circuit assembly it is essential that neurons connect with their specific synaptic partners. To facilitate this process, a common strategy in many organisms is the organization of brain regions, including the fly visual system, in layers and columns. The atypical-cadherin Flamingo (Fmi) and the receptor Golden Goal (Gogo) were proposed to regulate both the temporary and final layer selection of the R8 photoreceptor, through the cytoplasmic domain of Gogo. Our data suggests that Fmi intracellular signaling is also relevant for R8 final layer selection. The LIM-domain cytoplasmic molecule Espinas (Esn) binds Fmi, and they cooperatively control dendritic self-avoidance in sensory neurons. We observed defects in R8 layer selection in esn mutants with axons overshooting the final target layer, and we demonstrated that the LIM domain is necessary for layer selection. fmi knockdown in photoreceptors results in most R8 axons stalling at the temporary layer, however, we also detected R8 axons projecting past the final-target layer, and showed that fmi and esn genetically interact. Based on the previously described physical and genetic interactions between Fmi/Esn and the findings presented here, we propose that Esn signals downstream of Fmi to stabilize R8 axons in their final target layer.

Hepatitis C virus (HCV) propagation is highly dependent on cellular proteins. To identify the host factors involved in HCV propagation, we previously performed protein microarray assays and identified the LIM and SH3 domain protein 1 (LASP-1) as an HCV NS5A-interacting partner. LASP-1 plays an important role in the regulation of cell proliferation, migration, and protein-protein interactions. Alteration of LASP-1 expression has been implicated in hepatocellular carcinoma. However, the functional involvement of LASP1 in HCV propagation and HCV-induced pathogenesis has not been elucidated. Here, we first verified the protein interaction of NS5A and LASP-1 by both in vitro pulldown and coimmunoprecipitation assays. We further showed that NS5A and LASP-1 were colocalized in the cytoplasm of HCV infected cells. NS5A interacted with LASP-1 through the proline motif in domain I of NS5A and the tryptophan residue in the SH3 domain of LASP-1. Knockdown of LASP-1 increased HCV replication in both HCV-infected cells and HCV subgenomic replicon cells. LASP-1 negatively regulated viral propagation and thereby overexpression of LASP-1 decreased HCV replication. Moreover, HCV propagation was decreased by wild-type LASP-1 but not by an NS5A binding-defective mutant of LASP-1. We further demonstrated that LASP-1 was involved in the replication stage of the HCV life cycle. Importantly, LASP-1 expression levels were increased in persistently infected cells with HCV. These data suggest that HCV modulates LASP-1 via NS5A in order to regulate virion levels and maintain a persistent infection.