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The cellular protein GBF1, an activator of Arf GTPases (ArfGEF: Arf guanine nucleotide exchange factor), is recruited to the replication organelles of enteroviruses through interaction with the viral protein 3A, and its ArfGEF activity is required for viral replication, however how GBF1-dependent Arf activation supports the infection remains enigmatic. Here, we investigated the development of resistance of poliovirus, a prototype enterovirus, to increasing concentrations of brefeldin A (BFA), an inhibitor of GBF1. High level of resistance required a gradual accumulation of multiple mutations in the viral protein 2C. The 2C mutations conferred BFA resistance even in the context of a 3A mutant previously shown to be defective in the recruitment of GBF1 to replication organelles, and in cells depleted of GBF1, suggesting a GBF1-independent replication mechanism. Still, activated Arfs accumulated on the replication organelles of this mutant even in the presence of BFA, its replication was inhibited by a pan-ArfGEF inhibitor LM11, and the BFA-resistant phenotype was compromised in Arf1-knockout cells. Importantly, the mutations strongly increased the interaction of 2C with the activated form of Arf1. Analysis of other enteroviruses revealed a particularly strong interaction of 2C of human rhinovirus 1A with activated Arf1. Accordingly, the replication of this virus was significantly less sensitive to BFA than that of poliovirus. Thus, our data demonstrate that enterovirus 2Cs may behave like Arf1 effector proteins and that GBF1 but not Arf activation can be dispensable for enterovirus replication. These findings have important implications for the development of host-targeted anti-viral therapeutics. Author summary: As ultimate intracellular parasites, viruses rely on cellular proteins for every step of their life cycle. This provides a possibility of controlling viral replication by targeting the cellular factors. Such therapeutics are expected to be more difficult to develop resistance to since cellular proteins do not change. Yet, enteroviruses, a large group of small positive RNA viruses that are associated with multiple human pathologies, rapidly develop resistance to virtually all experimental therapeutics including compounds targeting host factors essential for the viral replication. Here we investigated in detail the development of enterovirus resistance to one of such compounds targeting a host protein GBF1 that activates small GTPases of the Arf family. We found that while the mutations in a resistant virus make it insensitive to the inhibition of GBF1, the virus acquired the capability of obtaining activated Arfs from other cellular sources and that the interaction of one of the viral proteins with activated Arfs is likely essential for the functioning of the replication complex. Thus, our work revealed that it is not GBF1 per se, but activated Arfs that are indispensable for viral replication, which provides new opportunities for the development of anti-enterovirus therapeutics refractory to the development of resistance. [ABSTRACT FROM AUTHOR]

As ultimate parasites, viruses depend on host factors for every step of their life cycle. On the other hand, cells evolved multiple mechanisms of detecting and interfering with viral replication. Yet, our understanding of the complex ensembles of pro- and anti-viral factors is very limited in virtually every virus-cell system. Here we investigated the proteins recruited to the replication organelles of poliovirus, a representative of the genus Enterovirus of the Picornaviridae family. We took advantage of a strict dependence of enterovirus replication on a host protein GBF1, and established a stable cell line expressing a truncated GBF1 fused to APEX2 peroxidase that effectively supported viral replication upon inhibition of the endogenous GBF1. This construct biotinylated multiple host and viral proteins on the replication organelles. Among the viral proteins, the polyprotein cleavage intermediates were overrepresented, suggesting that the GBF1 environment is linked to viral polyprotein processing. The proteomics characterization of biotinylated host proteins identified multiple proteins previously associated with enterovirus replication, as well as more than 200 new factors recruited to the replication organelles. RNA metabolism proteins, many of which normally localize in the nucleus, constituted the largest group, underscoring the massive release of nuclear factors into the cytoplasm of infected cells and their involvement in viral replication. Functional analysis of several newly identified proteins revealed both pro- and anti-viral factors, including a novel component of infection-induced stress granules. Depletion of these proteins similarly affected the replication of diverse enteroviruses indicating broad conservation of the replication mechanisms. Thus, our data significantly expand the knowledge of the composition of enterovirus replication organelles, provide new insights into viral replication, and offer a novel resource for identifying targets for anti-viral interventions. Author summary: Enteroviruses are arguably the most numerous group of viral pathogens infecting humans. Enterovirus infection can cause multiple complications including the development of asthma, type I diabetes, myocarditis, encephalitis, paralysis, and many others. Yet, there are no approved drugs against any of the pathogenic enteroviruses. The lack of effective anti-enterovirus measures is due in large part to insufficient understanding of molecular mechanisms supporting the development of enterovirus infections. Here, we developed a novel probe based on a host protein GBF1 that is universally required for enterovirus RNA replication to identify the proteins localized on enterovirus replication organelles, specialized membranous structures harboring the viral RNA replication complexes. Among the host proteins, we identified factors that promote and suppress the replication of diverse enteroviruses. Thus, our research provides new insights into molecular mechanisms of enterovirus replication and presents an important resource for the development of host-targeted anti-viral interventions. [ABSTRACT FROM AUTHOR]

Introduction: The formation of normal bone and bone healing requires the cAMP-responsive element binding protein 3-like-1 (Creb3l1) transmembrane transcription factor, as deletion of the murine CREB3L1 results in osteopenic animals with limited capacity to repair bone after a fracture. Creb3l1 undergoes regulated intramembrane proteolysis (RIP) to release the N-terminal transcription activating (TA) fragment that enters the nucleus and regulates the expression of target genes., Methods: To expand our understanding of Creb3l1's role in skeletal development and skeletal patterning, we aimed to generate animals expressing only the TA fragment of Creb3l1 lacking the transmembrane domain and thereby not regulated through RIP. However, the CRISPR/Cas9-mediated genome editing in zebrafish Danio rerio caused a frameshift mutation that added 56 random amino acids at the C-terminus of the TA fragment (TA+), making it unable to enter the nucleus. Thus, TA+ does not regulate transcription, and the creb3l1TA+/TA+ fish do not mediate creb3l1-dependent transcription., Results: We document that the creb3l1TA+/TA+ fish exhibit defects in the patterning of caudal fin lepidotrichia, with significantly distalized points of proximal bifurcation and decreased secondary bifurcations. Moreover, using the caudal fin amputation model, we show that creb3l1TA+/TA+ fish have decreased regeneration and that their regenerates replicate the distalization and bifurcation defects observed in intact fins of creb3l1TA+/TA+ animals. These defects correlate with altered expression of the shha and ptch2 components of the Sonic Hedgehog signaling pathway in creb3l1TA+/TA+ regenerates., Conclusion: Together, our results uncover a previously unknown intersection between Creb3l1 and the Sonic Hedgehog pathway and document a novel role of Creb3l1 in tissue patterning., (© 2024 S. Karger AG, Basel.)

Background: The gene cAMP-Responsive Element Binding protein 3-like-1 (CREB3L1) has been implicated in bone development in mice, with CREB3L1 knock-out mice exhibiting fragile bones, and in humans, with CREB3L1 mutations linked to osteogenesis imperfecta. However, the mechanism through which Creb3l1 regulates bone development is not fully understood., Results: To probe the role of Creb3l1 in organismal physiology, we used CRISPR-Cas9 genome editing to generate a Danio rerio (zebrafish) model of Creb3l1 deficiency. In contrast to mammalian phenotypes, the Creb3l1 deficient fish do not display abnormalities in osteogenesis, except for a decrease in the bifurcation pattern of caudal fin. Both, skeletal morphology and overall bone density appear normal in the mutant fish. However, the regeneration of caudal fin postamputation is significantly affected, with decreased overall regenerate and mineralized bone area. Moreover, the mutant fish exhibit a severe patterning defect during regeneration, with a significant decrease in bifurcation complexity of the fin rays and distalization of the bifurcation sites. Analysis of genes implicated in bone development showed aberrant patterning of shha and ptch2 in Creb3l1 deficient fish, linking Creb3l1 with Sonic Hedgehog signaling during fin regeneration., Conclusions: Our results uncover a novel role for Creb3l1 in regulating tissue growth and patterning during regeneration., (© 2024 The Author(s). Developmental Dynamics published by Wiley Periodicals LLC on behalf of American Association for Anatomy.)

Golgi homeostasis require the activation of Arf GTPases by the guanine-nucleotide exchange factor requires GBF1, whose recruitment to the Golgi represents a rate limiting step in the process. GBF1 contains a conserved, catalytic, Sec7 domain (Sec7d) and five additional (DCB, HUS, HDS1-3) domains. Herein, we identify the HDS3 domain as essential for GBF1 membrane association in mammalian cells and document the critical role of HDS3 during the development of Drosophila melanogaster . We show that upon binding to Golgi membranes, GBF1 undergoes conformational changes in regions bracketing the catalytic Sec7d. We illuminate GBF1 interdomain arrangements by negative staining electron microscopy of full-length human GBF1 to show that GBF1 forms an anti-parallel dimer held together by the paired central DCB-HUS core, with two sets of HDS1-3 arms extending outward in opposite directions. The catalytic Sec7d protrudes from the central core as a largely independent domain, but is closely opposed to a previously unassigned α-helix from the HDS1 domain. Based on our data, we propose models of GBF1 engagement on the membrane to provide a paradigm for understanding GBF1-mediated Arf activation required for cellular and organismal function., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Meissner, Akhmetova, Szul, Viktorova, Sha, Bhatt, Lee, Kahn, Belov, Chesnokov and Sztul.)