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Ladak, R. J.

Publications and source records attributed to Ladak, R. J..

5 recordsLinked to original sources

Viral inhibition of the anaphase promoting complex enhances replication by elevating nucleotide pools

The anaphase promoting complex/cyclosome (APC/C) is a large, ubiquitin ligase and a central regulator of cell cycle progression. By targeting key substrates for degradation during mitosis and G1 phase, the APC/C coordinates metabolic fluctuations that occur during the cell cycle. A diverse range of viruses have convergently evolved mechanisms to bind and inhibit the APC/C; however, a molecular understanding for these interactions has never been demonstrated. Here, we use chicken anemia virus (CAV), a small single-stranded DNA virus encoding only three proteins, to demonstrate the importance of viral APC/C inhibition during replication. We show that the Vp3 protein of CAV inhibits the APC/C, causing a dramatic mitotic arrest during infection. Mutant virus lacking Vp3 are defective for replication and can be rescued by APC/C inhibition. Metabolomic profiling during CAV infection revealed that Vp3 expression mediates a broad increase in nucleotide pools. Moreover, viral inhibition of the APC/C resulted in stabilization of enzymes required for nucleotide biosynthesis. These findings suggest that the APC/C is a general target of many viruses to elevate nucleotide levels and facilitate viral genome replication. Author SummaryViruses dramatically reshape the cells they infect in order to replicate. To do this efficiently, many viruses disrupt the hosts normal control systems, particularly those that regulate cell growth and division. One such system is the anaphase promoting complex/cyclosome (APC/C), a key cellular machine that controls when specific proteins are broken down during the cell cycle. Surprisingly, a wide range of viruses--including adenovirus, HIV, human papillomavirus, herpesvirus, and others--have been shown to interfere with the APC/C. Until now, however, it was unclear why so many unrelated viruses target this same pathway. In our study, we used chicken anemia virus (CAV), one of the smallest known animal viruses, as a simple model to uncover the underlying mechanism. CAV produces just three proteins, one of which--called VP3--strongly inhibits the APC/C. We found that this inhibition increases the availability of nucleotides, the basic building blocks needed to copy viral DNA. By disrupting normal cell cycle control, the virus effectively redirects cellular resources toward its own replication. These findings reveal APC/C inhibition as a common and powerful strategy used by diverse viruses, offering new insight into how viruses exploit host cells and highlighting potential targets for future antiviral therapies.

microbiology↗

Codon-dependent regulation of mRNA translation and stability by ZC3H7A and ZC3H7B RNA-binding proteins

Decelerated translation elongation caused by non-optimal codons can reduce mRNA stability through codon optimality-mediated mRNA degradation. A key element of this process is the coupling of sensing the mRNA codon usage with the regulation of translation efficiency and stability. We report that two paralog RNA-binding proteins (ZC3H7A and ZC3H7B), which are only found in Chordates, preferentially bind to and reduce the stability and translation of mRNAs enriched in non-optimal codons with A/U at their wobble sites (A/U3 codons). ZC3H7A/B engage with ribosomes that lack elongation factors and induce mRNA degradation or block translation initiation through their interactions with the CCR4-NOT and the GIGYF2/4EHP translation repressor complex, respectively. Depletion of ZC3H7A/B or 4EHP impairs the repression of non-optimal A/U3-rich mRNAs. This study provides insights into a unique mechanism in higher eukaryotes that couples codon usage with the regulation of translation efficiency and mRNA stability.

cell biology↗

No evidence that human GIGYF2 interacts with growth factor receptor-bound protein 10 (GRB10): implication for human disease

GIGYF2 (Growth factor receptor bound protein 10 (GRB10)-interacting GYF (glycine-tyrosine-phenylalanine) protein 2) reduces mRNA stability and translation via microRNAs, ribosome quality control, and several RNA-binding proteins. GIGYF2 was first identified in mouse cell lines as an interacting partner with Growth factor receptor-bound protein 10 (GRB10), which binds to the insulin receptor and insulin-like growth-factor receptor 1. Mutations in the human GIGYF2 gene were reported in autism spectrum disorder and Parkinsons disease. In mouse models, mutations in the gene encoding GIGYF2 exhibited disease phenotypes. It was, therefore, thought that the GIGYF2-associated disease in humans is caused by defective GRB10 signaling. We show here that GIGYF2 does not interact with GRB10 in human cell lines, as determined by proximity ligation and co-immunoprecipitation assays. Size exclusion chromatography assay further confirmed these findings. The lack of interaction is explained by the absence of the critical GYF-domain binding PPG{Phi} sequence in the human GRB10 protein. These results are in contrast with the current understanding that a GIGYF2/GRB10 complex is associated with human disease via IR and IGF-1R signaling and underscore alternative mechanisms responsible for the observed phenotypes related to mutations in the human GIGYF2 gene.

molecular biology↗

Repression of mRNA translation initiation by GIGYF1 via blocking the eIF3-eIF4G1 interaction

Viruses commonly interfere with the function of the eukaryotic translation initiation factor 4G1 (eIF4G1), a pivotal factor in the recruitment of the eIF3 complex and ribosome to the mRNA. This results in the inhibition of general host protein synthesis and redirecting ribosomes toward viral mRNAs. Certain viruses also selectively repress the translation of mRNAs involved in the host antiviral response. GIGYF2 and its interacting cap-binding protein 4EHP enable the transcript-specific repression of mRNA translation mediated by microRNAs and RNA-binding proteins (RBPs). RNA viruses, such as SARS-CoV-2, exploit the GIGYF2/4EHP complex to selectively repress the translation of transcripts such as Ifnb1 mRNA, which encodes the antiviral cytokine Interferon {beta} (IFN-{beta}). Herein, we reveal that GIGYF1, a paralogue of GIGYF2, robustly represses cellular mRNA translation through a distinct mechanism independent of 4EHP. Upon recruitment to a target mRNA by RBPs, the C-terminal region of GIGYF1 binds to subunits of eIF3 at the interaction interface of eIF3-eIF4G1. This disrupts binding of eIF3 to eIF4G1, resulting in mRNA-specific translational repression. This mechanism exerts profound influences on the host cells response to viral infection. Depletion of GIGYF1 induces a robust immune response by derepressing Ifnb1 mRNA translation. Overall, our study highlights a unique mechanism of translational regulation by GIGYF1 that involves sequestering eIF3 and abrogating its binding to eIF4G1. This mechanism can be utilized by RBPs that interact with GIGYF1 to specifically repress the translation of their target mRNAs, significantly affecting critical biological processes, including host-pathogen interactions.

cell biology↗

SARS-CoV-2 protein NSP2 enhances microRNA-mediated translational repression

microRNAs (miRNAs) inhibit mRNA translation initiation by recruiting the GIGYF2/4EHP translation repressor complex to the mRNA 5 cap structure. Viruses utilise miRNAs to impair the host antiviral immune system and facilitate viral infection by expressing their own miRNAs or co-opting cellular miRNAs. We recently reported that the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) encoded non-structural protein 2 (NSP2) interacts with GIGYF2. This interaction is critical for blocking translation of the Ifn1-b mRNA that encodes the cytokine Interferon-{beta}, and thereby impairs the host antiviral immune response. However, it is not known whether NSP2 also affects miRNA-mediated silencing. Here, we demonstrate the pervasive augmentation of the miRNA-mediated translational repression of cellular mRNAs by NSP2. We show that NSP2 interacts with Argonaute 2, the core component of the miRNA-Induced Silencing Complex (miRISC) and enhances the translational repression mediated by natural miRNA binding sites in the 3 UTR of cellular mRNAs. Our data reveal an additional layer of the complex mechanism by which SARS-CoV-2 and likely other coronaviruses manipulate the host gene expression program through co-opting the host miRNA-mediated silencing machinery.

cell biology↗