10 research outputs found
Cloning and characterization of the Schizosaccharomyces pombe homologs of the human protein Translin and the Translin-associated protein TRAX
Translin is a human octameric protein that specifically binds the single-stranded microsatellite repeats d(GT)(n) and the corresponding transcripts (GU)(n). It also binds, with lesser affinities, other single-stranded G-rich DNA and RNA sequences. TRAX is a human protein that bears a homology to Translin and interacts with it. Translin and TRAX have been proposed to be involved in DNA recombination, chromosomal translocation and mRNA transport and translation. Both proteins are highly conserved in eukaryotes, including the fission yeast Schizosaccharomyces pombe, which is amenable to genetic analysis. Here, we report the first study of the S.pombe Translin and TRAX homologs. We have deleted the genes encoding Translin and TRAX in S.pombe and found that the proliferation of the mutant cells was slightly stimulated, suggesting that these genes are not essential for the fission yeast. We have also shown that the S.pombe Translin and TRAX interact. Biochemical analysis of the S.pombe Translin, which was cloned and expressed in Escherichia coli, revealed that it is octameric and that it selectively binds d(GT)(n) and d(GTT)(n) microsatellite repeats. However, unlike the human protein, it has much higher affinities for the homologous RNA sequences (GU)(n) and (GUU)(n). These data suggest that the S.pombe Translin is primarily involved in functions related to RNA metabolism
The COG complex interacts directly with Syntaxin 6 and positively regulates endosome-to-TGN retrograde transport
The conserved oligomeric Golgi (COG) complex interacts with the t-SNARE Syntaxin 6 and promotes endosome-to-TGN retrograde trafficking
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Viral Generated Inter-Organelle Contacts Redirect Lipid Flux for Genome Replication
Positive-stranded RNA viruses extensively remodel host cell architecture to enable viral replication. Here, we examined the poorly understood formation of specialized membrane compartments that are critical sites for the synthesis of the viral genome. We show that the replication compartments (RCs) of enteroviruses are created through novel membrane contact sites that recruit host lipid droplets (LDs) to the RCs. Viral proteins tether the RCs to the LDs and interact with the host lipolysis machinery to enable transfer of fatty acids from LDs, thereby providing lipids essential for RC biogenesis. Inhibiting the formation of the membrane contact sites between LDs and RCs or inhibition of the lipolysis pathway disrupts RC biogenesis and enterovirus replication. Our data illuminate mechanistic and functional aspects of organelle remodeling in viral infection and establish that pharmacological targeting of contact sites linking viral and host compartments is a potential strategy for antiviral development
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Enterovirus pathogenesis requires the host methyltransferase SETD3.
Enteroviruses (EVs) comprise a large genus of positive-sense, single-stranded RNA viruses whose members cause a number of important and widespread human diseases, including poliomyelitis, myocarditis, acute flaccid myelitis and the common cold. How EVs co-opt cellular functions to promote replication and spread is incompletely understood. Here, using genome-scale CRISPR screens, we identify the actin histidine methyltransferase SET domain containing 3 (SETD3) as critically important for viral infection by a broad panel of EVs, including rhinoviruses and non-polio EVs increasingly linked to severe neurological disease such as acute flaccid myelitis (EV-D68) and viral encephalitis (EV-A71). We show that cytosolic SETD3, independent of its methylation activity, is required for the RNA replication step in the viral life cycle. Using quantitative affinity purification-mass spectrometry, we show that SETD3 specifically interacts with the viral 2A protease of multiple enteroviral species, and we map the residues in 2A that mediate this interaction. 2A mutants that retain protease activity but are unable to interact with SETD3 are severely compromised in RNA replication. These data suggest a role of the viral 2A protein in RNA replication beyond facilitating proteolytic cleavage. Finally, we show that SETD3 is essential for in vivo replication and pathogenesis in multiple mouse models for EV infection, including CV-A10, EV-A71 and EV-D68. Our results reveal a crucial role of a host protein in viral pathogenesis, and suggest targeting SETD3 as a potential mechanism for controlling viral infections
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Enterovirus pathogenesis requires the host methyltransferase SETD3.
Enteroviruses (EVs) comprise a large genus of positive-sense, single-stranded RNA viruses whose members cause a number of important and widespread human diseases, including poliomyelitis, myocarditis, acute flaccid myelitis and the common cold. How EVs co-opt cellular functions to promote replication and spread is incompletely understood. Here, using genome-scale CRISPR screens, we identify the actin histidine methyltransferase SET domain containing 3 (SETD3) as critically important for viral infection by a broad panel of EVs, including rhinoviruses and non-polio EVs increasingly linked to severe neurological disease such as acute flaccid myelitis (EV-D68) and viral encephalitis (EV-A71). We show that cytosolic SETD3, independent of its methylation activity, is required for the RNA replication step in the viral life cycle. Using quantitative affinity purification-mass spectrometry, we show that SETD3 specifically interacts with the viral 2A protease of multiple enteroviral species, and we map the residues in 2A that mediate this interaction. 2A mutants that retain protease activity but are unable to interact with SETD3 are severely compromised in RNA replication. These data suggest a role of the viral 2A protein in RNA replication beyond facilitating proteolytic cleavage. Finally, we show that SETD3 is essential for in vivo replication and pathogenesis in multiple mouse models for EV infection, including CV-A10, EV-A71 and EV-D68. Our results reveal a crucial role of a host protein in viral pathogenesis, and suggest targeting SETD3 as a potential mechanism for controlling viral infections
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Comparative Flavivirus-Host Protein Interaction Mapping Reveals Mechanisms of Dengue and Zika Virus Pathogenesis
Mosquito-borne flaviviruses, including dengue virus (DENV) and Zika virus (ZIKV), are a growing public health concern. Systems-level analysis of how flaviviruses hijack cellular processes through virus-host protein-protein interactions (PPIs) provides information about their replication and pathogenic mechanisms. We used affinity purification-mass spectrometry (AP-MS) to compare flavivirus-host interactions for two viruses (DENV and ZIKV) in two hosts (human and mosquito). Conserved virus-host PPIs revealed that the flavivirus NS5 protein suppresses interferon stimulated genes by inhibiting recruitment of the transcription complex PAF1C and that chemical modulation of SEC61 inhibits DENV and ZIKV replication in human and mosquito cells. Finally, we identified a ZIKV-specific interaction between NS4A and ANKLE2, a gene linked to hereditary microcephaly, and showed that ZIKV NS4A causes microcephaly in Drosophila in an ANKLE2-dependent manner. Thus, comparative flavivirus-host PPI mapping provides biological insights and, when coupled with in vivo models, can be used to unravel pathogenic mechanisms