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Craig Mccormick - One of the best experts on this subject based on the ideXlab platform.
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Composition of Herpesvirus RibonucleoProtein Complexes
Proceedings, 2020Co-Authors: Eric S. Pringle, Craig MccormickAbstract:Herpesvirus genomes are decoded by host RNA polymerase enzymes, generating messenger ribonucleotides (mRNA) that are post-transcriptionally modified and exported to the cytoplasm through the combined work of host and Viral factors. These Viral mRNA bear 5′-m7GTP caps and poly(A) tails that should permit the assembly of canonical host eIF4F cap-binding complexes to initiate Protein Synthesis. However, the precise mechanisms of translation initiation remain to be investigated for Kaposi’s sarcoma-associated herpesvirus (KSHV) and other herpesviruses. During KSHV lytic replication in lymphoid cells, the activation of caspases leads to the cleavage of eIF4G and depletion of eIF4F. Translating mRNPs depleted of eIF4F retain Viral mRNA, suggesting that non-eIF4F translation initiation is sufficient to support Viral Protein Synthesis. To identify Proteins required to support Viral Protein Synthesis, we isolated and characterized actively translating messenger ribonucleoProtein (mRNP) complexes by ultracentrifugation and sucrose-gradient fractionation followed by quantitative mass spectrometry. The abundance of host translation initiation factors available to initiate Viral Protein Synthesis were comparable between cells undergoing KSHV lytic or latent replication. The translation initiation factors eIF4E2, NCBP1, eIF4G2, and eIF3d were detected in association with actively translating mRNP complexes during KSHV lytic replication, but their depletion by RNA silencing did not affect virion production. By contrast, the N6-methyladenosine methyltransferase METTL3 was required for optimal late gene expression and virion production, but was dispensable for genome replication. Furthermore, we detected several KSHV Proteins in actively translating mRNP complexes that had not previously been shown to play roles in Viral Protein Synthesis. We conclude that KSHV usurps distinct host translation initiation systems during latent and lytic phases of infection.
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Kaposi's Sarcoma-Associated Herpesvirus Lytic Replication Interferes with mTORC1 Regulation of Autophagy and Viral Protein Synthesis.
Journal of virology, 2019Co-Authors: Eric S. Pringle, Carolyn-ann Robinson, Craig MccormickAbstract:Mechanistic target of rapamycin complex 1 (mTORC1) is a master regulator of cellular metabolism. In nutrient-rich environments, mTORC1 kinase activity stimulates Protein Synthesis to meet cellular anabolic demands. Under nutrient-poor conditions or under stress, mTORC1 is rapidly inhibited, global Protein Synthesis is arrested, and a cellular catabolic process known as autophagy is activated. Kaposi's sarcoma-associated herpesvirus (KSHV) encodes multiple Proteins that stimulate mTORC1 activity or subvert autophagy, but precise roles for mTORC1 in different stages of KSHV infection remain incompletely understood. Here, we report that during latent and lytic stages of KSHV infection, chemical inhibition of mTORC1 caused eukaryotic initiation factor 4F (eIF4F) disassembly and diminished global Protein Synthesis, which indicated that mTORC1-mediated control of translation initiation was largely intact. We observed that mTORC1 was required for Synthesis of the replication and transcription activator (RTA) lytic switch Protein and reactivation from latency, but once early lytic gene expression had begun, mTORC1 was not required for genome replication, late gene expression, or the release of infectious progeny. Moreover, mTORC1 control of autophagy was dysregulated during lytic replication, whereby chemical inhibition of mTORC1 prevented ULK1 phosphorylation but did not affect autophagosome formation or rates of autophagic flux. Together, these findings suggest that mTORC1 is dispensable for Viral Protein Synthesis and Viral control of autophagy during lytic infection and that KSHV undermines mTORC1-dependent cellular processes during the lytic cycle to ensure efficient Viral replication.IMPORTANCE All viruses require host cell machinery to synthesize Viral Proteins. A host cell Protein complex known as mechanistic target of rapamycin complex 1 (mTORC1) is a master regulator of Protein Synthesis. Under nutrient-rich conditions, mTORC1 is active and promotes Protein Synthesis to meet cellular anabolic demands. Under nutrient-poor conditions or under stress, mTORC1 is rapidly inhibited, global Protein Synthesis is arrested, and a cellular catabolic process known as autophagy is activated. Kaposi's sarcoma-associated herpesvirus (KSHV) stimulates mTORC1 activity and utilizes host machinery to synthesize Viral Proteins. However, we discovered that mTORC1 activity was largely dispensable for Viral Protein Synthesis, genome replication, and the release of infectious progeny. Likewise, during lytic replication, mTORC1 was no longer able to control autophagy. These findings suggest that KSHV undermines mTORC1-dependent cellular processes during the lytic cycle to ensure efficient Viral replication.
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KSHV lytic mRNA is efficiently translated in the absence of eIF4F
2018Co-Authors: Eric S. Pringle, Carolyn-ann Robinson, Nicolas Crapoulet, A.l. Monjo, Katrina Bouzanis, Andrew M. Leidal, Stephen M. Lewis, Daniel Gaston, James Uniacke, Craig MccormickAbstract:Herpesvirus genomes are decoded by host RNA polymerase II, generating messenger ribonucleic acids (mRNAs) that are post-transcriptionally modified and exported to the cytoplasm. These Viral mRNAs have 59-m7GTP caps and poly(A) tails that should permit assembly of canonical eIF4F cap-binding complexes to initiate Protein Synthesis. However, we have shown that chemical disruption of eIF4F does not impede KSHV lytic replication, suggesting that alternative translation initiation mechanisms support Viral Protein Synthesis. Here, using polysome profiling analysis, we confirmed that eIF4F disassembly did not affect the efficient translation of Viral mRNAs during lytic replication, whereas a large fraction of host mRNAs remained eIF4F-dependent. Lytic replication altered multiple host translation initiation factors (TIFs), causing caspase-dependent cleavage of eIF2alpha; and eIF4G1 and decreasing levels of eIF4G2 and eIF4G3. Non-eIF4F TIFs NCBP1, eIF4E2 and eIF4G2 associated with actively translating messenger ribonucleoProtein (mRNP) complexes during KSHV lytic replication, but their depletion by RNA silencing did not affect virion production, suggesting that the virus does not exclusively rely on one of these alternative TIFs for efficient Viral Protein Synthesis. METTL3, an N6-methyladenosine (m6A) methyltransferase that modifies mRNAs and influences translational efficiency, was dispensable for early Viral gene expression and genome replication but required for late gene expression and virion production. METTL3 was also subject to caspase-dependent degradation during lytic replication, suggesting that its positive effect on KSHV late gene expression may be indirect. Taken together, our findings reveal extensive remodelling of TIFs during lytic replication, which may help sustain efficient Viral Protein Synthesis in the context of host shutoff.
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stress granule inducing eukaryotic translation initiation factor 4a inhibitors block influenza a virus replication
Viruses, 2017Co-Authors: Patrick D Slaine, Mariel Klee, Natha K Smith, Denys A Khaperskyy, Craig MccormickAbstract:Eukaryotic translation initiation factor 4A (eIF4A) is a helicase that facilitates assembly of the translation preinitiation complex by unwinding structured mRNA 5' untranslated regions. Pateamine A (PatA) and silvestrol are natural products that disrupt eIF4A function and arrest translation, thereby triggering the formation of cytoplasmic aggregates of stalled preinitiation complexes known as stress granules (SGs). Here we examined the effects of eIF4A inhibition by PatA and silvestrol on influenza A virus (IAV) Protein Synthesis and replication in cell culture. Treatment of infected cells with either PatA or silvestrol at early times post-infection resulted in SG formation, arrest of Viral Protein Synthesis and failure to replicate the Viral genome. PatA, which irreversibly binds to eIF4A, sustained long-term blockade of IAV replication following drug withdrawal, and inhibited IAV replication at concentrations that had minimal cytotoxicity. By contrast, the antiViral effects of silvestrol were fully reversible; drug withdrawal caused rapid SG dissolution and resumption of Viral Protein Synthesis. IAV inhibition by silvestrol was invariably associated with cytotoxicity. PatA blocked replication of genetically divergent IAV strains, suggesting common dependence on host eIF4A activity. This study demonstrates that the core host Protein Synthesis machinery can be targeted to block Viral replication.
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stress granule inducing eukaryotic translation initiation factor 4a inhibitors block influenza a virus replication
bioRxiv, 2017Co-Authors: Patrick D Slaine, Mariel Klee, Natha K Smith, Denys A Khaperskyy, Craig MccormickAbstract:Eukaryotic translation initiation factor 4A (eIF4A) is a helicase that facilitates assembly of the translation preinitiation complex by unwinding structured mRNA 5-prime untranslated regions. Pateamine A (PatA) and silvestrol are natural products that disrupt eIF4A function and arrest translation, thereby triggering the formation of cytoplasmic aggregates of stalled preinitiation complexes known as stress granules (SGs). Here we examined the effects of eIF4A inhibition by PatA and silvestrol on influenza A virus (IAV) Protein Synthesis and replication in cell culture. Treatment of infected cells with either PatA or silvestrol at early times post-infection results in SG formation, arrest of Viral Protein Synthesis and failure to replicate the Viral genome. PatA, which irreversibly binds to eIF4A, sustained long-term blockade of IAV replication following drug withdrawal, and inhibited IAV replication at concentrations that had minimal cytotoxicity. By contrast, the antiViral effects of silvestrol were fully reversible; drug withdrawal caused rapid SG dissolution and resumption of Viral Protein Synthesis. IAV inhibition by silvestrol was invariably associated with cytotoxicity. PatA blocked replication of genetically divergent IAV strains, suggesting common dependence on host eIF4A activity. This study demonstrates the feasibility of targeting core host Protein Synthesis machinery to prevent Viral replication.
A Greco - One of the best experts on this subject based on the ideXlab platform.
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translational control of Viral and host Protein Synthesis during the course of herpes simplex virus type 1 infection evidence that initiation of translation is the limiting step
Journal of General Virology, 1998Co-Authors: A M Laurent, J J Madjar, A GrecoAbstract:Herpes simplex virus type 1 (HSV-1) infection induces the selective shut-off of host Protein Synthesis, other than ribosomal Proteins, and the successive Synthesis of Viral Proteins. Because Viral mRNAs persist in the cytoplasm after Viral Protein Synthesis has been inhibited, we hypothesized that Viral gene expression may be under translational control. Expression of genes encoding immediate early ICP27, early DBP and late US11 Proteins, together with glyceraldehyde-3-phosphate dehydrogenase (GAPDH), was monitored over the course of infection at the level of mRNA and Protein Synthesis. After an efficient Synthesis beginning with the appearance of successive Viral mRNAs in the cytoplasm, Synthesis of Viral Proteins was shut off similarly to the Synthesis of GAPDH. This shut-off was not achieved by mRNA degradation but by progressive shifts of Viral mRNAs from large polyribosomes to smaller ones, then to 40S ribosomal subunits. Transient expression of the UL41 gene alone, directing Synthesis of virion-associated host shut-off (VHS) Protein, induced efficient mRNA degradation, but did not impair recruitment of the remaining GAPDH and beta-actin mRNAs into polyribosomes. These results indicate that HSV-1 induces a selective repression of initiation of mRNA translation which is probably the main cause of the shut-off of Viral Protein Synthesis, and which contributes to the repression of host Protein Synthesis. VHS Protein is not directly involved in this repression, at least in the absence of other Viral Proteins.
Peter Staeheli - One of the best experts on this subject based on the ideXlab platform.
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human and mouse mx Proteins inhibit different steps of the influenza virus multiplication cycle
Journal of Virology, 1992Co-Authors: Jovan Pavlovic, Otto Haller, Peter StaeheliAbstract:Human MxA and mouse Mx1 are interferon-induced Proteins capable of inhibiting the multiplication of influenza virus. MxA Protein is localized in the cytoplasm, whereas Mx1 Protein accumulates in the nucleus. Taking advantage of stably transfected cell lines that constitutively express either MxA or Mx1 Protein, we examined the steps at which these Proteins block influenza A viruses. In infected cells expressing MxA Protein, all Viral mRNAs synthesized as a result of primary transcription in the nucleus by the virion-associated RNA polymerase accumulated to normal levels. These primary Viral transcripts were polyadenylated, were active in directing Viral Protein Synthesis in vitro, and appeared to be efficiently transported to the cell cytoplasm. Yet Viral Protein Synthesis and genome amplification were strongly inhibited, suggesting that MxA Protein interfered with either intracytoplasmic transport of Viral mRNAs, Viral Protein Synthesis, or translocation of newly synthesized Viral Proteins to the cell nucleus. However, in infected cells expressing Mx1 Protein, the concentrations of the longest primary transcripts encoding the three influenza virus polymerase Proteins PB1, PB2, and PA were at least 50-fold reduced. Accumulation of the shorter primary transcripts encoding the other Viral Proteins was also inhibited but to a lesser extent. These results demonstrate that the mouse Mx1 Protein interferes with primary transcription of influenza virus in the nucleus, whereas the human MxA Protein inhibits a subsequent step that presumably takes place in the cytoplasm of infected cells. Images
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Human and mouse Mx Proteins inhibit different steps of the influenza virus multiplication cycle.
Journal of virology, 1992Co-Authors: Jovan Pavlovic, Otto Haller, Peter StaeheliAbstract:Human MxA and mouse Mx1 are interferon-induced Proteins capable of inhibiting the multiplication of influenza virus. MxA Protein is localized in the cytoplasm, whereas Mx1 Protein accumulates in the nucleus. Taking advantage of stably transfected cell lines that constitutively express either MxA or Mx1 Protein, we examined the steps at which these Proteins block influenza A viruses. In infected cells expressing MxA Protein, all Viral mRNAs synthesized as a result of primary transcription in the nucleus by the virion-associated RNA polymerase accumulated to normal levels. These primary Viral transcripts were polyadenylated, were active in directing Viral Protein Synthesis in vitro, and appeared to be efficiently transported to the cell cytoplasm. Yet Viral Protein Synthesis and genome amplification were strongly inhibited, suggesting that MxA Protein interfered with either intracytoplasmic transport of Viral mRNAs, Viral Protein Synthesis, or translocation of newly synthesized Viral Proteins to the cell nucleus. However, in infected cells expressing Mx1 Protein, the concentrations of the longest primary transcripts encoding the three influenza virus polymerase Proteins PB1, PB2, and PA were at least 50-fold reduced. Accumulation of the shorter primary transcripts encoding the other Viral Proteins was also inhibited but to a lesser extent. These results demonstrate that the mouse Mx1 Protein interferes with primary transcription of influenza virus in the nucleus, whereas the human MxA Protein inhibits a subsequent step that presumably takes place in the cytoplasm of infected cells.
Solon L Rhode - One of the best experts on this subject based on the ideXlab platform.
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The Parvovirus H-1 NS2 Protein Affects Viral Gene Expression through Sequences in the 3′ Untranslated Region
Virology, 1993Co-Authors: Solon L RhodeAbstract:Abstract We reported previously that an NS2 null mutant of parvovirus H-1 (H-1SA) was capable of lytic growth in human and hamster cells, but not in rat cells (Li and Rhode, 1991). The host-range phenotype of H-1SA was also manifested in newborn rats and was associated with a reduction of Viral Protein Synthesis to about 10% of wild-type virus and an absence of virions in cultured rat fibroblasts. However, the H-1SA mRNAs for NS1 and capsid Proteins, R1 and R3, accumulated to wild-type levels and translated well with a cell free rabbit reticulocyte lysate. These results indicate that NS2 plays an important role in the regulation of Viral Protein Synthesis in rat cells in vivo and in vitro , but NS2 is largely dispensable in other types of cells, such as human and hamster cells. To analyze whether the 5′ and 3′ untranslated regions (UTR) of Viral RNA are involved in the regulation by NS2, the Viral VP2 gene was replaced by a reporter gene, firefly luciferase, in a plasmid clone of Viral sequences and the Protein Synthesis under the control of P38 was evaluated by luciferase assay. Cells were transfected with luciferase expressing plasmids and subsequently infected with wild-type H-1 or H-1SA. We were able to mimic the defect in expression that we observed in cultured cells and animals with virus infection. Luciferase activity in H-1SA-infected rat cells was about 10-fold lower than that in H-1 -infected rat cells, but only 2-fold lower or less in H-1SA-infected human cells and hamster cells compared to wild-type H-1. These results are consistent with our previous data that NS2 has a host-range phenotype in the natural host of H-1, the rat. Deletion of 5′ UTR sequences from P38 transcripts reduced the overall P38-luc expression but expression was NS2 independent, whereas deletion of the terminal 3′ UTR sequences of Viral RNA reduced NS2-dependent expression in rat cells. These results suggest that the regulation of Viral Protein Synthesis by NS2 depends on RNA sequences in the 3′ UTR.
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nonstructural Protein ns2 of parvovirus h 1 is required for efficient Viral Protein Synthesis and virus production in rat cellsin vivo andin vitro
Virology, 1991Co-Authors: Xu Li, Solon L RhodeAbstract:Abstract We generated a mutation in the gene for the nonstructural Protein NS2 of parvovirus H-1 in which the highly conserved dinucleotide AG at the 3′ splice acceptor site of NS2 intron 1 was mutated to CG. The mutation does not change the amino acid sequence for NS1. The splice acceptor (SA) mutant gene was introduced into the H-1 virus (H-1 SA) and an infectious clone of Will (pLuH1 SA). The R2 transcripts encoding NS2 were absent by both Northern blot and primer extension analysis in the LuH1 SA or H-1 SA virus-infected cells and the NS2 Protein was undetectable in the infected cell lysate by immunoprecipitation. These NS2 null mutant viruses were capable of lytic growth in cell lines that were derived from human, hamster, and dog, but they produced lower virus titers than wild-type H-1. The H-1SA virus nonproductively infected Rat2 rat fibroblasts and transformed Rat2 cell lines. Analysis of synchronized infections of rat fibroblasts demonstrated that H-1 SA Viral duplex replicatioe form DNA replication was reduced and that single-stranded progeny DNA was deficient compared to wild-type H-1. In addition, H-1 SA Viral Protein Synthesis was about 10% of wild-type virus and virions were not detectable in rat fibroblasts. However, H-1 SA mRNAs R1 and R3 accumulated to wild-type levels. NS2 was also required for productive infection in newborn rats but not in newborn hamsters. These results indicate that NS2 plays an important role in the regulation of Viral Protein Synthesis in rat cells in vivo and in vitro .
Patrick D Slaine - One of the best experts on this subject based on the ideXlab platform.
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stress granule inducing eukaryotic translation initiation factor 4a inhibitors block influenza a virus replication
Viruses, 2017Co-Authors: Patrick D Slaine, Mariel Klee, Natha K Smith, Denys A Khaperskyy, Craig MccormickAbstract:Eukaryotic translation initiation factor 4A (eIF4A) is a helicase that facilitates assembly of the translation preinitiation complex by unwinding structured mRNA 5' untranslated regions. Pateamine A (PatA) and silvestrol are natural products that disrupt eIF4A function and arrest translation, thereby triggering the formation of cytoplasmic aggregates of stalled preinitiation complexes known as stress granules (SGs). Here we examined the effects of eIF4A inhibition by PatA and silvestrol on influenza A virus (IAV) Protein Synthesis and replication in cell culture. Treatment of infected cells with either PatA or silvestrol at early times post-infection resulted in SG formation, arrest of Viral Protein Synthesis and failure to replicate the Viral genome. PatA, which irreversibly binds to eIF4A, sustained long-term blockade of IAV replication following drug withdrawal, and inhibited IAV replication at concentrations that had minimal cytotoxicity. By contrast, the antiViral effects of silvestrol were fully reversible; drug withdrawal caused rapid SG dissolution and resumption of Viral Protein Synthesis. IAV inhibition by silvestrol was invariably associated with cytotoxicity. PatA blocked replication of genetically divergent IAV strains, suggesting common dependence on host eIF4A activity. This study demonstrates that the core host Protein Synthesis machinery can be targeted to block Viral replication.
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stress granule inducing eukaryotic translation initiation factor 4a inhibitors block influenza a virus replication
bioRxiv, 2017Co-Authors: Patrick D Slaine, Mariel Klee, Natha K Smith, Denys A Khaperskyy, Craig MccormickAbstract:Eukaryotic translation initiation factor 4A (eIF4A) is a helicase that facilitates assembly of the translation preinitiation complex by unwinding structured mRNA 5-prime untranslated regions. Pateamine A (PatA) and silvestrol are natural products that disrupt eIF4A function and arrest translation, thereby triggering the formation of cytoplasmic aggregates of stalled preinitiation complexes known as stress granules (SGs). Here we examined the effects of eIF4A inhibition by PatA and silvestrol on influenza A virus (IAV) Protein Synthesis and replication in cell culture. Treatment of infected cells with either PatA or silvestrol at early times post-infection results in SG formation, arrest of Viral Protein Synthesis and failure to replicate the Viral genome. PatA, which irreversibly binds to eIF4A, sustained long-term blockade of IAV replication following drug withdrawal, and inhibited IAV replication at concentrations that had minimal cytotoxicity. By contrast, the antiViral effects of silvestrol were fully reversible; drug withdrawal caused rapid SG dissolution and resumption of Viral Protein Synthesis. IAV inhibition by silvestrol was invariably associated with cytotoxicity. PatA blocked replication of genetically divergent IAV strains, suggesting common dependence on host eIF4A activity. This study demonstrates the feasibility of targeting core host Protein Synthesis machinery to prevent Viral replication.
