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1.
PLoS Pathog ; 15(5): e1007798, 2019 05.
Article in English | MEDLINE | ID: mdl-31116799

ABSTRACT

Cellular antiviral programs encode molecules capable of targeting multiple steps in the virus lifecycle. Zinc-finger antiviral protein (ZAP) is a central and general regulator of antiviral activity that targets pathogen mRNA stability and translation. ZAP is diffusely cytoplasmic, but upon infection ZAP is targeted to particular cytoplasmic structures, termed stress granules (SGs). However, it remains unclear if ZAP's antiviral activity correlates with SG localization, and what molecular cues are required to induce this localization event. Here, we use Sindbis virus (SINV) as a model infection and find that ZAP's localization to SGs can be transient. Sometimes no apparent viral infection follows ZAP SG localization but ZAP SG localization always precedes accumulation of SINV non-structural protein, suggesting virus replication processes trigger SG formation and ZAP recruitment. Data from single-molecule RNA FISH corroborates this finding as the majority of cells with ZAP localization in SGs contain low levels of viral RNA. Furthermore, ZAP recruitment to SGs occurred in ZAP-expressing cells when co-cultured with cells replicating full-length SINV, but not when co-cultured with cells replicating a SINV replicon. ZAP recruitment to SGs is functionally important as a panel of alanine ZAP mutants indicate that the anti-SINV activity is correlated with ZAP's ability to localize to SGs. As ZAP is a central component of the cellular antiviral programs, these data provide further evidence that SGs are an important cytoplasmic antiviral hub. These findings provide insight into how antiviral components are regulated upon virus infection to inhibit virus spread.


Subject(s)
Alphavirus Infections/prevention & control , Antiviral Agents/pharmacology , Cytoplasmic Granules/metabolism , RNA-Binding Proteins/pharmacology , Sindbis Virus/pathogenicity , Stress, Physiological , Virus Replication/drug effects , Alphavirus Infections/metabolism , Alphavirus Infections/virology , Antiviral Agents/metabolism , Bone Neoplasms/drug therapy , Bone Neoplasms/metabolism , Bone Neoplasms/virology , Humans , Osteosarcoma/drug therapy , Osteosarcoma/metabolism , Osteosarcoma/virology , Protein Transport , RNA Interference , RNA, Messenger/genetics , RNA, Messenger/metabolism , RNA-Binding Proteins/metabolism , Tumor Cells, Cultured
2.
J Hepatol ; 59(2): 336-43, 2013 Aug.
Article in English | MEDLINE | ID: mdl-23542347

ABSTRACT

BACKGROUND & AIMS: Very low density lipoproteins (VLDLs) are triacylglycerol (TG)-rich lipoproteins produced by the human liver. VLDLs derive the majority of their TG cargo from the lipolysis of TG stored in hepatocellular lipid droplets (LDs). Important roles for LDs and the VLDL secretory pathway in the cell culture production of infectious hepatitis C virus (HCV) have been established. We hypothesized that TG lipolysis and VLDL production are impaired during HCV infection so that these cellular processes can be diverted towards HCV production. METHODS: We used an HCV permissive cell culture system (JFH-1/HuH7.5 cells) to examine the relationship between TG lipolysis, VLDL assembly, and the HCV lifecycle using standard biochemical approaches. RESULTS: Lipolysis of cellular TG and VLDL production were impaired in HCV infected cells during the early peak of viral infection. This was partially explained by an apparent deficiency of a putative TG lipase, arylacetamide deacetylase (AADAC). The re-introduction of AADAC to infected cells restored cellular TG lipolysis, indicating a role for HCV-mediated downregulation of AADAC in this process. Defective lipolysis of cellular TG stores and VLDL production were also observed in HuH7.5 cells stably expressing a short hairpin RNA targeting AADAC expression, proving AADAC deficiency contributes to these defective pathways. Finally, impaired production of HCV was observed with AADAC knockdown cells, demonstrating a role for AADAC in the HCV lifecycle. CONCLUSIONS: This insight into the biology of HCV infection and possibly pathogenesis identifies AADAC as a novel and translationally relevant therapeutic target.


Subject(s)
Carboxylic Ester Hydrolases/metabolism , Hepacivirus/physiology , Lipoproteins, VLDL/metabolism , Triglycerides/metabolism , Apolipoproteins B/metabolism , Carboxylic Ester Hydrolases/antagonists & inhibitors , Carboxylic Ester Hydrolases/genetics , Cell Line , Gene Knockdown Techniques , Hepacivirus/growth & development , Hepacivirus/pathogenicity , Host-Pathogen Interactions , Humans , Lipolysis , Models, Biological , Virulence , Virus Replication
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