Chlorovirus Skp1-Binding Ankyrin Repeat Protein Interplay and Mimicry of Cellular Ubiquitin Ligase Machinery

Noel, Eric A.; Kang, Ming; Adamec, Jiric; Etten, James L. Van; Oyler, George A.

doi:10.1128/jvi.02109-14

Cited by 8 publications

(6 citation statements)

References 57 publications

(70 reference statements)

Supporting

Mentioning

Contrasting

Order By: Relevance

“…This observation implies functions carried out mainly outside the factories. The latter conjecture is supported by the previous findings indicating that ankyrin repeat-containing proteins in vaccinia virus (39) and in Paramecium bursaria chlorella virus 1 (PBCV-1) virus (40) are involved in ubiquitination of host proteins in the host cytoplasm. This finding substantiates the notion suggested above that protein regulation through protein degradation is a crucial process throughout viral infection both inside and outside the viral factories.…”

Section: Resultssupporting

confidence: 77%

Efficiency in Complexity: Composition and Dynamic Nature of Mimivirus Replication Factories

Fridmann-Sirkis

Milrot

Mutsafi

et al. 2016

J Virol

View full text Add to dashboard Cite

The recent discovery of multiple giant double-stranded DNA (dsDNA) viruses blurred the consensual distinction between viruses and cells due to their size, as well as to their structural and genetic complexity. A dramatic feature revealed by these viruses as well as by many positive-strand RNA viruses is their ability to rapidly form elaborate intracellular organelles, termed "viral factories," where viral progeny are continuously generated. Here we report the first isolation of viral factories at progressive postinfection time points. The isolated factories were subjected to mass spectrometry-based proteomics, bioinformatics, and imaging analyses. These analyses revealed that numerous viral proteins are present in the factories but not in mature virions, thus implying that multiple and diverse proteins are required to promote the efficiency of viral factories as "production lines" of viral progeny. Moreover, our results highlight the dynamic and highly complex nature of viral factories, provide new and general insights into viral infection, and substantiate the intriguing notion that viral factories may represent the living state of viruses. IMPORTANCELarge dsDNA viruses such as vaccinia virus and the giant mimivirus, as well as many positive-strand RNA viruses, generate elaborate cytoplasmic organelles in which the multiple and diverse transactions required for viral replication and assembly occur. These organelles, which were termed "viral factories," are attracting much interest due to the increasing realization that the rapid and continuous production of viral progeny is a direct outcome of the elaborate structure and composition of the factories, which act as efficient production lines. To get new insights into the nature and function of viral factories, we devised a method that allows, for the first time, the isolation of these organelles. Analyses of the isolated factories generated at different times postinfection by mass spectrometry-based proteomics provide new perceptions of their role and reveal the highly dynamic nature of these organelles.A n exciting recent development in cellular biology is the realization that intracellular organelles previously considered to be randomly organized are in fact exquisitely ordered and that this order crucially affects their function. A prominent example is provided by replication cycles of positive-strand RNA [(ϩ)RNA] viruses, which were shown to involve massive reorganization of the host cytoskeleton and membrane networks into well-defined cytoplasmic platforms, termed "viral factories" (VFs), within which genome replication and virion assembly are effectively coordinated (1-4). An additional example of highly ordered virus-generated intracellular organelles is the replication cycle of nucleocytoplasmic large DNA viruses (NCLDVs), which include Poxviridae, Phycodnaviridae, Iridoviridae, Asfarviridae, Mimiviridae, and Marseilleviridae (5-7). Specifically, these viruses were shown to partially or exclusively replicate within large and elaborate cytoplasmic ...

show abstract

Section: Resultssupporting

confidence: 77%

Efficiency in Complexity: Composition and Dynamic Nature of Mimivirus Replication Factories

Fridmann-Sirkis

Milrot

Mutsafi

et al. 2016

J Virol

View full text Add to dashboard Cite

show abstract

“…As a first step in identifying such domains, 30 constructs were generated for expressing N-terminally Flag-tagged versions of either protein lacking the N-terminus, one or every possible combination of multiple ankyrin repeats (including the potential Ank1 ankyrin repeat that aligns with the fourth ankyrin repeat of Ank6; S5 Fig and S1 Table ; hereafter referred to as Ank1 ankyrin repeat four), putative NES of Ank6 (or corresponding Ank1 amino acids 143 to 160), ISR, or F-box ( Fig 14 ). This approach has proven effective for identifying protein-protein interaction domains of many other microbial F-box containing Ank proteins [ 22 , 45 , 54 – 64 ]. Next, the abilities of these proteins to co-immunoprecipitate p65, importin β1, and exportin 1 were examined.…”

Section: Resultsmentioning

confidence: 99%

Orientia tsutsugamushi uses two Ank effectors to modulate NF-κB p65 nuclear transport and inhibit NF-κB transcriptional activation

Evans¹,

Rodino²,

Adcox³

et al. 2018

PLoS Pathog

View full text Add to dashboard Cite

Orientia tsutsugamushi causes scrub typhus, a potentially fatal infection that threatens over one billion people. Nuclear translocation of the transcription factor, NF-κB, is the central initiating cellular event in the antimicrobial response. Here, we report that NF-κB p65 nuclear accumulation and NF-κB-dependent transcription are inhibited in O. tsutsugamushi infected HeLa cells and/or primary macrophages, even in the presence of TNFα. The bacterium modulates p65 subcellular localization by neither degrading it nor inhibiting IκBα degradation. Rather, it exploits host exportin 1 to mediate p65 nuclear export, as this phenomenon is leptomycin B-sensitive. O. tsutsugamushi antagonizes NF-κB-activated transcription even when exportin 1 is inhibited and NF-κB consequently remains in the nucleus. Two ankyrin repeat-containing effectors (Anks), Ank1 and Ank6, each of which possess a C-terminal F-box and exhibit 58.5% amino acid identity, are linked to the pathogen’s ability to modulate NF-κB. When ectopically expressed, both translocate to the nucleus, abrogate NF-κB-activated transcription in an exportin 1-independent manner, and pronouncedly reduce TNFα-induced p65 nuclear levels by exportin 1-dependent means. Flag-tagged Ank 1 and Ank6 co-immunoprecipitate p65 and exportin 1. Both also bind importin β1, a host protein that is essential for the classical nuclear import pathway. Importazole, which blocks importin β1 activity, abrogates Ank1 and Ank6 nuclear translocation. The Ank1 and Ank6 regions that bind importin β1 also mediate their transport into the nucleus. Yet, these regions are distinct from those that bind p65/exportin 1. The Ank1 and Ank6 F-box and the region that lies between it and the ankyrin repeat domain are essential for blocking p65 nuclear accumulation. These data reveal a novel mechanism by which O. tsutsugamushi modulates the activity and nuclear transport of NF-κB p65 and identify the first microbial proteins that co-opt both importin β1 and exportin 1 to antagonize a critical arm of the antimicrobial response.

show abstract

“…numerous viruses (particularly poxviruses) (39,42,(47)(48)(49)(50)(51)(52)(53)(54)(55)(56)(57)(58)(59); various plant bacterial pathogens (22,60,61); and several intracellular bacteria that infect mammals, such as Legionella pneumophila (20-22, 31, 34, 35), Coxiella burnetii (22,32,37), Salmonella enterica serovar Typhimurium (36), and Chlamydia spp. (22,32,33).…”

Section: Discussionmentioning

confidence: 99%

Orientia tsutsugamushi Strain Ikeda Ankyrin Repeat-Containing Proteins Recruit SCF1 Ubiquitin Ligase Machinery via Poxvirus-Like F-Box Motifs

et al. 2015

View full text Add to dashboard Cite

A rising theme among intracellular microbes is the delivery of ankyrin repeat-containing effectors (Anks) that interact with target proteins to co-opt host cell functions. Orientia tsutsugamushi, an obligate intracellular bacterium and the etiologic agent of scrub typhus, encodes one of the largest Ank repertoires of any sequenced microorganism. They have been previously identified as type 1 secretion system substrates. Here, in silico and manual sequence analyses revealed that a large proportion of O. tsutsugamushi strain Ikeda Anks bear a eukaryotic/poxvirus-like F-box motif, which is known to recruit host cell SCF1 ubiquitin ligase machinery. We assessed the Anks for the ability to serve as F-box proteins. Coimmunoprecipitation assays demonstrated that F-box-containing Anks interact with overexpressed and/or endogenous SCF1 components. When coexpressed with FLAGAnk4_01 or FLAG-Ank9, a glutathione S-transferase (GST)-tagged version of the SCF1 component SKP1 localized to subcellular sites of FLAG-Ank accumulation. The abilities of recombinant Anks to interact and colocalize with SKP1 were F-box dependent. GST-SKP1 precipitated O. tsutsugamushi-derived Ank9 from infected host cells, verifying both that the pathogen expresses Ank9 during infection and the protein's capability to bind SKP1. Aligning O. tsutsugamushi, poxviral, and eukaryotic F-box sequences delineated three F-box residues that are highly conserved and likely to be functionally important. Substitution of these residues ablated the ability of GFP-Ank9 to interact with GST-SKP1. These results demonstrate that O. tsutsugamushi strain Ikeda Anks can co-opt host cell polyubiquitination machinery, provide the first evidence that an O. tsutsugamushi Ank does so during infection, and advance overall understanding of microbial F-box proteins. IMPORTANCEAnkyrin repeat-containing proteins (Anks) are important virulence factors of intracellular bacteria that mediate protein-protein interactions with host cell targets. Orientia tsutsugamushi, which causes a debilitating infection called scrub typhus in one of the most densely populated regions of the world, encodes one of the largest Ank armamentariums of any sequenced bacterium. This study demonstrates that O. tsutsugamushi strain Ikeda Anks also bear F-box motifs that interact with host cell polyubiquitination machinery. By proving that an Orientia-derived Ank interacts with SKP1 in infected cells, this evidences the first bona fide Orientia effector and the first example of an endogenous F-box-containing Ank-mammalian-host ligand interaction for any intracellular bacterium. Also, importantly, this work identifies key residues that are essential for microbial F-box function.T he obligate intracellular bacterium Orientia tsutsugamushi is the causative agent of scrub typhus, a potentially fatal disease transmitted to humans through the bite of an infected trombiculid mite. O. tsutsugamushi invades phagocytic cells of the immune system, as well as endothelial cells of the skin and major organs (1-4). Di...

show abstract

Chlorovirus Skp1-Binding Ankyrin Repeat Protein Interplay and Mimicry of Cellular Ubiquitin Ligase Machinery

Cited by 8 publications

References 57 publications

Efficiency in Complexity: Composition and Dynamic Nature of Mimivirus Replication Factories

Efficiency in Complexity: Composition and Dynamic Nature of Mimivirus Replication Factories

Orientia tsutsugamushi uses two Ank effectors to modulate NF-κB p65 nuclear transport and inhibit NF-κB transcriptional activation

Orientia tsutsugamushi Strain Ikeda Ankyrin Repeat-Containing Proteins Recruit SCF1 Ubiquitin Ligase Machinery via Poxvirus-Like F-Box Motifs

Contact Info

Product

Resources

About