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Herpes simplex virus 1 protein kinase US3 hyperphosphorylates p65/RelA and dampens NF-?B activation.

ABSTRACT: Nuclear factor ?B (NF-?B) plays important roles in innate immune responses by regulating the expression of a large number of target genes involved in the immune and inflammatory response, apoptosis, cell proliferation, differentiation, and survival. To survive in the host cells, viruses have evolved multiple strategies to evade and subvert the host immune response. Herpes simplex virus 1 (HSV-1) bears a large DNA genome, with the capacity to encode many different viral proteins to counteract the host immune responses. In the present study, we demonstrated that HSV-1 protein kinase US3 significantly inhibited NF-?B activation and decreased the expression of inflammatory chemokine interleukin-8 (IL-8). US3 was also shown to hyperphosphorylate p65 at serine 75 and block its nuclear translocation. Two US3 mutants, K220M and D305A, still interacted with p65; however, they could not hyperphosphorylate p65, indicating that the kinase activity of US3 was indispensable for the function. The attenuation of NF-?B activation by HSV-1 US3 protein kinase may represent a critical adaptation to enable virus persistence within the host. Importance: This study demonstrated that HSV-1 protein kinase US3 significantly inhibited NF-?B activation and decreased the expression of inflammatory chemokine interleukin-8 (IL-8). US3 hyperphosphorylated p65 at serine 75 to inhibit NF-?B activation. The kinase activity of US3 was indispensable for its hyperphosphorylation of p65 and abrogation of the nuclear translocation of p65. The present study elaborated a novel mechanism of HSV-1 US3 to evade the host innate immunity.

SUBMITTER: Wang K

PROVIDER: S-EPMC4097809 | biostudies-literature | 2014 Jul

REPOSITORIES: biostudies-literature

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Herpes simplex virus 1 protein kinase US3 hyperphosphorylates p65/RelA and dampens NF-κB activation.

Wang Kezhen K Ni Liwen L Wang Shuai S Zheng Chunfu C

Journal of virology 20140507 14

Nuclear factor κB (NF-κB) plays important roles in innate immune responses by regulating the expression of a large number of target genes involved in the immune and inflammatory response, apoptosis, cell proliferation, differentiation, and survival. To survive in the host cells, viruses have evolved multiple strategies to evade and subvert the host immune response. Herpes simplex virus 1 (HSV-1) bears a large DNA genome, with the capacity to encode many different viral proteins to counteract the ...[more]

PMID: 24807716

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Project description:The NF-κB signaling pathway is central to the innate and adaptive immune responses. Upon their detection of pathogen-associated molecular patterns, Toll-like receptors on the cell surface initiate signal transduction and activate the NF-κB pathway, leading to the production of a wide array of inflammatory cytokines, in attempt to eradicate the invaders. As a countermeasure, pathogens have evolved ways to subvert and manipulate this system to their advantage. Enteropathogenic and enterohemorrhagic Escherichia coli (EPEC and EHEC) are closely related bacteria responsible for major food-borne diseases worldwide. Via a needle-like protein complex called the type three secretion system (T3SS), these pathogens deliver virulence factors directly to host cells and modify cellular functions, including by suppressing the inflammatory response. Using gain- and loss-of-function screenings, we identified two bacterial effectors, NleC and NleE, that down-regulate the NF-κB signal upon being injected into a host cell via the T3SS. A recent report showed that NleE inhibits NF-κB activation, although an NleE-deficient pathogen was still immune-suppressive, indicating that other anti-inflammatory effectors are involved. In agreement, our present results showed that NleC was also required to inhibit inflammation. We found that NleC is a zinc protease that disrupts NF-κB activation by the direct cleavage of NF-κB's p65 subunit in the cytoplasm, thereby decreasing the available p65 and reducing the total nuclear entry of active p65. More importantly, we showed that a mutant EPEC/EHEC lacking both NleC and NleE (ΔnleC ΔnleE) caused greater inflammatory response than bacteria carrying ΔnleC or ΔnleE alone. This effect was similar to that of a T3SS-defective mutant. In conclusion, we found that NleC is an anti-inflammatory bacterial zinc protease, and that the cooperative function of NleE and NleC disrupts the NF-κB pathway and accounts for most of the immune suppression caused by EHEC/EPEC.

Project description:Background: Capsids of herpes simplex virus 1 (HSV-1) are assembled in the nucleus, translocated either to the perinuclear space by budding at the inner nuclear membrane acquiring tegument and envelope, or released to the cytosol in a "naked" state via impaired nuclear pores that finally results in impairment of the nuclear envelope. The Us3 gene encodes a protein acting as a kinase, which is responsible for phosphorylation of numerous viral and cellular substrates. The Us3 kinase plays a crucial role in nucleus to cytoplasm capsid translocation. We thus investigate the nuclear surface in order to evaluate the significance of Us3 in maintenance of the nuclear envelope during HSV-1 infection. Methods: To address alterations of the nuclear envelope and capsid nucleus to cytoplasm translocation related to the function of the Us3 kinase we investigated cells infected with wild type HSV-1 or the Us3 deletion mutant R7041(∆Us3) by transmission electron microscopy, focused ion-beam electron scanning microscopy, cryo-field emission scanning electron microscopy, confocal super resolution light microscopy, and polyacrylamide gel electrophoresis. Results: Confocal super resolution microscopy and cryo-field emission scanning electron microscopy revealed decrement in pore numbers in infected cells. Number and degree of pore impairment was significantly reduced after infection with R7041(∆Us3) compared to infection with wild type HSV-1. The nuclear surface was significantly enlarged in cells infected with any of the viruses. Morphometric analysis revealed that additional nuclear membranes were produced forming multiple folds and caveolae, in which virions accumulated as documented by three-dimensional reconstruction after ion-beam scanning electron microscopy. Finally, significantly more R7041(∆Us3) capsids were retained in the nucleus than wild-type capsids whereas the number of R7041(∆Us3) capsids in the cytosol was significantly lower. Conclusions: The data indicate that Us3 kinase is involved in facilitation of nuclear pore impairment and, concomitantly, in capsid release through impaired nuclear envelope.

Project description:BackgroundThe NF-κB family of transcription factors and associated signalling pathways are abundant and ubiquitous in human immune responses. Activation of NF-κB transcription factors by viral pathogen-associated molecular patterns, such as viral RNA and DNA, is fundamental to anti-viral innate immune defences and pro-inflammatory cytokine production that steers adaptive immune responses. Diverse non-viral stimuli, such as lipopolysaccharide and cytokines, also activate NF-κB and the same anti-pathogen gene networks. Viruses adapted to human cells often encode multiple proteins targeting the NF-κB pathway to mitigate the anti-viral effects of NF-κB-dependent host immunity.ResultsIn this study we have demonstrated using a variety of assays, in a number of different cell types including primary cells, that plasmid-encoded or virus-delivered simian immunodeficiency virus (SIV) accessory protein Vpx is a broad antagonist of NF-κB signalling active against diverse innate NF-κB agonists. Using targeted Vpx mutagenesis, we showed that this novel Vpx phenotype is independent of known Vpx cofactor DCAF1 and other cellular binding partners, including SAMHD1, STING and the HUSH complex. We found that Vpx co-immunoprecipitated with canonical NF-κB transcription factor p65, but not NF-κB family members p50 or p100, preventing nuclear translocation of p65. We found that broad antagonism of NF-κB activation by Vpx was conserved across distantly related lentiviruses as well as for Vpr from SIV Mona monkey (SIVmon), which has Vpx-like SAMHD1-degradation activity.ConclusionsWe have discovered a novel mechanism by which lentiviruses antagonise NF-κB activation by targeting p65. These findings extend our knowledge of how lentiviruses manipulate universal regulators of immunity to avoid the anti-viral sequelae of pro-inflammatory gene expression stimulated by both viral and extra-viral agonists. Importantly our findings are also relevant to the gene therapy field where virus-like particle associated Vpx is routinely used to enhance vector transduction through antagonism of SAMHD1, and perhaps also through manipulation of NF-κB.

Dataset Information

Herpes simplex virus 1 protein kinase US3 hyperphosphorylates p65/RelA and dampens NF-?B activation.

Publications

Herpes simplex virus 1 protein kinase US3 hyperphosphorylates p65/RelA and dampens NF-κB activation.

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