Pseudorabies Virus DNA Polymerase Processivity Factor UL42 Inhibits Type I IFN Response by Preventing ISGF3-ISRE Interaction.
Zhang Rui,Chen Shifan,Zhang Ying,Wang Mengdong,Qin Chao,Yu Cuilian,Zhang Yunfan,Li Yue,Chen Liankai,Zhang Xinrui,Yuan Xiufang,Tang Jun
Journal of immunology (Baltimore, Md. : 1950)
Alphaherpesviruses are large dsDNA viruses with an ability to establish persistent infection in hosts, which rely partly on their ability to evade host innate immune responses, notably the type I IFN response. However, the relevant molecular mechanisms are not well understood. In this study, we report the UL42 proteins of alphaherpesvirus pseudorabies virus (PRV) and HSV type 1 (HSV1) as a potent antagonist of the IFN-I-induced JAK-STAT signaling pathway. We found that ectopic expression of UL42 in porcine macrophage CRL and human HeLa cells significantly suppresses IFN-α-mediated activation of the IFN-stimulated response element (ISRE), leading to a decreased transcription and expression of IFN-stimulated genes (ISGs). Mechanistically, UL42 directly interacts with ISRE and interferes with ISG factor 3 (ISGF3) from binding to ISRE for efficient gene transcription, and four conserved DNA-binding sites of UL42 are required for this interaction. The substitution of these DNA-binding sites with alanines results in reduced ISRE-binding ability of UL42 and impairs for PRV to evade the IFN response. Knockdown of UL42 in PRV remarkably attenuates the antagonism of virus to IFN in porcine kidney PK15 cells. Our results indicate that the UL42 protein of alphaherpesviruses possesses the ability to suppress IFN-I signaling by preventing the association of ISGF3 and ISRE, thereby contributing to immune evasion. This finding reveals UL42 as a potential antiviral target.
10.4049/jimmunol.2001306
Host Interferon-Stimulated Gene 20 Inhibits Pseudorabies Virus Proliferation.
Virologica Sinica
Host interferon-stimulated gene 20 (ISG20) exerts antiviral effects on viruses by degrading viral RNA or by enhancing IFN signaling. Here, we examined the role of ISG20 during pseudorabies virus (PRV) proliferation. We found that ISG20 modulates PRV replication by enhancing IFN signaling. Further, ISG20 expression was upregulated following PRV infection and poly(I:C) treatment. Ectopic expression of ISG20 inhibited PRV proliferation in PK15 cells, whereas knockdown of ISG20 promoted PRV proliferation. In addition, ISG20 expression upregulated IFN-β expression and enhanced IFN downstream signaling during PRV infection. Notably, PRV UL24 suppressed the transcription of ISG20, thus antagonizing its antiviral effect. Further domain mapping analysis showed that the N terminus (amino acids 1-90) of UL24 was responsible for the inhibition of ISG20 transcription. Collectively, these findings characterize the role of ISG20 in suppressing PRV replication and increase the understanding of host-PRV interplay.
10.1007/s12250-021-00380-0
A monoclonal antibody neutralizes pesudorabies virus by blocking gD binding to the receptor nectin-1.
Zhang Teng,Liu Yunchao,Chen Yumei,Wang Jucai,Feng Hua,Wei Qiang,Zhao Shuangshuang,Yang Suzhen,Liu Dongmin,Zhang Gaiping
International journal of biological macromolecules
Pseudorabies virus (PRV) was isolated from some human cases recently and the infected patients manifested respiratory dysfunction and acute neurological symptoms. However, no effective drug or vaccine, preventing the progression of PRV infection, is available. Nectin-1 was the only reported receptor for PRV cell entry both swine and human origin, representing an excellent target to block PRV infection, and especially its transmission from pigs to humans. A PRV-gD specific mAbs (10B6) was isolated from hybridomas and its neutralizing activities in vitro and in vivo were determined. 10B6 exhibited effective neutralizing activities in vitro with IC = 2.514 μg/ml and 4.297 μg/ml in the presence and absence of complement. And in vivo, 10B6 provided 100% protection against PRV lethal challenge with a dose of 15 mg/kg. Further, 10B6 could bind to a conserved epitope, QPAEPFP, locating in gD pro-fusion domain, and finally blocks the binding of PRV-gD to nectin-1. Moreover, 10B6 showed an effective inhibition on PRV cell-attachment in a cell type-independent manner and could also block the virus spreading among cells. 10B6 exhibited effectively neutralizing activities to Chinese PRV variant strain in vitro and in vivo by blocking gD binding to nectin-1, implied both prophylactic and therapeutic interventions against PRV infections.
10.1016/j.ijbiomac.2021.07.170
A Novel Human Acute Encephalitis Caused by Pseudorabies Virus Variant Strain.
Liu Qingyun,Wang Xiaojuan,Xie Caihua,Ding Shifang,Yang Hongna,Guo Shibang,Li Jixuan,Qin Lingzhi,Ban Fuguo,Wang Dongfang,Wang Cui,Feng Lingxiao,Ma Haichang,Wu Bin,Zhang Liping,Dong Changxian,Xing Li,Zhang Jiewen,Chen Huanchun,Yan Ruoqian,Wang Xiangru,Li Wei
Clinical infectious diseases : an official publication of the Infectious Diseases Society of America
BACKGROUND:Pseudorabies virus (PRV) is a common pathogen in multiple animal species, particularly in pigs. However, PRV infection in humans is rare and, to the best of our knowledge, PRV has never been isolated from human cases before. METHODS:Four acute encephalitis cases in humans were confirmed as PRV infection based on clinical symptoms, laboratory diagnosis, and metagenomic next-generation sequencing (mNGS). Cerebrospinal fluid (CSF) samples were collected and applied for virus isolation. Etiological and genetic characteristics of this PRV human isolate were further determined. RESULTS:The patients manifested respiratory dysfunction and acute neurological symptoms. The mNGS revealed PRV-specific nucleotide sequences in patients' CSF samples (7-6198 reads and 0.2446%-80.58% coverage). The PRV envelope glycoprotein B antibody, glycoprotein E antibody, and neutralizing antibody were positively detected. For the first time, a PRV strain, designated hSD-1/2019, was isolated and identified from a CSF sample, and transmission electron microscopy revealed that hSD-1/2019 had typical morphology similar to that of swine PRV. Phylogenetic analysis illustrated that hSD-1/2019 was genetically closest to those PRV variant strains currently circulating in pigs in China, and this strain showed similar etiological characteristics to Chinese PRV variant strains, while different from Chinese classical strain. Moreover, hSD-1/2019 showed high pathogenicity and induced acute neurological symptoms in pigs. CONCLUSIONS:A PRV strain was isolated from an acute human encephalitis case. This isolate showed close phylogenetic relationships and similar etiological characteristics to Chinese PRV variant strains, implying the great risk of PRV transmission from pigs to humans.
10.1093/cid/ciaa987
PRV-encoded UL13 protein kinase acts as an antagonist of innate immunity by targeting IRF3-signaling pathways.
Lv Lin,Cao Mingzhu,Bai Juan,Jin Ling,Wang Xianwei,Gao Yanni,Liu Xuewei,Jiang Ping
Veterinary microbiology
Pseudorabies virus (PRV), a porcine alphaherpesvirus, causes neurological disorders and reproductive failure in swine. It is capable of avoiding host antiviral responses, resulting in viral latency in infected animals. The mechanisms by which many PRV proteins help the virus to evade immune surveillance are poorly understood. In this study, we found that the PRV protein kinase, UL13, inhibits the IFN-β signaling pathway by targeting interferon regulatory factor 3 (IRF3) for ubiquitination and degradation. PRV with mutant of UL13 is impaired in its ability to hinder IRF3 and interferon-β (IFN-β) activation, and has significantly less pathogenesis in mice that wild-type PRV. Our findings reveal an as yet undescribed mechanism utilized by PRV to evade host immune responses. PRV UL13 is a potential target for attenuated vaccines and antiviral drugs.
10.1016/j.vetmic.2020.108860
Hydroquinone inhibits PRV infection in neurons in vitro and in vivo.
Fang Linlin,Gao Yanni,Lan Min,Jiang Ping,Bai Juan,Li Yufeng,Wang XianWei
Veterinary microbiology
Pseudorabies virus (PRV) is a prevalent and endemic swine pathogen that causes significant economic losses in the global swine industry. Due to the emergence of PRV mutant strains in recent years, vaccines can't completely prevent and control PRV infection. Therefore, research and development of new vaccines and drugs with inhibitory effects on PRV are of great significance in the prevention and treatment of PR. In this study, we firstly screened a library of 44 FDA-approved drugs and found that hydroquinone (HQ) displayed high anti-PRV activity by inhibiting PRV adsorption onto and internalization into cells. This study revealed that hydroquinone treatment stimulated genes associated with the PI3K-AKT signal pathway. HQ increased AKT mRNA production and activated AKT phosphorylation in N2a cells. This finding suggests that HQ significantly inhibits PRV replication by activating the phosphorylation of AKT. We also conducted in vivo experiments in mice. Hydroquinone significantly reduced the viral loads in mouse tissues and the mortality after PRV infection. The above results indicate that hydroquinone significantly inhibits the replication of PRV mutant strain ZJ01 in ICR mice and has an inhibitory effect on PRV. This study will contribute to the development of a novel prophylactic and therapeutic strategy against PRV infection.
10.1016/j.vetmic.2020.108864
PRV UL13 inhibits cGAS-STING-mediated IFN-β production by phosphorylating IRF3.
Bo Zongyi,Miao Yurun,Xi Rui,Zhong Qiuping,Bao Chenyi,Chen Huan,Sun Liumei,Qian Yingjuan,Jung Yong-Sam,Dai Jianjun
Veterinary research
Cyclic GMP-AMP (cGAMP) synthase (cGAS) is an intracellular sensor of cytoplasmic viral DNA created during virus infection, which subsequently activates the stimulator of interferon gene (STING)-dependent type I interferon response to eliminate pathogens. In contrast, viruses have developed different strategies to modulate this signalling pathway. Pseudorabies virus (PRV), an alphaherpesvirus, is the causative agent of Aujeszky's disease (AD), a notable disease that causes substantial economic loss to the swine industry globally. Previous reports have shown that PRV infection induces cGAS-dependent IFN-β production, conversely hydrolysing cGAMP, a second messenger synthesized by cGAS, and attenuates PRV-induced IRF3 activation and IFN-β secretion. However, it is not clear whether PRV open reading frames (ORFs) modulate the cGAS-STING-IRF3 pathway. Here, 50 PRV ORFs were screened, showing that PRV UL13 serine/threonine kinase blocks the cGAS-STING-IRF3-, poly(I:C)- or VSV-mediated transcriptional activation of the IFN-β gene. Importantly, it was discovered that UL13 phosphorylates IRF3, and its kinase activity is indispensable for such an inhibitory effect. Moreover, UL13 does not affect IRF3 dimerization, nuclear translocation or association with CREB-binding protein (CBP) but attenuates the binding of IRF3 to the IRF3-responsive promoter. Consistent with this, it was discovered that UL13 inhibits the expression of multiple interferon-stimulated genes (ISGs) induced by cGAS-STING or poly(I:C). Finally, it was determined that PRV infection can activate IRF3 by recruiting it to the nucleus, and PRVΔUL13 mutants enhance the transactivation level of the IFN-β gene. Taken together, the data from the present study demonstrated that PRV UL13 inhibits cGAS-STING-mediated IFN-β production by phosphorylating IRF3.
10.1186/s13567-020-00843-4
Identifying Cellular and Viral Factor Recruitment to Herpes Simplex Virus Type 1 Replication Forks.
FASEB journal : official publication of the Federation of American Societies for Experimental Biology
Herpes simplex virus type 1 (HSV-1) is a ubiquitous pathogen that replicates the 152kbp viral genome within the nucleus of host cells. Replication of the HSV-1 genome is catalyzed by viral replication machinery consisting of a DNA polymerase (UL30), processivity factor (UL42), helicase-primase complex, origin binding protein (UL9), and a single stranded DNA binding protein. Despite encoding a viral DNA polymerase processivity factor, we found that host Proliferating Cell Nuclear Antigen (PCNA) interacts with HSV-1 DNA at replication forks and associates with viral DNA in a replication-dependent manner. PCNA is a homotrimer that provides processivity to cellular DNA polymerases and selectively recruits DNA damage response and DNA repair factors to cellular replication forks. We therefore hypothesized that PCNA associates at viral replication forks to promote HSV-1 replication while also tethering cellular DNA repair proteins to replicating viral DNA. To test this, we used two models to identify how associated factors change as a function of PCNA: a known PCNA inhibitor, PCNA-I1, and a cell line that was engineered to inducibly express a shRNA targeting PCNA. We performed a technique adapted from isolation of proteins on nascent DNA (iPOND) to isolate HSV-1 DNA from infected cells and identified associated viral and cellular proteins. Cells were infected and replicating viral DNA was selectively labeled with EdC. Labeled DNA was specifically and irreversibly tagged via the covalent attachment of biotin azide via click reaction. Biotin-tagged DNA was purified on streptavidin-coated beads and associated proteins were eluted and identified by mass spectrometry. As a complimentary approach, we also performed immunofluorescence (IF) to confirm iPOND data. We found that PCNA is associated with viral DNA despite treatment with PCNA-I1, an observation that is consistent with IF imaging data. Viral replication proteins UL30, UL42, and UL9 decreased in the presence of PCNA-I1. DNA damage response proteins such as mismatch repair proteins and RECQL both decreased up to 10-fold. Of note, however, proteins that make up the MRN complex, a class of proteins that functions in DNA break repair, restart of stalled replication forks, and viral DNA infection, increased in response to PCNA-I1 treatment. Given this data, PCNA may be involved in tethering viral replication proteins, cellular DNA repair proteins, and/or virion assembly proteins to viral replication forks.
10.1096/fasebj.2022.36.S1.R2044