Staphylococcus aureus (S. aureus) is one of major pathogens in animal production. S. aureus-induced mastitis poses a serious threat to animal health and leads to significant economic losses. The objective of this study was to isolate and characterize bacteriophage(s) specific to S. aureus and assess its (their) in vitro antibacterial activity. A strain of S. aureus bacteriophage, designated JXAU-SA-P1, was isolated from sewage in a commercial pig farm. Morphological analysis revealed that JXAU-SA-P1 belongs to the Caudovimetes class. The genome of JXAU-SA-P1 is 134.23 kb in length with a GC content of 29.99

Virus infection activates the host's innate immune responses, which is a very precise and complex biological process and will lead to the immediate transcription of type I interferon. The general transcriptional activator proteins such as IRF3, ATF2/c-Jun, and NF-κB can be induced to form a stable enhanceosome in the transcriptional regulatory region of IFN-β promoter. Several cellular factors have recently been reported to be involved in the transcriptional regulation of IFN-β under certain physiological conditions. Here, we identified four and a half LIM domains protein 2 (FHL2) as an interacting protein of the Human Cytomegalovirus (HCMV) replication-related protein UL84 and determined that FHL2 plays an architectural role in enhancing the transcription of IFN-β induced by HCMV infection and many other viruses. Firstly, after thevirus binds to the host cell, the signal is transmitted to protein kinases, causing the cytoplasmic FHL2 to be phosphorylated and translocated into the nucleus. Then, the phosphorylated FHL2 promotes the formation of the transcription preinitiation complex (PIC) of the IFN-β promoter. Simultaneously, FHL2 is also crucial for the recruitment of TFIID to the TATA-box for initial transcription. Interestingly, during HCMV infection, HCMV replication-related protein UL84 was determined to interact with FHL2 to help the virus evade innate immune response and promote viral lytic origin (oriLyt) dependent DNA replication. Our results highlight the FHL2 as part of a signaling cascade during viral invasion, and its important regulatory effect in type I interferon synthesis, as well as provide theoretical support for the development of candidate anti-HCMV drugs acting specifically on a novel UL84 target.

High-throughput yeast two-hybrid (YTH) screening systems are essential technologies for understanding the viral life cycle, pathogenesis, and development of antiviral therapies. They are commonly used in molecular virology to identify and analyze the complex molecular interactions between viruses and host cells. This chapter details the relevant information and experimental procedures for systematically screening and identifying interactions between viral and host proteins via high-throughput YTH technology.

The mortality rate of cardiovascular and cerebrovascular diseases ranks first among all causes. This study elucidated the role and potential mechanism of the NLRC5 gene in atherosclerosis (AS). We enrolled patients (number = 30) diagnosed with AS and healthy volunteers (number = 30) as controls from our hospital. In patients with AS, the levels of serum NLRC5 were up-regulated (Fig. 1A) and positively correlated with CIMT/CRP. In a mouse model of AS, the expression of serum NLRC5 mRNA was increased at 6 or 12 weeks after inducing AS. The expression of NLRC5 protein was found to be elevated in a mouse model of AS. The inhibition of NLRC5 reduced development of AS in ApoE–/– Mice. Reducing NLRC5 inhibited the polarization of M2 macrophages and shifted macrophages towards proinflammatory M1 phenotype. STAT3 was identified as a target of NLRC5, with NLRC5 protein expression shown to reduce STAT3 ubiquitination. Methylation promoted NLRC5 DNA stability in vitro model of AS. Sh-NLRC5 increased M1/M2 macrophage ratio, foam cell formation and ox-LDL uptake. STAT3 reduced the effects of sh-NLRC5-mediated M1/M2 macrophage ratio in model of AS. These data confirmed that NLRC5 in macrophages promotes atherosclerosis in acute coronary syndrome by regulating STAT3 expression. This suggests that NLRC5 could be a potential target for the treatment of premature AS.

Yeast two-hybrid (YTH) technology is a powerful tool for studying protein interactions and has been widely used in various fields of molecular biology, including the study of antiviral innate immunity. This chapter presents detailed information and experimental procedures for identifying virus-host protein interactions involved in immune regulation using yeast two-hybrid technology.

Auxin, as a central phytohormone and signaling molecule, plays a crucial role in plant growth and development. The activity of auxin is tightly regulated by the auxin-responsive GH3 gene family. In this study, a total of 40 GH3 genes in A. thaliana, S. miltiorrhiza, and O. sativa were identified and subjected to comprehensive study. Phylogenetic analysis revealed that those GH3 genes can be classified into three distinct subgroups, with 11 pairs of paralogs identified. Genetic divergence analysis indicated that the GH3 gene family had predominantly experienced purifying selection as evidenced by the Ka/Ks ratio being less than 1 for all 11 paralogs pairs. Positive selection analysis with the site and branch-site models further suggested that SmGH3 AtGH3 and OsGH3 genes had gone through purifying selective pressure for adaptive evolution. Motif analysis indicated that group-specific motifs may contribute to functional divergence across species and subgroups. Functional divergence analysis confirmed that subgroup-specific genes have experienced functional divergence during evolution, and elucidated the molecular mechanisms underlying their divergent functions. The tissue-specific expression analysis of SmGH3 AtGH3 and OsGH3 genes revealed that these genes might perform distinct functions in different tissues. This study performed a comprehensive bioinformatics analysis of the GH3 gene family, offering valuable information to further elucidate the functional roles of GH3 genes.

Human cytomegalovirus (HCMV) is a typical opportunistic human pathogen, which can endanger the lives of individuals with immune insufficiency or low immune function. One of the most effective immune mechanisms against HCMV in host cells is the production of antiviral cytokines. Chemokines are small secreted proteins produced by cell immune responses to inflammatory stimuli or viral infection and act as potent chemoattractants for granulocytes, monocytes, lymphocytes and other leukocytes, and thus play a significant role in antiviral defence. Viruses have also evolved multiple strategies to resist the host’s immune system while coexisting with the host. In this study, based on RNA sequencing transcriptome differential analysis, we found that HCMV encoded UL23 May specifically down-regulate chemokines Chemokine ligand 2 (CCL2) and Chemokine ligand 5 (CCL5). Next, we determined that UL23 could inhibit the expression of Chemokine CCL2 and CCL5 by mainly affecting the phosphorylation of IRF-3, and then inhibited the migration of immune cells and blocked the antiviral immune responses in the migration and co-culture assays of HCMV-infected cells with immune migration-related cells. In conclusion, these results highlight that UL23 plays an important role in the immune evasion of HCMV by specially inhibiting the expression of chemokines CCL2 and CCL5, impairing the recruitment of immune cells by infected host cells and helping the virus escape immune killing.

The viral protein mutations can modify virus-host interactions during virus evolution, and thus alter the extent of infection or pathogenicity. Studies indicate that nucleocapsid (N) protein of SARS-CoV-2 participates in viral genome assembly, intracellular signal regulation and immune interference. However, its biological function in viral evolution is not well understood. SARS-CoV-2 N protein mutations were analyzed in Delta, Omicron, and original strains. Two mutations with a methionine (M) residue at site 203 and a tyrosine (Y) residue at site 377 of the N protein were found in Delta strain but not in Omicron and original strains, and promoted SARS-CoV-2 infection therein. Those mutations, R203M and D377Y, enhanced the inhibitory impact of N protein on the impairment of RIG-I-mediated antiviral signaling, such as IRF3 phosphorylation and IFN-β activation. The viral RNA-binding activity of N protein was promoted by these mutations, effectively attenuating the recognition and interaction of RIG-I with viral RNA compared to the original or other variants. The R203M/D377Y mutations thus enhanced the suppressive activity of the N protein on RIG-I-mediated interferon induction both in vitro and in vivo, which in turn promoted viral replication. This study helps to understand the variability of SARS-CoV-2 in regulating host immunity.

Human cytomegalovirus (HCMV), also known as human herpesvirus 5, infects the majority of human populations worldwide and causes a range of diseases, particularly in immunocompromised individuals. HCMV can establish life-long latency in infected hematopoietic cells, maintaining the viral genome as an episome that can reactivate to produce viral progeny upon appropriate stimulation. Understanding the establishment, maintenance, and reactivation of HCMV latency is crucial for developing targeted therapeutic strategies against HCMV infection and HCMV-induced diseases. Here, we discuss the recent advances in the mechanisms by which HCMV maintains its genome in infected cells, the cellular factors or viral antigens that modulate its reactivation, and the development of anti-HCMV therapeutics or vaccines. Overall, these insights may pave the way for the development of novel therapies that specifically and efficiently target HCMV-associated diseases.

Early studies have shown that epigenetic modifications play an important role in the establishment of latent human cytomegalovirus (HCMV) infection. However, the specific regulatory mechanisms remain unclear, especially regarding how HCMV overcomes these silencing effects of epigenetic modifications during viral reactivation from latency. Here, we showed that HCMV reactivation from latency is indeed regulated by histone H3K27 trimethylation. Histone H3K27 methyltransferase EZH2, the core catalytic enzyme of Polycomb Repressive Complex 2 (PRC2), inhibited HCMV reactivation from latency by silencing the transcription of HCMV major immediate early promoter (MIEP). More interestingly, our findings provided the first evidence that UL82 (pp71) can interact with EZH2, leading to its ubiquitin-dependent degradation. Further experiments showed that pp71 directly interacted with EZH2 to eliminate the effects of epigenetic modifications and promoted the reactivation from latency of the virus. Our results highlight the important role and molecular interaction mechanisms of HCMV tegument protein pp71 in elimination of the influences of histone H3K27 epigenetic modifications during the critical process of viral reactivation from latency.

Human Cytomegalovirus (HCMV) is a commonly infected double-stranded DNA virus of the β-herpesviridae subfamily that typically establishes lifelong latency or persistent infection following primary infection. The regulation of HCMV latency and reactivation is governed by the chromatin structure at the viral major immediate early promoter (MIEP) within myeloid cells. Both cellular and viral factors play a role in regulating the reactivation of latent HCMV. Recently, it has been found that severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) promotes HCMV reactivation in the clinic; however, the mechanism remains unclear. In this study, we found that SARS-CoV-2 ORF3a can activate HCMV MIEP by interacting with Yin Yang 1 (YY1), an inhibitor of MIEP. This interaction leads to YY1 ubiquitin-dependent degradation and subsequently promotes the reactivation of latent HCMV, as well as the replication and proliferation of the virus. These findings reveal the molecular mechanism underlying the interaction between SARS-CoV-2 and HCMV during co-infection, providing a new theoretical basis for future prevention and treatment strategies against the co-infection of these two viruses.

The NACHT, leucine-rich repeat, and pyrin domains-containing protein 3 (collectively known as NLRP3) inflammasome activation plays a critical role in innate immune and pathogenic microorganism infections. However, excessive activation of NLRP3 inflammasome will lead to cellular inflammation and tissue damage, and naturally it must be precisely controlled in the host. Here, we discovered that solute carrier family 25 member 3 (SLC25A3), a mitochondrial phosphate carrier protein, plays an important role in negatively regulating NLRP3 inflammasome activation. We found that SLC25A3 could interact with NLRP3, overexpression of SLC25A3 and knockdown of SLC25A3 could regulate NLRP3 inflammasome activation, and the interaction of NLRP3 and SLC25A3 is significantly boosted in the mitochondria when the NLRP3 inflammasome is activated. Our detailed investigation demonstrated that the interaction between NLRP3 and SLC25A3 disrupted the interaction of NLRP3-NEK7, promoted ubiquitination of NLRP3, and negatively regulated NLRP3 inflammasome activation. Thus, these findings uncovered a new regulatory mechanism of NLRP3 inflammasome activation, which provides a new perspective for the therapy of NLRP3 inflammasome-associated inflammatory diseases.

ABSTRACT Human cytomegalovirus (HCMV), a widely prevalent human beta-herpesvirus, establishes lifelong persistence in the host following primary infection. In healthy individuals, the virus is effectively controlled by HCMV-specific T cells and typically exhibits asymptomatic. The T cell immune response plays a pivotal role in combating HCMV infection, while HCMV employs various strategies to counteract it within the host. Previously, we reported that UL23, a tegument protein of HCMV, facilitates viral immune evasion from interferon-gamma (IFN-γ) responses, and it is well known that IFN-γ is mainly derived from T cells. However, the involvement of UL23 in viral immune evasion from T cell-mediated immunity remains unclear. Herein, we present compelling evidence that UL23 significantly enhances viral resistance against T cell-mediated cytotoxicity during HCMV infection from the co-culture assays of HCMV-infected cells with T cells. We found that IFN-γ plays a major role in regulating T cell cytotoxicity mediated by UL23. More interestingly, we demonstrated that UL23 not only regulates the IFN-γ downstream responses but also modulates the IFN-γ secretion by regulating T cell activities. Further experiments indicate that UL23 upregulates the expression and signaling of programmed death ligand 1 (PD-L1), which is responsible for inhibiting multiple aspects of T cell activities, including activation, apoptosis, and IFN-γ secretion, as determined through RNA-seq analysis and inhibitor-blocking experiments, ultimately facilitating viral replication and spread. Our findings highlight the potential role of UL23 as an alternative antagonist in suppressing T cell cytotoxicity and unveil a novel strategy for HCMV to evade T cell immunity. IMPORTANCE T cell immunity is pivotal in controlling primary human cytomegalovirus (HCMV) infection, restricting periodic reactivation, and preventing HCMV-associated diseases. Despite inducing a robust T cell immune response, HCMV has developed sophisticated immune evasion mechanisms that specifically target T cell responses. Although numerous studies have been conducted on HCMV-specific T cells, the primary focus has been on the impact of HCMV on T cell recognition via major histocompatibility complex molecules. Our studies show for the first time that HCMV exploits the programmed death ligand 1 (PD-L1) inhibitory signaling pathway to evade T cell immunity by modulating the activities of T cells and thereby blocking the secretion of IFN-γ, which is directly mediated by HCMV-encoded tegument protein UL23. While PD-L1 has been extensively studied in the context of tumors and viruses, its involvement in HCMV infection and viral immune evasion is rarely reported. We observed an upregulation of PD-L1 in normal cells during HCMV infection and provided strong evidence supporting its critical role in UL23-induced inhibition of T cell-mediated cytotoxicity. The novel strategy employed by HCMV to manipulate the inhibitory signaling pathway of T cell immune activation for viral evasion through its encoded protein offers valuable insights for the understanding of HCMV-mediated T cell immunomodulation and developing innovative antiviral treatment strategies.

Background Oxidative stress and inflammation can lead to apoptosis of ovarian granulosa cells (GCs), resulting in ovulation disorders and infertility. Baicalin (BAI) promotes cell proliferation and reduces inflammation and oxidative stress. However, the mechanisms by which BAI treatment affects oxidative stress and inflammation in GCs remain incompletely understood. Methods KGN cells were treated with hydrogen peroxide (H 2 O 2 ) to analyze the effect of oxidative stress on GCs in vitro. Subsequently, H 2 O 2 -stimulated KGN cells were treated with BAI. The levels of GSH-Px, CAT, and SOD were measured using an activity assay kit. The levels of MDA, IL-1β, IL-6, IL-8, and TNF-α were measured by ELISA. Proliferation, apoptosis, and mRNA and protein levels were measured using the CCK8, flow cytometry, qRT-PCR, and western blotting. Results H 2 O 2 treatment inhibited KGN cell proliferation and promoted apoptosis, accompanied by increased oxidative stress and inflammation. BAI promoted proliferation, inhibited apoptosis, and reduced oxidative stress and inflammation in H 2 O 2 -stimulated KGN cells. BAI treatment promoted USP48 protein expression, and USP48 knockdown abrogated the protective effects of BAI, indicating that USP48 is a downstream mediator of BAI. Conclusion BAI treatment enhanced cell proliferation and ameliorated oxidative stress and inflammation by enhancing USP48 protein expression. BAI, which is used clinically and as a dietary supplement, may alleviate oxidative stress-induced GC injury and ovarian disorders.

Human Cytomegalovirus Capsid Assembly Protein Precursor (pAP, UL80.5) plays a key role in capsid assembly by forming an internal protein scaffold with Major Capsid Protein (MCP, UL86) and other capsid subunits. In this study, we revealed UL80.5 as a novel SUMOylated viral protein. We confirmed that UL80.5 interacted with the SUMO E2 ligase UBC9 (58-93aa) and could be covalently modified by SUMO1/SUMO2/SUMO3 proteins. 371Lysine located within a ψKxE consensus motif on UL80.5 carboxy-terminal was the major SUMOylation site. Interestingly, the SUMOylation of UL80.5 restrained its interaction with UL86 but had no effects on translocating UL86 into the nucleus. Furthermore, we showed that the removal of the 371lysine SUMOylation site of UL80.5 inhibited viral replication. In conclusion, our data demonstrates that SUMOylation plays an important role in regulating UL80.5 functions and viral replication.

Interferon-γ (IFN-γ) is a critical component of innate immune responses in humans to combat infection by many viruses, including human cytomegalovirus (HCMV). IFN-γ exerts its biological effects by inducing hundreds of IFN-stimulated genes (ISGs). In this study, RNA-seq analyses revealed that HCMV tegument protein UL23 could regulate the expression of many ISGs under IFN-γ treatment or HCMV infection. We further confirmed that among these IFN-γ stimulated genes, individual APOL1 (Apolipoprotein-L1), CMPK2 (Cytidine/uridine monophosphate kinase 2), and LGALS9 (Galectin-9) could inhibit HCMV replication. Moreover, these three proteins exhibited a synergistic effect on HCMV replication. UL23-deficient HCMV mutants induced higher expression of APOL1, CMPK2, and LGALS9, and exhibited lower viral titers in IFN-γ treated cells compared with parental viruses expressing full functional UL23. Thus, UL23 appears to resist the antiviral effect of IFN-γ by downregulating the expression of APOL1, CMPK2, and LGALS9. This study highlights the roles of HCMV UL23 in facilitating viral immune escape from IFN-γ responses by specifically downregulating these ISGs.

Influenza virus has the ability to circumvent host innate immune system through regulating certain host factors for its effective propagation. However, the detailed mechanism is still not fully understood. Here, we report that a host sphingolipid metabolism-related factor, sphingosine kinase 2 (SPHK2), upregulated during influenza A virus (IAV) infection, promotes IAV infection in an enzymatic independent manner. The enhancement of the virus replication is not abolished in the catalytic-incompetent SPHK2 (G212E) overexpressing cells. Intriguingly, the sphingosine-1-phosphate (S1P) related factor HDAC1 also plays a crucial role in SPHK2-mediated IAV infection. We found that SPHK2 cannot facilitate IAV infection in HDAC1 deficient cells. More importantly, SPHK2 overexpression diminishes the IFN-β promoter activity upon IAV infection, resulting in the suppression of type I IFN signaling. Furthermore, ChIP-qPCR assay revealed that SPHK2 interacts with IFN-β promoter through the binding of demethylase TET3, but not with the other promoters regulated by TET3, such as TGF-β1 and IL6 promoters. The specific regulation of SPHK2 on IFN-β promoter through TET3 can in turn recruit HDAC1 to the IFN-β promoter, enhancing the deacetylation of IFN-β promoter, therefore leading to the inhibition of IFN-β transcription. These findings reveal an enzymatic independent mechanism on host SPHK2, which associates with TET3 and HDAC1 to negatively regulate type I IFN expression and thus facilitates IAV propagation.

BACKGROUND:Ankylosing spondylitis is a progressive, disabling joint disease that affects millions worldwide. Given its unclear etiology, studies of ankylosing spondylitis relied heavily on drug-induced or transgenic rodent models which retain only partial clinical features. There is obviously a lack of a useful disease model to conduct comprehensive mechanistic studies.METHODS:We followed a group of cynomolgus monkeys having joint lesions reported of spinal stiffness for 2 years by conducting hematological testing, radiographic examination, family aggregation analysis, pathological analysis, and genetic testing.RESULTS:The results confirmed that these diseased animals suffered from spontaneous ankylosing spondylitis with clinical features recapitulating human ankylosing spondylitis disease progression, manifested by pathological changes and biochemical indicators similar to that of ankylosing spondylitis patients.CONCLUSION:The study offers a promising non-human primate model for spontaneous ankylosing spondylitis which may serve as an excellent substitute for its pre-clinical research.

Controlled regulation of genomic DNA synthesis is a universally conserved process for all herpesviruses, including human cytomegalovirus (HCMV), and plays a key role in viral pathogenesis, such as persistent infections. HCMV DNA polymerase processivity factor UL44 plays an essential role in viral DNA replication. To better understand the biology of UL44, we performed a yeast two-hybrid screen for host proteins that could interact with UL44. The most frequently isolated result was the SUMO-conjugating enzyme UBC9, a protein involved in the sumoylation pathway. The UBC9-UL44 interaction was confirmed by in vitro His-tag pull-down and in vivo co-immunoprecipitation assays. Using deletion mutants of UL44, we mapped two small regions of UL44, aa 11–16, and 260–269, which might be critical for the interaction with UBC9. We then demonstrated that UL44 was a target for sumoylation by in vitro and in vivo sumoylation assays, as well as in HCMV-infected cells. We further confirmed that 410 lysine located within a ψKxE consensus motif on UL44 carboxy-terminal was the major sumoylation site of UL44. Interestingly, although 410 lysine had no effects on subcellular localization or protein stability of UL44, the removal of 410 lysine sumoylation site enhanced both viral DNA synthesis in transfection-replication assays and viral progeny production in infected cells for HCMV, suggesting sumoylation can attenuate HCMV replication through targeting UL44. Our results suggest that sumoylation plays a key role in regulating UL44 functions and viral replication, and reveal the crucial role of the carboxy-terminal of UL44, for which little function has been known before.

Lytic replication of human cytomegalovirus (HCMV), a member of β-herpesvirus, is a highly complicated and organized process that requires its DNA polymerase processivity factor, UL44, the first-reported HCMV replication protein subjected to SUMO post-translational modification (PTM). SUMOylation plays a pleiotropic role in protein functions of host cells and infecting viruses. Particularly, formation of herpesviral replication compartments (RCs) upon infection is induced in proximity to ND10 subnuclear domains, the host cell’s intrinsic antiviral immune devices and hot SUMOylation spots, relying just on SUMOylation of their protein components to become mature and functional in restriction of the viral replication. In this study, to unveil the exact role of SUMO PTM on UL44 involved in HCMV replication, we screened and identified PIAS3, an annotated E3 SUMO ligase, as a novel UL44-interacting protein engaged in cellular SUMOylation pathway. Co-existence of PIAS3 could enhance the UBC9-based SUMO modification of UL44 specifically at its conserved 410lysine residue lying within the single canonical ψKxE SUMO Conjugation Motif (SCM). Intriguingly, we found this SCM-specific SUMOylation contributes to UL44 co-localization and interaction with subnuclear ND10 domains during infection, which in turn exerts an inhibitory effect on HCMV replication and growth. Together, these results highlight the importance of SUMOylation in regulating viral protein subnuclear localization, representing a novel way of utilizing ND10-based restriction to achieve the self-controlled slower replication and reproduction of herpesviruses.