Herpes Simplex Virus 1 UL2 Inhibits the TNF-α-Mediated NF-κB Activity by Interacting With p65/p50.

Herpes simplex virus 1 (HSV-1) is a large double-stranded DNA virus that encodes at least 80 viral proteins, many of which are involved in the virus-host interaction and are beneficial to the viral survival and reproduction. However, the biological functions of some HSV-1-encoded proteins are not fully understood. Nuclear factor κB (NF-κB) activation is the major antiviral innate response, which can be triggered by various signals induced by cellular receptors from different pathways. Here, we demonstrated that HSV-1 UL2 protein could antagonize the tumor necrosis factor α (TNF-α)-mediated NF-κB activation. Co-immunoprecipitation assays showed that UL2 could interact with the NF-κB subunits p65 and p50, which also revealed the region of amino acids 9 to 17 of UL2 could suppress the NF-κB activation and interact with p65 and p50, and UL2 bound to the immunoglobulin-like plexin transcription factor functional domain of p65. However, UL2 did not affect the formation of p65/p50 dimerization and their nuclear localizations. Yet, UL2 was demonstrated to inhibit the NF-κB activity by attenuating TNF-α-induced p65 phosphorylation at Ser536 and therefore decreasing the expression of downstream inflammatory chemokine interleukin 8. Taken together, the attenuation of NF-κB activation by UL2 may contribute to the escape of host's antiviral innate immunity for HSV-1 during its infection.

Molecular anatomy of the subcellular localization and nuclear import mechanism of herpes simplex virus 1 UL6.

As an indispensable structure protein, the herpes simplex virus 1 (HSV-1) UL6 has been described to exert numerous roles in viral proliferation. However, its exact subcellular localization and subcellular transport mechanism is not well known. In the present study, by utilizing confocal fluorescent microscopy, UL6 was shown to mainly locate in the nucleus in enhanced yellow fluorescent protein or Flag tag fused expression plasmid-transfected cells or HSV-1-infected cells, whereas its predicted nuclear localization signal was nonfunctional. In addition, by exploiting dominant negative mutant and inhibitor of different nuclear import receptors, as well as co-immunoprecipitation and RNA interference assays, UL6 was established to interact with importin α1, importin α7 and transportin-1 to mediate its nuclear translocation under the help of Ran-mediated GTP hydrolysis. Accordingly, these results will advance the knowledge of UL6-mediated biological significances in HSV-1 infection cycle.

The nuclear localization signal-mediated nuclear targeting of herpes simplex virus 1 early protein UL2 is important for efficient viral production.

Herpes simplex virus 1 (HSV-1) is a representative alphaherpesvirus that can provoke a series of severe diseases to human being, but its exact pathogenesis is not perfectly understood. UL2, a uracil-DNA glycosylase involved in the process of HSV-1 DNA replication, has been shown to be predominantly targeted to the nuclei in our previous study, yet little is established regarding the subcellular localization signal or its related function of UL2 during HSV-1 propagation. Here, by creating a number of UL2 variants merged with enhanced yellow fluorescent protein, an authentic nuclear localization signal (NLS) of UL2 was, for the first time, identified and profiled to amino acids (aa) 1 to 17 (MKRACSRSPSPRRRPSS), and 12RRR14 was indispensable for its nuclear accumulation. Besides, the predicted nuclear export signal (aa 225 to 240) of UL2 was determined to be nonfunctional. Based on the HSV-1 bacterial artificial chromosome and homologous recombination technique, three recombinant viruses with mutations of the identified NLS, deletion and revertant of UL2 were constructed to assess the effect of UL2 nuclear targeting on HSV-1 replication. Compared to the wild type HSV-1, UL2 deletion remarkably restrained viral production, and mutation of NLS targeting UL2 to cytoplasm (pan-cellular distribution) in recombinant virus-infected cells showed a certain degree of deficiency in HSV-1 proliferation. Moreover, recombinant virus with UL2 deletion exhibited serious damages of viral DNA synthesis and mRNA expression, and these processes were partially disrupted in the recombinant virus with UL2 NLS mutation. Collectively, we had established a functional NLS in UL2 and showed that the NLS-mediated nuclear translocation of UL2 was important for efficient production of HSV-1. These data were of significance for further clarifying the biological function of UL2 during HSV-1 infection.

Intracellular distribution of pseudorabies virus UL2 and detection of its nuclear import mechanism.

Pseudorabies virus (PRV) UL2 (pUL2) is a multifunctional protein, which is homologous with herpes simplex virus 1 early protein UL2 (hUL2) and crucial for the viral propagation. Yet, how pUL2 executes its roles in the viral life cycle remain inadequately understood. In order to uncover its effect on the procedure of PRV infection, investigation was performed to examine the subcellular distribution of pUL2 and establish its trafficking mechanism. In the present study, enhanced yellow fluorescent protein or Myc tag fused pUL2 was transiently overexpressed in transfected cells and exhibited an absolutely nuclear accumulation without the existence of other PRV proteins. Additionally, the nuclear trafficking of pUL2 was proved to rely on Ran-, transportin-1, importin ß1, importin α1, α3 and α5. Accordingly, these data will benefit the knowledge of pUL2-mediated biological effects in PRV infection cycle.

Expression, Purification, and Antiserum Production of the Truncated UL31 Protein of Herpes Simplex Virus 1.

BACKGROUND: The UL31 protein of herpes simplex virus 1 (HSV-1) plays an important role in the HSV-1 replication, however, its pinpoint functions in the life cycle of the virus have yet to be adequately elucidated. OBJECTIVES: An antiserum specific for detecting HSV-1 UL31 was prepared as the foundation for future research on the role of UL31 in the course of HSV-1 infection. MATERIALS AND METHODS: Recombinant protein of UL31 was expressed in Escherichia coli, which was then purified and employed to raise the level of antiserum in mice. Subsequently, western blot and immunofluorescence assay (IFA) were utilized to detect the specific antiserum. RESULTS: The recombinant UL31 protein consisting of N-terminal 27 aa of UL31 was fused to EYFP and His-tag. It was expressed, purified, and applied to the preparation of the antiserum. Western blot analysis and IFA demonstrated that this antiserum could detect both the recombinant UL31 and the native UL31. CONCLUSIONS: Our results manifest that this antiserum could be conducive to further investigations concerning the roles of UL31 in the HSV-1 infection.

Epstein-Barr virus tegument protein BGLF2 inhibits NF-κB activity by preventing p65 Ser536 phosphorylation.

Epstein-Barr virus (EBV), a ubiquitous gammaherpesvirus, can regulate the antiviral response of NF-κB signaling, which is critical for cell survival, growth transformation, and virus latency. Here, we showed that tegument protein BGLF2 could inhibit TNF-α-induced NF-κB activity. BGLF2 was shown to interplay with the NF-κB subunits p65 and p50, and the Rel homology domain of p65 was the pivotal region to interact with BGLF2. Nonetheless, BGLF2 did not influence the development of p65-p50 dimerization. Yet, overexpression of BGLF2 inhibited the phosphorylation of p65 Ser536 (but not Ser276) and blocked the nuclear translocation of p65. In addition, knockdown of BGLF2 during EBV lytic replication elevated NF-κB activity and the phosphorylation of p65 Ser536. Taken together, these results suggest that the inhibition of NF-κB activation may serve as a strategy to escape the host's antiviral innate immunity to EBV during its lytic infection.-Chen, T., Wang, Y., Xu, Z., Zou, X., Wang, P., Ou, X., Li, Y., Peng, T., Chen, D., Li, M., Cai, M. Epstein-Barr virus tegument protein BGLF2 inhibits NF-κB activity by preventing p65 Ser536 phosphorylation.

Identification of the molecular determinants for nuclear import of PRV EP0.

Pseudorabies virus (PRV) early protein EP0 is a homologue of the herpes simplex virus 1 (HSV-1) immediate-early protein ICP0, which is a multifunctional protein and important for HSV-1 infection. However, the definite function of EP0 during PRV infection is not clear. In this study, to determine if EP0 might localize to the nucleus, as it is shown for its homologue in HSV-1, the subcellular localization pattern and molecular determinants for the nuclear import of EP0 were investigated. EP0 was demonstrated to predominantly target the nucleus in both PRV infected- and plasmid-transfected cells. Furthermore, the nuclear import of EP0 was shown to be dependent on the Ran-, importin α1-, α3-, α7-, ß1- and transportin-1-mediated multiple pathways. Taken together, these data will open up new horizons for portraying the biological roles of EP0 in the course of PRV lytic cycle.

The Interaction Mechanism Between Herpes Simplex Virus 1 Glycoprotein D and Host Antiviral Protein Viperin.

Viperin is an interferon-inducible protein that responsible for a variety of antiviral responses to different viruses. Our previous study has shown that the ribonuclease UL41 of herpes simplex virus 1 (HSV-1) can degrade the mRNA of viperin to promote HSV-1 replication. However, it is not clear whether other HSV-1 encoded proteins can regulate the function of viperin. Here, one novel viperin associated protein, glycoprotein D (gD), was identified. To verify the interaction between gD and viperin, gD and viperin expression plasmids were firstly co-transfected into COS-7 cells, and fluorescence microscope showed they co-localized at the perinuclear region, then this potential interaction was confirmed by co-immunoprecipitation (Co-IP) assays. Moreover, confocal microscopy demonstrated that gD and viperin co-localized at the Golgi body and lipid droplets. Furthermore, dual-luciferase reporter and Co-IP assays showed gD and viperin interaction leaded to the increase of IRF7-mediated IFN-ß expression through promoting viperin and IRAK1 interaction and facilitating K63-linked IRAK1 polyubiquitination. Nevertheless, gD inhibited TRAF6-induced NF-κB activity by decreasing the interaction of viperin and TRAF6. In addition, gD restrained viperin-mediated interaction between IRAK1 and TRAF6. Eventually, gD and viperin interaction was corroborated to significantly inhibit the proliferation of HSV-1. Taken together, this study would open up new avenues toward delineating the function and physiological significance of gD and viperin during HSV-1 replication cycle.

Characterization of the Nucleocytoplasmic Transport Mechanisms of Epstein-Barr Virus BFLF2.

BACKGROUND/AIMS: Epstein-Barr virus (EBV) BFLF2, the homologue of herpes simplex virus 1 (HSV-1) UL31, is crucial for the efficient viral DNA packaging and primary egress across the nuclear membrane. However, we still do not know its subcellular transport mechanisms. METHODS: Interspecies heterokaryon assays were utilized to detect the nucleocytoplasmic shuttling of BFLF2, and mutation analysis, plasmid transfection and fluorescence microscopy assays were performed to identify the functional nuclear localization sequence (NLS) and nuclear export sequence (NES) of BFLF2 in live cells. Furthermore, the nuclear import and export of BFLF2 were assessed by confocal microscopy, co-immunoprecipitation and immunoblot assays. RESULTS: BFLF2 was confirmed to shuttle between the nucleus and cytoplasm. Two predicted NESs were shown to be nonfunctional, yet we proved that the nuclear export of BFLF2 was mediated through transporter associated with antigen processing (TAP), but not chromosomal region maintenance 1 (CRM1) dependent pathway. Furthermore, one functional NLS, 22RRLMHPHHRNYTASKASAH40, was identified, and the aa22-23, aa22-25, aa28-30 and aa37-40 had an important role in the nuclear localization of BFLF2. Besides, the nuclear import of BFLF2 was demonstrated through Ran-, importin α7-, importin ß1- and transportin-1-dependent mechanism that does not require importin α1, α3 and α5. CONCLUSION: These works are of significance for the further study of the functions of BFLF2 during EBV infection, as well as for further insights into the design of new antiviral drug target and vaccine development against EBV.

Molecular Characterization of the Epstein-Barr Virus BGLF2 Gene, its Expression, and Subcellular Localization.

BACKGROUND: Epstein-Barr virus (EBV) is a universal herpes virus which can cause a life-long and largely asymptomatic infection in the human population. However, the exact pathogenesis of the EBV infection is not well known. OBJECTIVE: A comprehensive bioinformatics prediction was carried out for investigating the molecular properties of the BGLF2 and to afford a foundation for future research of the role and instrument of BGLF2 in the course of EBV infection. MATERIALS AND METHODS: A 1011-base-pair sequence of BGLF2 gene from the Epstein-Barr virus (EBV) Akata strain genome was amplified using polymerase chain reaction and was further characterized by cloning, sequencing, and subcellular localization in the COS-7 cells. RESULTS: The bioinformatics analysis demonstrated that EBV BGLF2 gene encodes a putative BGLF2 polypeptide which contains a conservative Herpes_UL16 domain. It was established that the polypeptide shows a close relationship with the Herpes UL16 tegument protein family and is extremely conserved among its homologues proteins encoded by UL16 genes. Multiple sequence alignments of the nucleic acid and amino acid sequence showed that the gene product of EBV BGLF2 contains a comparatively higher homology with the BGLF2-like proteins of the subfamily Gammaherpesvirinae than that of other subfamilies of the herpes virus. Moreover, the phylogenetic analyses suggested that EBV BGLF2 has a close genetic relationship with the member of Gammaherpesvirinae; in particular with the members of Cercopithecine herpesvirus 15 and Callitrichine herpesvirus 3. An antigen epitope analysis indicated that BGLF2 contains several potential B-cell epitopes. In addition, the secondary structure, as well as the three dimensional structure prediction suggests that BGLF2 consists of the both α-helix and ß-strand. Besides, the subcellular localization prediction revealed that BGLF2 localizes in both nucleus and cytoplasm. CONCLUSIONS: Illustrating the relevance of the molecular properties and genetic evolution of EBV, BGLF2 will offer the perspectives for further study on the role and mechanism of the BGLF2 in course of EBV infection. These works will also conduct our understanding of the EBV at the molecular level as well as enriching the herpesvirus database.

Characterization of the subcellular localization of Epstein-Barr virus encoded proteins in live cells.

Epstein-Barr virus (EBV) is the pathogenic factor of numerous human tumors, yet certain of its encoded proteins have not been studied. As a first step for functional identification, we presented the construction of a library of expression constructs for most of the EBV encoded proteins and an explicit subcellular localization map of 81 proteins encoded by EBV in mammalian cells. Viral open reading frames were fused with enhanced yellow fluorescent protein (EYFP) tag in eukaryotic expression plasmid then expressed in COS-7 live cells, and protein localizations were observed by fluorescence microscopy. As results, 34.57% (28 proteins) of all proteins showed pan-nuclear or subnuclear localization, 39.51% (32 proteins) exhibitted pan-cytoplasmic or subcytoplasmic localization, and 25.93% (21 proteins) were found in both the nucleus and cytoplasm. Interestingly, most envelope proteins presented pan-cytoplasmic or membranous localization, and most capsid proteins displayed enriched or complete localization in the nucleus, indicating that the subcellular localization of specific proteins are associated with their roles during viral replication. Taken together, the subcellular localization map of EBV proteins in live cells may lay the foundation for further illustrating the functions of EBV-encoded genes in human diseases especially in its relevant tumors.