PmrAB, the two-component system of Acinetobacter baumannii, controls the phosphoethanolamine modification of lipooligosaccharide in response to metal ions.

Acinetobacter baumannii is highly resistant to antimicrobial agents, and XDR strains have become widespread. A. baumannii has developed resistance to colistin, which is considered the last resort against XDR Gram-negative bacteria, mainly caused by lipooligosaccharide (LOS) phosphoethanolamine (pEtN) and/or galactosamine (GalN) modifications induced by mutations that activate the two-component system (TCS) pmrAB. Although PmrAB of A. baumannii has been recognized as a drug resistance factor, its function as TCS, including its regulatory genes and response factors, has not been fully elucidated. In this study, to clarify the function of PmrAB as TCS, we elucidated the regulatory genes (regulon) of PmrAB via transcriptome analysis using pmrAB-activated mutant strains. We discovered that PmrAB responds to low pH, Fe2+, Zn2+, and Al3+. A. baumannii selectively recognizes Fe2+ rather than Fe3+, and a novel region ExxxE, in addition to the ExxE motif sequence, is involved in the environmental response. Furthermore, PmrAB participates in the phosphoethanolamine modification of LOS on the bacterial surface in response to metal ions such as Al3+, contributing to the attenuation of Al3+ toxicity and development of resistance to colistin and polymyxin B in A. baumannii. This study demonstrates that PmrAB in A. baumannii not only regulates genes that play an important role in drug resistance but is also involved in responses to environmental stimuli such as metal ions and pH, and this stimulation induces LOS modification. This study reveals the importance of PmrAB in the environmental adaptation and antibacterial resistance emergence mechanisms of A. baumannii. IMPORTANCE: Antimicrobial resistance (AMR) is a pressing global issue in human health. Acinetobacter baumannii is notably high on the World Health Organization's list of bacteria for which new antimicrobial agents are urgently needed. Colistin is one of the last-resort drugs used against extensively drug-resistant (XDR) Gram-negative bacteria. However, A. baumannii has become increasingly resistant to colistin, primarily by modifying its lipooligosaccharide (LOS) via activating mutations in the two-component system (TCS) PmrAB. This study comprehensively elucidates the detailed mechanism of drug resistance of PmrAB in A. baumannii as well as its biological functions. Understanding the molecular biology of these molecules, which serve as drug resistance factors and are involved in environmental recognition mechanisms in bacteria, is crucial for developing fundamental solutions to the AMR problem.

Tyrosinase regulates the motility of human melanoma cell line A375 through its hydroxylase enzymatic activity.

Melanoma, originating from melanocytes, is a highly aggressive tumor. Tyrosinase is involved in melanin production in melanocytes, and its overexpression is noted in malignant melanomas. However, the role of tyrosinase in melanomas remains unclear. Therefore, this study aimed to evaluate the potential functions of tyrosinase in the human melanoma cell line A375. The expression level of tyrosinase in A375 cells was undetectable. However, markedly increased expression level was observed in the mouse melanoma cell line B16F10 and the human melanoma cell line WM266-4. Subsequently, we investigated the effect of ectopic tyrosinase expression on A375 cell motility using wound-healing assay. The overexpression of tyrosinase resulted in enhanced cell migration in both stable and transient tyrosinase expression cells. The levels of filamentous actin were decreased in tyrosinase-expressing A375 cells, suggesting that tyrosinase regulates cell motility by modulating actin polymerization. Histidine residues in tyrosinase are important for its enzymatic activity for synthesizing melanin. Substitution of these histidine residues to alanine residues mitigated the promotion of tyrosinase-induced A375 cell metastasis. Furthermore, melanin treatment enhanced A375 cell metastasis and phosphorylation of Cofilin. Thus, our findings suggest that tyrosinase increases the migration of A375 cells by regulating actin polymerization through its enzymatic activity.

Kaposi's Sarcoma-Associated Herpesvirus ORF67.5 Functions as a Component of the Terminase Complex.

Kaposi's sarcoma-associated herpesvirus (KSHV) is a double-stranded DNA (dsDNA) gammaherpesvirus with a poorly characterized lytic replication cycle. However, the lytic replication cycle of the alpha- and betaherpesviruses are well characterized. During lytic infection of alpha- and betaherpesviruses, the viral genome is replicated as a precursor form, which contains tandem genomes linked via terminal repeats (TRs). One genomic unit of the precursor form is packaged into a capsid and is cleaved at the TR by the terminase complex. While the alpha- and betaherpesvirus terminases are well characterized, the KSHV terminase remains poorly understood. KSHV open reading frame 7 (ORF7), ORF29, and ORF67.5 are presumed to be components of the terminase complex based on their homology to other terminase proteins. We previously reported that ORF7-deficient KSHV formed numerous immature soccer ball-like capsids and failed to cleave the TRs. ORF7 interacted with ORF29 and ORF67.5; however, ORF29 and ORF67.5 did not interact with each other. While these results suggested that ORF7 is important for KSHV terminase function and capsid formation, the function of ORF67.5 was completely unknown. Therefore, to analyze the function of ORF67.5, we constructed ORF67.5-deficient BAC16. ORF67.5-deficient KSHV failed to produce infectious virus and cleave the TRs, and numerous soccer ball-like capsids were observed in ORF67.5-deficient KSHV-harboring cells. Furthermore, ORF67.5 promoted the interaction between ORF7 and ORF29, and ORF29 increased the interaction between ORF67.5 and ORF7. Thus, our data indicated that ORF67.5 functions as a component of the KSHV terminase complex by contributing to TR cleavage, terminase complex formation, capsid formation, and virus production. IMPORTANCE Although the formation and function of the alpha- and betaherpesvirus terminase complexes are well understood, the Kaposi's sarcoma-associated herpesvirus (KSHV) terminase complex is still largely uncharacterized. This complex presumably contains KSHV open reading frame 7 (ORF7), ORF29, and ORF67.5. We were the first to report the presence of soccer ball-like capsids in ORF7-deficient KSHV-harboring lytic-induced cells. Here, we demonstrated that ORF67.5-deficient KSHV also formed soccer ball-like capsids in lytic-induced cells. Moreover, ORF67.5 was required for terminal repeat (TR) cleavage, infectious virus production, and enhancement of the interaction between ORF7 and ORF29. ORF67.5 has several highly conserved regions among its human herpesviral homologs. These regions were necessary for virus production and for the interaction of ORF67.5 with ORF7, which was supported by the artificial intelligence (AI)-predicted structure model. Importantly, our results provide the first evidence showing that ORF67.5 is essential for terminase complex formation and TR cleavage.

The Terminase Complex of Each Human Herpesvirus.

The human herpesviruses (HHVs) are classified into the following three subfamilies: Alphaherpesvirinae, Betaherpesvirinae, and Gammaherpesvirinae. These HHVs have distinct pathological features, while containing a highly conserved viral replication pathway. Among HHVs, the basic viral particle structure and the sequential processes of viral replication are nearly identical. In particular, the capsid formation mechanism has been proposed to be highly similar among herpesviruses, because the viral capsid-organizing proteins are highly conserved at the structural and functional levels. Herpesviruses form capsids containing the viral genome in the nucleus of infected cells during the lytic phase, and release infectious virus (i.e., virions) to the cell exterior. In the capsid formation process, a single-unit-length viral genome is encapsidated into a preformed capsid. The single-unit-length viral genome is produced by cleavage from a viral genome precursor in which multiple unit-length viral genomes are tandemly linked. This encapsidation and cleavage is carried out by the terminase complex, which is composed of viral proteins. Since the terminase complex-mediated encapsidation and cleavage is a virus-specific mechanism that does not exist in humans, it may be an excellent inhibitory target for anti-viral drugs with high virus specificity. This review provides an overview of the functions of the terminase complexes of HHVs.

Scraping Assay as a Novel Strategy to Evaluate Axonal Regeneration Using Human-Induced Pluripotent Stem Cell-Derived Neurons.

Neuronal regrowth after traumatic injury is strongly inhibited in the central nervous system (CNS) of adult mammals. Cell-intrinsic and extrinsic factors limit the regulation of axonal growth and regrowth of fibers is minimal despite nearly all neurons surviving. Developing medical drugs to promote neurological recovery is crucial since neuronal injuries have few palliative cares and no pharmacological interventions. Herein, we developed a novel in vitro axonal regeneration assay system to screen the chemical reagents using human-induced pluripotent stem cell (hiPSC)-derived neurons. These neurons were cultured in a 96-well plate to form a monolayer and were scraped using a floating metal pin tool for axotomy. The cell number and plate coating conditions were optimized to score the regenerating axon. Treatment using the Rho-associated kinase (ROCK) inhibitor Y-27632 enhanced axonal regeneration in this regeneration assay system with hiPSC-derived neurons. Therefore, our novel screening method is suitable for drug screening to identify the chemical compounds that promote axonal regeneration after axotomy under in vitro conditions.

Sofosbuvir Suppresses the Genome Replication of DENV1 in Human Hepatic Huh7 Cells.

Dengue virus (DENV) causes dengue fever and dengue hemorrhagic fever, and DENV infection kills 20,000 people annually worldwide. Therefore, the development of anti-DENV drugs is urgently needed. Sofosbuvir (SOF) is an effective drug for HCV-related diseases, and its triphosphorylated metabolite inhibits viral RNA synthesis by the RNA-dependent RNA polymerase (RdRp) of HCV. (2'R)-2'-Deoxy-2'-fluoro-2'-methyluridine (FMeU) is the dephosphorylated metabolite produced from SOF. The effects of SOF and FMeU on DENV1 replication were analyzed using two DENV1 replicon-based methods that we previously established. First, a replicon-harboring cell assay showed that DENV1 replicon replication in human hepatic Huh7 cells was decreased by SOF but not by FMeU. Second, a transient replicon assay showed that DENV1 replicon replication in Huh7 cells was decreased by SOF; however, in hamster kidney BHK-21 cells, it was not suppressed by SOF. Additionally, the replicon replication in Huh7 and BHK-21 cells was not affected by FMeU. Moreover, we assessed the effects of SOF on infectious DENV1 production. SOF suppressed infectious DENV1 production in Huh7 cells but not in monkey kidney Vero cells. To examine the substrate recognition of the HCV and DENV1 RdRps, the complex conformation of SOF-containing DENV1 RdRp or HCV RdRp was predicted using AlphaFold 2. These results indicate that SOF may be used as a treatment for DENV1 infection.

The Contribution of Kaposi's Sarcoma-Associated Herpesvirus ORF7 and Its Zinc-Finger Motif to Viral Genome Cleavage and Capsid Formation.

During Kaposi's sarcoma-associated herpesvirus (KSHV) lytic infection, lytic-related proteins are synthesized, viral genomes are replicated as a tandemly repeated form, and subsequently, capsids are assembled. The herpesvirus terminase complex is proposed to package an appropriate genome unit into an immature capsid, by cleavage of terminal repeats (TRs) flanking tandemly linked viral genomes. Although the mechanism of capsid formation in alpha- and betaherpesviruses are well-studied, in KSHV, it remains largely unknown. It has been proposed that KSHV ORF7 is a terminase subunit, and ORF7 harbors a zinc-finger motif, which is conserved among other herpesviral terminases. However, the biological significance of ORF7 is unknown. We previously reported that KSHV ORF17 is essential for the cleavage of inner scaffold proteins in capsid maturation, and ORF17 knockout (KO) induced capsid formation arrest between the procapsid and B-capsid stages. However, it remains unknown if ORF7-mediated viral DNA cleavage occurs before or after ORF17-mediated scaffold collapse. We analyzed the role of ORF7 during capsid formation using ORF7-KO-, ORF7&17-double-KO (DKO)-, and ORF7-zinc-finger motif mutant-KSHVs. We found that ORF7 acted after ORF17 in the capsid formation process, and ORF7-KO-KSHV produced incomplete capsids harboring nonspherical internal structures, which resembled soccer balls. This soccer ball-like capsid was formed after ORF17-mediated B-capsid formation. Moreover, ORF7-KO- and zinc-finger motif KO-KSHV failed to appropriately cleave the TR on replicated genome and had a defect in virion production. Interestingly, ORF17 function was also necessary for TR cleavage. Thus, our data revealed ORF7 contributes to terminase-mediated viral genome cleavage and capsid formation. IMPORTANCE In herpesviral capsid formation, the viral terminase complex cleaves the TR sites on newly synthesized tandemly repeating genomes and inserts an appropriate genomic unit into an immature capsid. Herpes simplex virus 1 (HSV-1) UL28 is a subunit of the terminase complex that cleaves the replicated viral genome. However, the physiological importance of the UL28 homolog, KSHV ORF7, remains poorly understood. Here, using several ORF7-deficient KSHVs, we found that ORF7 acted after ORF17-mediated scaffold collapse in the capsid maturation process. Moreover, ORF7 and its zinc-finger motif were essential for both cleavage of TR sites on the KSHV genome and virus production. ORF7-deficient KSHVs produced incomplete capsids that resembled a soccer ball. To our knowledge, this is the first report showing ORF7-KO-induced soccer ball-like capsids production and ORF7 function in the KSHV capsid assembly process. Our findings provide insights into the role of ORF7 in KSHV capsid formation.

A novel intramolecular negative regulation of mouse Jak3 activity by tyrosine 820.

Jak3, a member of the Janus kinase family, is essential for the cytokine receptor common gamma chain (γc)-mediated signaling. During activation of Jak3, tyrosine residues are phosphorylated and potentially regulate its kinase activity. We identified a novel tyrosine phosphorylation site within mouse Jak3, Y820, which is conserved in human Jak3, Y824. IL-2-induced tyrosine phosphorylation of Jak3 Y824 in human T cell line HuT78 cells was detected by using a phosphospecific, pY824, antibody. Mutation of mouse Jak3 Y820 to alanine (Y820A) showed increased autophosphorylation of Jak3 and enhanced signal transducer and activator of transcription 5 (STAT5) tyrosine phosphorylation and transcriptional activation. Stably expressed Jak3 Y820A in F7 cells, an IL-2 responsive mouse pro-B cell line Ba/F3, exhibited enhanced IL-2-dependent cell growth. Mechanistic studies demonstrated that interaction between Jak3 and STAT5 increased in Jak3 Y820A compared to wild-type Jak3. These data suggest that Jak3 Y820 plays a role in negative regulation of Jak3-mediated STAT5 signaling cascade upon IL-2-stimulation. We speculate that this occurs through an interaction promoted by the tyrosine phosphorylated Y820 or a conformational change by Y820 mutation with either the STAT directly or with the recruitment of molecules such as phosphatases via a SH2 interaction. Additional studies will focus on these interactions as Jak3 plays a crucial role in disease and health.

Kaposi's Sarcoma-Associated Herpesvirus ORF21 Enhances the Phosphorylation of MEK and the Infectivity of Progeny Virus.

Kaposi's sarcoma-associated herpesvirus (KSHV), also known as human herpesvirus-8, is the causative agent of Kaposi's sarcoma, Castleman's disease, and primary effusion lymphoma. Although the functions of the viral thymidine kinases (vTK) of herpes simplex virus-1/2 are well understood, that of KSHV ORF21 (an ortholog of vTK) is largely unknown. Here, we investigated the role of ORF21 in lytic replication and infection by generating two ORF21-mutated KSHV BAC clones: ORF21-kinase activity deficient KSHV (21KD) and stop codon-induced ORF21-deleted KSHV (21del). The results showed that both ORF21 mutations did not affect viral genome replication, lytic gene transcription, or the production of viral genome-encapsidated particles. The ORF21 molecule-dependent function, other than the kinase function of ORF21, was involved in the infectivity of the progeny virus. ORF21 was expressed 36 h after the induction of lytic replication, and endogenously expressed ORF21 was localized in the whole cytoplasm. Moreover, ORF21 upregulated the MEK phosphorylation and anchorage-independent cell growth. The inhibition of MEK signaling by U0126 in recipient target cells suppressed the number of progeny virus-infected cells. These suggest that ORF21 transmitted as a tegument protein in the progeny virus enhances the new infection through MEK up-regulation in the recipient cell. Our findings indicate that ORF21 plays key roles in the infection of KSHV through the manipulation of the cellular function.

The FAT10 post-translational modification is involved in the lytic replication of Kaposi's sarcoma-associated herpesvirus.

During Kaposi's sarcoma-associated herpesvirus (KSHV) lytic replication, host cell functions including protein expression and post-translational modification pathways are dysregulated by KSHV to promote virus production. Here, we attempted to identify key proteins for KSHV lytic replication by profiling protein expression in the latent and lytic phases using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Proteomic analysis, immunoblotting, and quantitative PCR demonstrated that antigen-F (HLA-F) adjacent transcript 10 (FAT10) and UBE1L2 (also known as ubiquitin-like modifier-activating enzyme 6, UBA6) were upregulated during lytic replication. FAT10 is a ubiquitin-like protein (UBL). UBE1L2 is the FAT10-activating enzyme (E1), which is essential for FAT10 modification (FAT10ylation). FAT10ylated proteins were immediately expressed after lytic induction and increased over time during lytic replication. Knockout of UBE1L2 suppressed KSHV production but not KSHV DNA synthesis. In order to isolate FAT10ylated proteins during KSHV lytic replication, we conducted immunoprecipitations using anti-FAT10 antibody and Ni-NTA chromatography of exogenously expressed His-tagged FAT10 from cells undergoing latent or lytic replication. LC-MS/MS was performed to identify FAT10ylated proteins. We identified KSHV ORF59 and ORF61 as FAT10ylation substrates. Our study revealed that the UBE1L2-FAT10 system is upregulated during KSHV lytic replication, and it contributes to viral propagation.ImportanceUbiquitin and UBL post-translational modifications, including FAT10, are utilized and dysregulated by viruses for achievement of effective infection and virion production. The UBE1L2-FAT10 system catalyzes FAT10ylation, where one or more FAT10 molecules are covalently linked to a substrate. FAT10ylation is catalyzed by the sequential actions of E1 (activation enzyme), E2 (conjugation enzyme), and E3 (ligase) enzymes. The E1 enzyme for FAT10ylation is UBE1L2, which activates FAT10 and transfers it to E2/USE1. FAT10ylation regulates the cell cycle, IFN signaling, and protein degradation; however, its primary biological function remains unknown. Here, we revealed that KSHV lytic replication induces UBE1L2 expression and production of FAT10ylated proteins including KSHV lytic proteins. Moreover, UBE1L2 knockout suppressed virus production during the lytic cycle. This is the first report demonstrating the contribution of the UBE1L2-FAT10 system to KSHV lytic replication. Our findings provide insight into the physiological function(s) of novel post-translational modifications in KSHV lytic replication.

Epstein-Barr virus tegument protein BGLF2 in exosomes released from virus-producing cells facilitates de novo infection.

BACKGROUND: Viruses must adapt to the environment of their host cells to establish infection and persist. Diverse mammalian cells, including virus-infected cells, release extracellular vesicles such as exosomes containing proteins and miRNAs, and use these vesicles to mediate intercellular communication. However, the roles of exosomes in viral infection remain unclear. RESULTS: We screened viral proteins to identify those responsible for the exosome-mediated enhancement of Epstein-Barr virus (EBV) infection. We identified BGLF2 protein encapsulated in exosomes, which were released by EBV-infected cells. BGLF2 protein is a tegument protein that exists in the space between the envelope and nucleocapsid, and it is released into the cytoplasm shortly after infection. BGLF2 protein-containing exosomes enhanced viral gene expression and repressed innate immunity, thereby supporting the EBV infection. CONCLUSIONS: The EBV tegument protein BGLF2 is encapsulated in exosomes and released by infected cells to facilitate the establishment of EBV infection. These findings suggest that tegument proteins support viral infection not only between the envelope and nucleocapsid, as well as in extraviral particles such as exosomes. Video abstract.

Herpes simplex virus 1 infection induces ubiquitination of UBE1a.

Herpes simplex virus 1 (HSV-1) is a human DNA virus that causes cold sores, keratitis, meningitis, and encephalitis. Ubiquitination is a post-translational protein modification essential for regulation of cellular events, such as proteasomal degradation, signal transduction, and protein trafficking. The process is also involved in events for establishing viral infection and replication. The first step in ubiquitination involves ubiquitin (Ub) binding with Ub-activating enzyme (E1, also termed UBE1) via a thioester linkage. Our results show that HSV-1 infection alters protein ubiquitination pattern in host cells, as evidenced by MS spectra and co-immunoprecipitation assays. HSV-1 induced ubiquitination of UBE1a isoform via an isopeptide bond with Lys604. Moreover, we show that ubiquitination of K604 in UBE1a enhances UBE1a activity; that is, the activity of ubiquitin-transfer to E2 enzyme. Subsequently, we investigated the functional role of UBE1a and ubiquitination of K604 in UBE1a. We found that UBE1-knockdown increased HSV-1 DNA replication and viral production. Furthermore, overexpression of UBE1a, but not a UBE1a K604A mutant, suppressed viral replication. Furthermore, we found that UBE1a and ubiquitination at K604 in UBE1a retarded expression of HSV-1 major capsid protein, ICP5. Our findings show that UBE1a functions as an antiviral factor that becomes activated upon ubiquitination at Lys604.

Biomolecular Fluorescence Complementation Profiling and Artificial Intelligence Structure Prediction of the Kaposi's Sarcoma-Associated Herpesvirus ORF18 and ORF30 Interaction.

Kaposi's sarcoma-associated herpesvirus (KSHV) is the etiologic agent of Kaposi's sarcoma, primary effusion lymphoma (PEL), and multicentric Castleman's disease. During KSHV lytic infection, lytic-related genes, categorized as immediate-early, early, and late genes, are expressed in a temporal manner. The transcription of late genes requires the virus-specific pre-initiation complex (vPIC), which consists of viral transcription factors. However, the protein-protein interactions of the vPIC factors have not been completely elucidated. KSHV ORF18 is one of the vPIC factors, and its interaction with other viral proteins has not been sufficiently revealed. In order to clarify these issues, we analyzed the interaction between ORF18 and another vPIC factor, ORF30, in living cells using the bimolecular fluorescence complementation (BiFC) assay. We identified four amino-acid residues (Leu29, Glu36, His41, and Trp170) of ORF18 that were responsible for its interaction with ORF30. Pull-down assays also showed that these four residues were required for the ORF18-ORF30 interaction. The artificial intelligence (AI) system AlphaFold2 predicted that the identified four residues are localized on the surface of ORF18 and are in proximity to each other. Thus, our AI-predicted model supports the importance of the four residues for binding ORF18 to ORF30. These results indicated that wet experiments in combination with AI may enhance the structural characterization of vPIC protein-protein interactions.

Human hepatitis B virus-derived virus-like particle as a drug and DNA delivery carrier.

The controlled release of medications using nanoparticle-based drug delivery carriers is a promising method to increase the efficacy of pharmacotherapy and gene therapy. One critical issue that needs to be overcome with these drug delivery carriers is their target specificity. We focused on the cell tropism of a virus to solve this issue, i.e., we attempted to apply hepatitis B virus-like particle (HBV-VLP) as a novel hepatic cell-selective carrier for medication and DNA. To prepare HBV-VLP, 293T cells were transfected with expression plasmids carrying HBV envelope surface proteins, large envelope protein (L), and small envelope protein (S). After 72 h post-transfection, VLP-containing culture supernatants were harvested, and HBV-VLP was labeled with red fluorescent dye (DiI) and was purified by sucrose gradient ultracentrifugation. An anticancer drugs (geldanamycin or doxorubicin) and GFP-expressing plasmid DNA were incorporated into HBV-VLP, and medication- and plasmid DNA-loaded VLPs were prepared. We evaluated their delivery capabilities into hepatocytes, other organ-derived cells, and hepatocytes expressing sodium taurocholate cotransporting polypeptide (NTCP), which functions as the cellular receptor for HBV by binding to HBV L protein. HBV-VLP selectively delivered both anticancer drugs and plasmid DNA not into HepG2, Huh7, and other organ cells but into HepG2 cells expressing NTCP. In summary, we developed a novel delivery nanocarrier using HBV-VLP that could be used as a hepatitis selective drug- and DNA-carrier for cancer treatment and gene therapy.

Kaposi's Sarcoma-Associated Herpesvirus ORF66 Is Essential for Late Gene Expression and Virus Production via Interaction with ORF34.

Kaposi's sarcoma-associated herpesvirus (KSHV) is closely associated with B-cell and endothelial cell malignancies. After the initial infection, KSHV retains its viral genome in the nucleus of the host cell and establishes a lifelong latency. During lytic infection, KSHV-encoded lytic-related proteins are expressed in a sequential manner and are classified as immediate early, early, and late (L) gene transcripts. The transcriptional initiation of KSHV late genes is thought to require the complex formation of the viral preinitiation complex (vPIC), which may consist of at least 6 transcription factors (ORF18, -24, -30, -31, -34, and -66). However, the functional role of ORF66 in vPIC during KSHV replication remains largely unclear. Here, we generated ORF66-deficient KSHV using a bacterial artificial chromosome (BAC) system to evaluate its role during viral replication. While ORF66-deficient KSHV demonstrated mainly attenuated late gene expression and decreased virus production, viral DNA replication was unaffected. Chromatin immunoprecipitation analysis showed that ORF66 bound to the promoters of a late gene (K8.1) but did not bind to those of a latent gene (ORF72), an immediate early gene (ORF16), or an early gene (ORF46/47). Furthermore, we found that three highly conserved C-X-X-C sequences and a conserved leucine repeat in the C-terminal region of ORF66 were essential for the interaction with ORF34, the transcription of K8.1, and virus production. The interaction between ORF66 and ORF34 occurred in a zinc-dependent manner. Our data support a model in which ORF66 serves as a critical vPIC component to promote late viral gene expression and virus production.IMPORTANCE KSHV ORF66 is expressed during the early stages of lytic infection, and ORF66 and vPIC are thought to contribute significantly to late gene expression. However, the physiological importance of ORF66 in terms of vPIC formation remains poorly understood. Therefore, we generated an ORF66-deficient BAC clone and evaluated its viral replication. The results showed that ORF66 plays a critical role in virus production and the transcription of L genes. To our knowledge, this is the first report showing the function of ORF66 in virus replication using ORF66-deficient KSHV. We also clarified that ORF66 interacts with the transcription start site of the K8.1 gene, a late gene. Furthermore, we identified the ORF34-binding motifs in the ORF66 C terminus: three C-X-X-C sequences and a leucine-repeat sequence, which are highly conserved among beta- and gammaherpesviruses. Our study provides insights into the regulatory mechanisms of not only the late gene expression of KSHV but also those of other herpesviruses.

The Lysosome Pathway Degrades CD81 on the Cell Surface by Poly-ubiquitination and Clathrin-Mediated Endocytosis.

CD81 is a highly conserved four-transmembrane protein in mammals and widely expressed on many tissues. It belongs to the tetraspanin family and forms complexes with various cell surface membrane proteins. It also functions in cell migration and B-cell activation, which is induced by CD81 complexing with CD19, CD21 and the B-cell receptor. Thus, CD81 is thought to play a key role in regulating cell function and fate. However, little is known about the degradation mechanism of CD81. Here we found that CD81 on the plasma membrane is degraded by the lysosome pathway via endocytosis. The expression levels of CD81 in HEK293T cells treated with a proteasome inhibitor (lactacystin) and lysosome inhibitors (chloroquine and bafilomycin A1) were analyzed by flow cytometry. The expression of CD81 on the cell surface was increased by the lysosome inhibitors, but not lactacystin. A pulldown assay revealed that CD81 was conjugated with a K63- and K29-linked poly-ubiquitin chain before its degradation, and the poly-ubiquitination site was Lys8 at the N-terminal intracellular domain of CD81. Furthermore, mutant CD81, in which Lys8 was substituted with alanine (Ala), extended the CD81 half-life compared with wildtype. CD81 was mainly localized on the plasma membrane in normal cells, but also co-localized with lysosomal LAMP1 and early endosomal EEA1 in chloroquine-treated cells. Furthermore, a clathrin-mediated endocytosis inhibitor, chlorpromazine, stabilized CD81 expression on the cell surface. Hence, we demonstrated that CD81 is internalized by clathrin-mediated endocytosis and subsequently degraded via a lysosome pathway requiring the K63- and K29-linked poly-ubiquitination of CD81.

Synthesis and biological evaluation of novel imidazole nucleosides as potential anti-dengue virus agents.

In this work, we developed imidazole nucleoside derivatives with anti-dengue virus (DENV) activity was examined. First, compounds in a nucleosides library were screened to find lead compounds which inhibit replication of DENV. As a result, 5-ethynyl-(1-ß-d-ribofuranosyl)imidazole-4-carboxamide (1; EICAR) and its 4-carbonitrile derivative EICNR (2) were selected as promising antiviral compounds. However, both of them also exhibited cytotoxicity. In order to develop an effective and less toxic compound, 4'-thio and 4'-seleno derivatives of EICAR and EICNR 3-6 were prepared. The resulting 4'-thioEICAR and 4'-thioEICNR showed inhibitory effect on DENV replication without cytotoxicity as potent as ribavirin, a positive control.

Capsaicin Induces ATF4 Translation with Upregulation of CHOP, GADD34 and PUMA.

Primary Effusion Lymphoma (PEL) is a rare and aggressive B-lymphoma caused by Kaposi's sarcoma-associated herpes virus (KSHV) infection that occurs in immunocompromised patients. PEL patients have a poor prognosis. KSHV modulates various cellular signaling pathways to maintain latent infection, and causes malignant conversion of host cells. We previously reported that capsaicin suppressed extracellular signal-regulated kinase (ERK) and p38 mitogen-activated protein kinase (MAPK) signaling and induced apoptosis in PEL. Generally, cellular stress such as nutrient starvation, oxidation and virus infection induce CCAAT/enhancer binding protein (C/EBP) homologous protein (CHOP) expression by activating transcription factor 4 (ATF4), however endoplasmic reticulum (ER) stress induces CHOP expression by both ATF4 and ATF6. CHOP is associated with apoptosis induction and upregulates growth arrest and DNA damage-inducible protein 34 (GADD34) and p53 up-regulated modulator of apoptosis (PUMA) mRNA expression. In this study, we found a new mechanism in which capsaicin induces apoptosis via ATF4-CHOP-PUMA. Capsaicin promoted transcriptional activation of CHOP, which increased mRNA expression of GADD34 and PUMA, resulting in PEL apoptosis. Furthermore, capsaicin increased ATF4 protein levels by promoting ATF4 translation, not transcription, and had no effect on ATF6-dependent transcriptional activation. In sum, capsaicin promotes ATF4 translation and transcriptional induction of CHOP, which results in PUMA expression and apoptosis in PEL cells.

Sulforaphane Exhibits Cytotoxic Effects against Primary Effusion Lymphoma Cells by Suppressing p38MAPK and AKT Phosphorylation.

Primary effusion lymphoma (PEL) is a rare subtype of non-Hodgkin's B-cell lymphoma and is caused by Kaposi's sarcoma-associated herpesvirus (KSHV) in immunosuppressed patients. PEL is an aggressive lymphoma and is frequently resistant to conventional chemotherapies. Sulforaphane (SFN), a natural compound found in cruciferous vegetables and broccoli sprouts, modulates signaling pathways and epigenetic gene expression. However, the anti-proliferative effects of SFN on PEL cells and the underlying mechanisms have not been identified. Here, we found that SFN decreased the viability of KSHV-infected PEL cells compared to KSHV-uninfected B-lymphoma cells. The anti-proliferative effects of SFN on PEL cells were mediated by apoptosis with activating caspases. In addition, SFN inhibited the phosphorylation of p38 mitogen-activated protein kinase (p38MAPK) and AKT in PEL cells. We also showed that p38MAPK and AKT inhibitors reduced PEL cell growth. The constitutive and/or transient activation of p38MAPK and AKT signaling are necessary for the survival and proliferation of PEL cells. Our data and previous literature indicate that SFN represses the phosphorylation of p38MAPK and AKT, which results in PEL cell apoptosis. Moreover, we investigated whether MG132 or sangivamycin (Sangi) in combination with SFN potentiated the cytotoxic effects of SFN on PEL cells. Compared to treatment with SFN alone, the addition of MG132 or Sangi enhanced the cytotoxic activity of SFN in a synergistic manner. In conclusion, the anti-proliferative effects of SFN indicate its potential as a new substance for the treatment of PEL.

Signal Transduction Pathways Associated with KSHV-Related Tumors.

Signal transduction pathways play a key role in the regulation of cell growth, cell differentiation, cell survival, apoptosis, and immune responses. Bacterial and viral pathogens utilize the cell signal pathways by encoding their own proteins or noncoding RNAs to serve their survival and replication in infected cells. Kaposi's sarcoma-associated herpesvirus (KSHV), also known as human herpesvirus 8 (HHV-8), is classified as a rhadinovirus in the γ-herpesvirus subfamily and was the eighth human herpesvirus to be discovered from Kaposi's sarcoma specimens. KSHV is closely associated with an endothelial cell malignancy, Kaposi's sarcoma, and B-cell malignancies, primary effusion lymphoma, and multicentric Castleman's disease. Recent studies have revealed that KSHV manipulates the cellular signaling pathways to achieve persistent infection, viral replication, cell proliferation, anti-apoptosis, and evasion of immune surveillance in infected cells. This chapter summarizes recent developments in our understanding of the molecular mechanisms used by KSHV to interact with the cell signaling machinery.