Highly sensitive and quantitative HiBiT-tagged Nipah virus-like particles: A platform for rapid antibody neutralization studies.

Biocontainment regulations restrict the research on NiV to BSL-4 laboratories, thus limiting the mechanistic studies related to viral entry and allied pathogenesis. Understanding the precise process of viral-particle production and host cell entry is critical for designing targeted therapies or particle-based vaccines. In this study, we have synthesized HiBiT-tagged-NiV-VLPs to ease in-vitro BSL-2 particle handling. We propose a simple yet effective approach of generating substantial amount of HiBiT-tagged NiV-VLPs in vitro by co-expressing viral structural proteins in HEK293T cells. Though homologous to parent virus, the incapacitated replication potential facilitates a BSL-2 handling of these particles. The inclusion of a highly sensitive HiBiT tag on these VLPs allows for a quick detection of viral binding and entry, as well as in assessing the efficiency of neutralizing antibodies in vitro using the NanoBiT technology. The HiBiT-tag binds in high affinity with LgBiT (Large BiT an 18 kDa fusion protein and complementary subunit of HiBiT peptide), and the resultant complex elicits high intensity luminescence in the presence of substrate. The VLPs produced were morphologically and functionally identical to the native virus, and the HiBiT-tag permitted their quick application in viral binding, entry, and antibody neutralization assays. "Thus, we report a simple setting for generating HiBiT-NiV VLPs which can be utilized in a BSL-2 laboratory, to concurrently quantify features of NiV assembly, binding and entry. This also offers an alternate-safe and effective platform for viral based antibody neutralization assays in vitro".

Biology of Variola Virus.

Variola virus is an anthroponotic agent that belongs to the orthopoxvirus family. It is an etiological agent of smallpox, an ancient disease that caused massive mortality of human populations. Twentieth century has witnessed the death of about 300 million people due to the unavailability of an effective vaccine. Early detection is the primary strategy to prevent an outbreak of smallpox. Variola virus forms the characteristic pus-filled pustules and centrifugal rash distribution in the infected patients while transmission occurs mainly through respiratory droplets during the early stage of infection. No antiviral drugs are approved for variola virus till date. Generation of first-generation vaccines helped in the eradication of smallpox which was declared by the World Health Organization.

Epstein-Barr Virus: Human Interactome Reveals New Molecular Insights into Viral Pathogenesis for Potential Therapeutics and Antiviral Drug Discovery.

Host-virus Protein-Protein Interactions (PPIs) play pivotal roles in biological processes crucial for viral pathogenesis and by extension, inform antiviral drug discovery and therapeutics innovations. Despite efforts to develop the Epstein-Barr virus (EBV)-host PPI network, there remain significant knowledge gaps and a limited number of interacting human proteins deciphered. Furthermore, understanding the dynamics of the EBV-host PPI network in the distinct lytic and latent viral stages remains elusive. In this study, we report a comprehensive map of the EBV-human protein interactions, encompassing 1752 human and 61 EBV proteins by integrating data from the public repository HPIDB (v3.0) as well as curated high-throughput proteomic data from the literature. To address the stage-specific nature of EBV infection, we generated two detailed subset networks representing the latent and lytic stages, comprising 747 and 481 human proteins, respectively. Functional and pathway enrichment analysis of these subsets uncovered the profound impact of EBV proteins on cancer. The identification of highly connected proteins and the characterization of intrinsically disordered and cancer-related proteins provide valuable insights into potential therapeutic targets. Moreover, the exploration of drug-protein interactions revealed notable associations between hub proteins and anticancer drugs, offering novel perspectives for controlling EBV pathogenesis. This study represents, to the best of our knowledge, the first comprehensive investigation of the two distinct stages of EBV infection using high-throughput datasets. This makes a contribution to our understanding of EBV-host interactions and provides a foundation for future drug discovery and therapeutic interventions.

Epstein-Barr virus infection controls the concentration of the intracellular antioxidant glutathione by upregulation of the glutamate transporter EAAT3 in tumor cells.

Epstein-Barr virus or human herpesvirus 4 (EBV/HHV-4) is an omnipresent oncovirus etiologically associated with various B-cell lymphomas and epithelial cancers. The malignant transformation associated with the persistent expression of viral proteins often deregulates the host cellular machinery and EBV infection is coupled to elevated levels of reactive oxygen species. Here, we investigated the role that the glutamate transporter EAAT3 plays in regulating the antioxidant system as a protective mechanism of EBV-infected cells against the virus-induced oxidative stress. Our study demonstrated that the expression of EAAT3 was upregulated and localized to the plasma membrane in EBV latently infected and de novo EBV-infected cells. EAAT3 was regulated by the transcription factor NFAT5 in the infected cells. Membrane localized EAAT3 was found to be involved in the transportation of glutamate from the extracellular space into the cell, as EAAT3 and NFAT5 inhibitors markedly reduced the levels of intracellular glutamate levels in EBV latently infected cells. Additionally, our data demonstrated a notable decrease in the intracellular glutathione levels following treatment with an EAAT3 inhibitor. Collectively, our results suggest that upregulation of the glutamate transporter EAAT3 is an adaptation of EBV-infected cells to maintain cellular redox homeostasis against the virus-induced oxidative stress, and that this cellular balance could be therapeutically destroyed by targeting EAAT3 to impede EBV-associated cancers.

Approaches and advances in the development of potential therapeutic targets and antiviral agents for the management of SARS-CoV-2 infection.

Virus onslaughts continue to spread fear and cause rampage across the world every now and then. The twenty first century is yet again witnessing a gross global pandemic, Coronavirus Disease 2019 (COVID-19) caused by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). Globally no vaccines or drug specific to COVID-19 is available. Corona viruses have been in mutual relationship with humans and other hosts over many decades though aggressive zoonotic strains have caused havoc. Zoonotic emergent corona viruses prior to SARS-COV-2 included severe acute respiratory syndrome coronavirus (SARS-CoV) and Middle East respiratory syndrome coronavirus (MERS-CoV), with the former leading to aggressive infectious spread and the later with high mortality rate. Although they emerged in the early period of the twenty first century, resilient biomedical and expertise in pharmaceutical domain could not appropriate any proprietary therapeutics. Studies envisaged towards curtailing their spread employed different stages of the virus life cycle with all zoonotic coronaviruses (CoVs) sharing genomic and structural similarities. Hence the strategies against SARS-CoV and MERS-CoV could prove effective against the recent outbreak of SAR-CoV-2. The review unravels key events involved in the lifecycle of SARS-CoV-2 while highlighting the possible avenues of therapy. The review also holds the scope in better understanding a broad-spectrum antivirals, monoclonal antibodies and small molecule inhibitors against viral glycoproteins, host cell receptor, viral mRNA synthesis, RNA-dependent RNA polymerase (RdRp) and viral proteases in order to design and develop antiviral drugs for SARS-CoV-2.

Proximity Ligation Assay (PLA) to Determine the Endosomal Localization of ESCRT Subunit in Virus-Infected Cells.

Proximity ligation assay (PLA) is a newly developed technique that outperforms the traditional immunoassays for visualizing the in situ endogenous protein-protein interactions and localizations and the activation of proteins in cell culture systems as well as in tissue sections. PLA, when combined with cellular marker staining, becomes a powerful approach to identify differential interaction of the proteins of endosomal sorting complex required for transport (ESCRT) at distinct stages of virus infection. In this chapter, we describe a PLA protocol to study the localization and interaction between the ESCRT protein TSG101 and endosomal markers in early stages of viral endocytosis in in vitro infected cells.

Insight into the Roles of E3 Ubiquitin Ligase c-Cbl, ESCRT Machinery, and Host Cell Signaling in Kaposi's Sarcoma-Associated Herpesvirus Entry and Trafficking.

Kaposi's sarcoma-associated herpesvirus (KSHV) in vitro infection of dermal endothelial cells begins with its binding to host cell surface receptor molecules such as heparan sulfate (HS), integrins (α3ß1, αVß3, and αVß5), xCT, and EphA2 receptor tyrosine kinase (EphA2R). These initial events initiate dynamic host protein-protein interactions involving a multimolecular complex of receptors, signal molecules (focal adhesion kinase [FAK], Src, phosphatidylinositol 3-kinase [PI3-K], and RhoA-GTPase), adaptors (c-Cbl, CIB1, Crk, p130Cas, and GEF-C3G), actin, and myosin II light chain that lead to virus entry via macropinocytosis. Here we discuss how KSHV hijacks c-Cbl, an E3 ubiquitin ligase, to monoubiquitinate the receptors and actin, which acts like a marker for trafficking (similar to zip codes), resulting in the recruitment of the members of the host endosomal sorting complexes required for transport (ESCRT) Hrs, Tsg101, EAP45, and the CHMP5 and -6 proteins (zip code readers) recognizing the ubiquitinated protein and adaptor machinery to traffic through the different endosomal compartments in the cytoplasm to initiate the macropinocytic process and infection.

ESCRT-I Protein Tsg101 Plays a Role in the Post-macropinocytic Trafficking and Infection of Endothelial Cells by Kaposi's Sarcoma-Associated Herpesvirus.

Kaposi's sarcoma-associated herpesvirus (KSHV) binding to the endothelial cell surface heparan sulfate is followed by sequential interactions with α3ß1, αVß3 and αVß5 integrins and Ephrin A2 receptor tyrosine kinase (EphA2R). These interactions activate host cell pre-existing FAK, Src, PI3-K and RhoGTPase signaling cascades, c-Cbl mediated ubiquitination of receptors, recruitment of CIB1, p130Cas and Crk adaptor molecules, and membrane bleb formation leading to lipid raft dependent macropinocytosis of KSHV into human microvascular dermal endothelial (HMVEC-d) cells. The Endosomal Sorting Complexes Required for Transport (ESCRT) proteins, ESCRT-0, -I, -II, and-III, play a central role in clathrin-mediated internalized ubiquitinated receptor endosomal trafficking and sorting. ESCRT proteins have also been shown to play roles in viral egress. We have recently shown that ESCRT-0 component Hrs protein associates with the plasma membrane during macropinocytosis and mediates KSHV entry via ROCK1 mediated phosphorylation of NHE1 and local membrane pH change. Here, we demonstrate that the ESCRT-I complex Tsg101 protein also participates in the macropinocytosis of KSHV and plays a role in KSHV trafficking. Knockdown of Tsg101 did not affect virus entry in HMVEC-d and human umbilical vein endothelial (HUVEC) cells but significantly inhibited the KSHV genome entry into the nucleus and consequently viral gene expression in these cells. Double and triple immunofluorescence, proximity ligation immunofluorescence and co-immuoprecipitation studies revealed the association of Tsg101 with the KSHV containing macropinosomes, and increased levels of Tsg101 association/interactions with EphA2R, c-Cbl, p130Cas and Crk signal molecules, as well as with upstream and downstream ESCRT components such as Hrs (ESCRT-0), EAP45 (ESCRT-II), CHMP6 (ESCRT-III) and CHMP5 (ESCRT-III) in the KSHV infected cells. Tsg101 was also associated with early (Rab5) and late endosomal (Rab7) stages of KSHV intracellular trafficking, and CHMP5 (ESCRT-III) was also associated with Rab 5 and Rab 7. Knockdown of Tsg101 significantly inhibited the transition of virus from early to late endosomes. Collectively, our studies reveal that Tsg101 plays a role in the trafficking of macropinocytosed KSHV in the endothelial cells which is essential for the successful viral genome delivery into the nucleus, viral gene expression and infection. Thus, ESCRT molecules could serve as therapeutic targets to combat KSHV infection.

Histone H2B-IFI16 Recognition of Nuclear Herpesviral Genome Induces Cytoplasmic Interferon-ß Responses.

IFI16 (gamma-interferon-inducible protein 16), a predominantly nuclear protein involved in transcriptional regulation, also functions as an innate immune response DNA sensor and induces the IL-1ß and antiviral type-1 interferon-ß (IFN-ß) cytokines. We have shown that IFI16, in association with BRCA1, functions as a sequence independent nuclear sensor of episomal dsDNA genomes of KSHV, EBV and HSV-1. Recognition of these herpesvirus genomes resulted in IFI16 acetylation, BRCA1-IFI16-ASC-procaspase-1 inflammasome formation, cytoplasmic translocation, and IL-1ß generation. Acetylated IFI16 also interacted with cytoplasmic STING and induced IFN-ß. However, the identity of IFI16 associated nuclear proteins involved in STING activation and the mechanism is not known. Mass spectrometry of proteins precipitated by anti-IFI16 antibodies from uninfected endothelial cell nuclear lysate revealed that histone H2B interacts with IFI16. Single and double proximity ligation microscopy, immunoprecipitation, EdU-genome labeled virus infection, and chromatin immunoprecipitation studies demonstrated that H2B is associated with IFI16 and BRCA1 in the nucleus in physiological conditions. De novo KSHV and HSV-1 infection as well as latent KSHV and EBV infection induces the cytoplasmic distribution of H2B-IFI16, H2B-BRCA1 and IFI16-ASC complexes. Vaccinia virus (dsDNA) cytoplasmic replication didn't induce the redistribution of nuclear H2B-IFI16 or H2B into the cytoplasm. H2B is critical in KSHV and HSV-1 genome recognition by IFI16 during de novo infection. Viral genome sensing by IFI16-H2B-BRCA1 leads to BRCA1 dependent recruitment of p300, and acetylation of H2B and IFI16. BRCA1 knockdown or inhibition of p300 abrogated the acetylation of H2B-IFI16 or H2B. Ran-GTP protein mediated the translocation of acetylated H2B and IFI16 to the cytoplasm along with BRCA1 that is independent of IFI16-ASC inflammasome. ASC knockdown didn't affect the acetylation of H2B, its cytoplasmic transportation, and the association of STING with IFI16 and H2B during KSHV infection. Absence of H2B didn't affect IFI16-ASC association and cytoplasmic distribution and thus demonstrating that IFI16-H2B complex is independent of IFI16-ASC-procaspase-1-inflammasome complex formed during infection. The H2B-IFI16-BRCA1 complex interacted with cGAS and STING in the cytoplasm leading to TBK1 and IRF3 phosphorylation, nuclear translocation of pIRF3 and IFN-ß production. Silencing of H2B, cGAS and STING inhibited IFN-ß induction but not IL-1ß secretion, and cGAMP activity is significantly reduced by H2B and IFI16 knockdown during infection. Silencing of ASC inhibited IL-1ß secretion but not IFN-ß secretion during de novo KSHV and HSV-1 infection. These studies identify H2B as an innate nuclear sensor mediating a novel extra chromosomal function, and reveal that two IFI16 complexes mediate KSHV and HSV-1 genome recognition responses, with recognition by the IFI16-BRCA1-H2B complex resulting in IFN-ß responses and recognition by IFI16-BRCA1 resulting in inflammasome responses.

ESCRT-0 Component Hrs Promotes Macropinocytosis of Kaposi's Sarcoma-Associated Herpesvirus in Human Dermal Microvascular Endothelial Cells.

UNLABELLED: Kaposi's sarcoma-associated herpesvirus (KSHV) enters human dermal microvascular endothelial cells (HMVEC-d), its naturalin vivotarget cells, by lipid raft-dependent macropinocytosis. The internalized viral envelope fuses with the macropinocytic membrane, and released capsid is transported to the nuclear vicinity, resulting in the nuclear entry of viral DNA. The endosomal sorting complexes required for transport (ESCRT) proteins, which include ESCRT-0, -I, -II, and -III, play a central role in endosomal trafficking and sorting of internalized and ubiquitinated receptors. Here, we examined the role of ESCRT-0 component Hrs (hepatocyte growth factor-regulated tyrosine kinase substrate) in KSHV entry into HMVEC-d by macropinocytosis. Knockdown of Hrs by short hairpin RNA (shRNA) transduction resulted in significant decreases in KSHV entry and viral gene expression. Immunofluorescence analysis (IFA) and plasma membrane isolation and proximity ligation assay (PLA) demonstrated the translocation of Hrs from the cytosol to the plasma membrane of infected cells and association with α-actinin-4. In addition, infection induced the plasma membrane translocation and activation of the serine/threonine kinase ROCK1, a downstream target of the RhoA GTPase. Hrs knockdown reduced these associations, suggesting that the recruitment of ROCK1 is an Hrs-mediated event. Interaction between Hrs and ROCK1 is essential for the ROCK1-induced phosphorylation of NHE1 (Na(+)/H(+)exchanger 1), which is involved in the regulation of intracellular pH. Thus, our studies demonstrate the plasma membrane association of ESCRT protein Hrs during macropinocytosis and suggest that KSHV entry requires both Hrs- and ROCK1-dependent mechanisms and that ROCK1-mediated phosphorylation of NHE1 and pH change is an essential event required for the macropinocytosis of KSHV. IMPORTANCE: Macropinocytosis is the major entry pathway of KSHV in human dermal microvascular endothelial cells, the natural target cells of KSHV. Although the role of ESCRT protein Hrs has been extensively studied with respect to endosomal movement and sorting of ubiquitinated proteins into lysosomes, its function in macropinocytosis is not known. In the present study, we demonstrate for the first time that upon KSHV infection, the endogenous Hrs localizes to the plasma membrane and the membrane-associated Hrs facilitates assembly of signaling molecules, macropinocytosis, and virus entry. Hrs recruits ROCK1 to the membrane, which is required for the activation of NHE1 and an increase in submembranous intracellular pH occurring during macropinocytosis. These studies demonstrate that the localization of Hrs from the cytosol to the plasma membrane is important for coupling membrane dynamics to the cytosolic signaling events during macropinocytosis of KSHV.

Herpesvirus Genome Recognition Induced Acetylation of Nuclear IFI16 Is Essential for Its Cytoplasmic Translocation, Inflammasome and IFN-ß Responses.

The IL-1ß and type I interferon-ß (IFN-ß) molecules are important inflammatory cytokines elicited by the eukaryotic host as innate immune responses against invading pathogens and danger signals. Recently, a predominantly nuclear gamma-interferon-inducible protein 16 (IFI16) involved in transcriptional regulation has emerged as an innate DNA sensor which induced IL-1ß and IFN-ß production through inflammasome and STING activation, respectively. Herpesvirus (KSHV, EBV, and HSV-1) episomal dsDNA genome recognition by IFI16 leads to IFI16-ASC-procaspase-1 inflammasome association, cytoplasmic translocation and IL-1ß production. Independent of ASC, HSV-1 genome recognition results in IFI16 interaction with STING in the cytoplasm to induce interferon-ß production. However, the mechanisms of IFI16-inflammasome formation, cytoplasmic redistribution and STING activation are not known. Our studies here demonstrate that recognition of herpesvirus genomes in the nucleus by IFI16 leads into its interaction with histone acetyltransferase p300 and IFI16 acetylation resulting in IFI16-ASC interaction, inflammasome assembly, increased interaction with Ran-GTPase, cytoplasmic redistribution, caspase-1 activation, IL-1ß production, and interaction with STING which results in IRF-3 phosphorylation, nuclear pIRF-3 localization and interferon-ß production. ASC and STING knockdowns did not affect IFI16 acetylation indicating that this modification is upstream of inflammasome-assembly and STING-activation. Vaccinia virus replicating in the cytoplasm did not induce nuclear IFI16 acetylation and cytoplasmic translocation. IFI16 physically associates with KSHV and HSV-1 genomes as revealed by proximity ligation microscopy and chromatin-immunoprecipitation studies which is not hampered by the inhibition of acetylation, thus suggesting that acetylation of IFI16 is not required for its innate sensing of nuclear viral genomes. Collectively, these studies identify the increased nuclear acetylation of IFI16 as a dynamic essential post-genome recognition event in the nucleus that is common to the IFI16-mediated innate responses of inflammasome induction and IFN-ß production during herpesvirus (KSHV, EBV, HSV-1) infections.

BRCA1 Regulates IFI16 Mediated Nuclear Innate Sensing of Herpes Viral DNA and Subsequent Induction of the Innate Inflammasome and Interferon-ß Responses.

The innate immune system pattern recognition receptors (PRR) are the first line of host defenses recognizing the various pathogen- or danger-associated molecular patterns and eliciting defenses by regulating the production of pro-inflammatory cytokines such as IL-1ß, IL-18 or interferon ß (IFN-ß). NOD-like receptors (NLRs) and AIM2-like receptors (ALRs) are cytoplasmic inflammasome sensors of foreign molecules, including DNA. IFI16, a sequence-independent nuclear innate sensor ALR, recognizes episomal dsDNA genomes of herpes viruses such as KSHV, EBV, and HSV-1 in the infected cell nuclei, forms an inflammasome complex with ASC and procaspase1, and relocates into the cytoplasm leading into Caspase-1 and IL-1ß generation. IFI16 also induces IFN-ß during HSV-1 infection via the cytoplasmic STING-TBK1-IRF3 pathway. Thus far, whether IFI16 recognizes foreign DNA directly or utilizes other host protein(s) is unknown. Here, we demonstrate that BRCA1, a DNA damage repair sensor and transcription regulator, is in complex with IFI16 in the host cell nucleus, and their association increases in the presence of nuclear viral genomes during de novo KSHV, EBV and HSV-1 infection, and in latent KSHV or EBV infection, but not by DNA damage responses (DDR) induced by bleomycin and vaccinia virus cytoplasmic dsDNA. BRCA1 is a constituent of the triggered IFI16-inflammasome and is translocated into the cytoplasm after genome recognition along with the IFI16-inflammasome. The absence of BRCA1 abrogated IFI16-viral genome association, inflammasome assembly, IFI16 cytoplasmic localization, and Caspase-1 and IL-1ß production. The absence of BRCA1 also abolished the cytoplasmic IFI16-STING interaction, downstream IRF3 phosphorylation, nuclear translocation of pIRF3 and IFN-ß production during de novo KSHV and HSV-1 infection. These findings highlight that BRCA1 plays a hitherto unidentified innate immunomodulatory role by facilitating nuclear foreign DNA sensing by IFI16, subsequent assembly and cytoplasmic distribution of IFI16-inflammasomes leading into IL-1ß formation and the induction of IFN-ß via cytoplasmic signaling through IFI16-STING, TBK1 and IRF3.

Activated Nrf2 Interacts with Kaposi's Sarcoma-Associated Herpesvirus Latency Protein LANA-1 and Host Protein KAP1 To Mediate Global Lytic Gene Repression.

UNLABELLED: Kaposi's sarcoma-associated herpesvirus (KSHV) is etiologically associated with Kaposi's sarcoma (KS), primary effusion lymphoma (PEL), and multicentric Castleman's disease. We have previously shown that KSHV utilizes the host transcription factor Nrf2 to aid in infection of endothelial cells and oncogenesis. Here, we investigate the role of Nrf2 in PEL and PEL-derived cell lines and show that KSHV latency induces Nrf2 protein levels and transcriptional activity through the COX-2/PGE2/EP4/PKCζ axis. Next-generation sequencing of KSHV transcripts in the PEL-derived BCBL-1 cell line revealed that knockdown of this activated Nrf2 results in global elevation of lytic genes. Nrf2 inhibition by the chemical brusatol also induces lytic gene expression. Both Nrf2 knockdown and brusatol-mediated inhibition induced KSHV lytic reactivation in BCBL-1 cells. In a series of follow-up experiments, we characterized the mechanism of Nrf2-mediated regulation of KSHV lytic repression during latency. Biochemical assays showed that Nrf2 interacted with KSHV latency-associated nuclear antigen 1 (LANA-1) and the host transcriptional repressor KAP1, which together have been shown to repress lytic gene expression. Promoter studies showed that although Nrf2 alone induces the open reading frame 50 (ORF50) promoter, its association with LANA-1 and KAP1 abrogates this effect. Interestingly, LANA-1 is crucial for efficient KAP1/Nrf2 association, while Nrf2 is essential for LANA-1 and KAP1 recruitment to the ORF50 promoter and its repression. Overall, these results suggest that activated Nrf2, LANA-1, and KAP1 assemble on the ORF50 promoter in a temporal fashion. Initially, Nrf2 binds to and activates the ORF50 promoter during early de novo infection, an effect that is exploited during latency by LANA-1-mediated recruitment of the host transcriptional repressor KAP1 on Nrf2. Cell death assays further showed that Nrf2 and KAP1 knockdown induce significant cell death in PEL cell lines. Our studies suggest that Nrf2 modulation through available oral agents is a promising therapeutic approach in the treatment of KSHV-associated malignancies. IMPORTANCE: KS and PEL are aggressive KSHV-associated malignancies with moderately effective, highly toxic chemotherapies. Other than ganciclovir and alpha interferon (IFN-α) prophylaxis, no KSHV-associated chemotherapy targets the underlying infection, a major oncogenic force. Hence, drugs that selectively target KSHV infection are necessary to eradicate the malignancy while sparing healthy cells. We recently showed that KSHV infection of endothelial cells activates the transcription factor Nrf2 to promote an environment conducive to infection and oncogenesis. Nrf2 is modulated through several well-tolerated oral agents and may be an important target in KSHV biology. Here, we investigate the role of Nrf2 in PEL and demonstrate that Nrf2 plays an important role in KSHV gene expression, lytic reactivation, and cell survival by interacting with the host transcriptional repressor KAP1 and the viral latency-associated protein LANA-1 to mediate global lytic gene repression and thus cell survival. Hence, targeting Nrf2 with available therapies is a viable approach in the treatment of KSHV malignancies.

Kaposi's sarcoma-associated herpesvirus induces Nrf2 activation in latently infected endothelial cells through SQSTM1 phosphorylation and interaction with polyubiquitinated Keap1.

UNLABELLED: Nuclear factor erythroid 2-related factor 2 (Nrf2), the cellular master regulator of the antioxidant response, dissociates from its inhibitor Keap1 when activated by stress signals and participates in the pathogenesis of viral infections and tumorigenesis. Early during de novo infection of endothelial cells, KSHV induces Nrf2 through an intricate mechanism involving reactive oxygen species (ROS) and prostaglandin E2 (PGE2). When we investigated the Nrf2 activity during latent KSHV infection, we observed increased nuclear serine-40-phosphorylated Nrf2 in human KS lesions compared to that in healthy tissues. Using KSHV long-term-infected endothelial cells (LTC) as a cellular model for KS, we demonstrated that KSHV infection induces Nrf2 constitutively by extending its half-life, increasing its phosphorylation by protein kinase Cζ (PKCζ) via the infection-induced cyclooxygenase-2 (COX-2)/PGE2 axis and inducing its nuclear localization. Nrf2 knockdown in LTC decreased expression of antioxidant genes and genes involved in KS pathogenesis such as the NAD(P)H quinone oxidase 1 (NQO1), gamma glutamylcysteine synthase heavy unit (γGCSH), the cysteine transporter (xCT), interleukin 6 (IL-6), and vascular endothelial growth factor A (VEGF-A) genes. Nrf2 activation was independent of oxidative stress but dependent on the autophagic protein sequestosome-1 (SQSTM1; p62). SQSTM1 levels were elevated in LTC, a consequence of protein accumulation due to decreased autophagy and Nrf2-mediated transcriptional activation. SQSTM1 was phosphorylated on serine-351 and -403, while Keap1 was polyubiquitinated with lysine-63-ubiquitin chains, modifications known to increase their mutual affinity and interaction, leading to Keap1 degradation and Nrf2 activation. The latent KSHV protein Fas-associated death domain-like interleukin-1ß-converting enzyme-inhibitory protein (vFLIP) increased SQSTM1 expression and activated Nrf2. Collectively, these results demonstrate that KSHV induces SQSTM1 to constitutively activate Nrf2, which is involved in the regulation of genes participating in KSHV oncogenesis. IMPORTANCE: The transcription factor Nrf2 is activated by stress signals, including viral infection, and responds by activating the transcription of cytoprotective genes. Recently, Nrf2 has been implicated in oncogenesis and was shown to be activated during de novo KSHV infection of endothelial cells through ROS-dependent pathways. The present study was undertaken to determine the mechanism of Nrf2 activation during prolonged latent infection of endothelial cells, using an endothelial cell line latently infected with KSHV. We show that Nrf2 activation was elevated in KSHV latently infected endothelial cells independently of oxidative stress but dependent on the autophagic protein sequestosome-1 (SQSTM1), which was involved in the degradation of the Nrf2 inhibitor Keap1. Furthermore, our results indicated that the KSHV latent protein vFLIP participates in Nrf2 activation. This study suggests that KSHV hijacks the host's autophagic protein SQSTM1 to induce Nrf2 activation, thereby manipulating the infected host gene regulation to promote KS pathogenesis.

Interaction of KSHV with host cell surface receptors and cell entry.

Virus entry is a complex process characterized by a sequence of events. Since the discovery of KSHV in 1994, tremendous progress has been made in our understanding of KSHV entry into its in vitro target cells. KSHV entry is a complex multistep process involving viral envelope glycoproteins and several cell surface molecules that is utilized by KSHV for its attachment and entry. KSHV has a broad cell tropism and the attachment and receptor engagement on target cells have an important role in determining the cell type-specific mode of entry. KSHV utilizes heparan sulfate, integrins and EphrinA2 molecules as receptors which results in the activation of host cell pre-existing signal pathways that facilitate the subsequent cascade of events resulting in the rapid entry of virus particles, trafficking towards the nucleus followed by viral and host gene expression. KSHV enters human fibroblast cells by dynamin dependant clathrin mediated endocytosis and by dynamin independent macropinocytosis in dermal endothelial cells. Once internalized into endosomes, fusion of the viral envelope with the endosomal membranes in an acidification dependent manner results in the release of capsids which subsequently reaches the nuclear pore vicinity leading to the delivery of viral DNA into the nucleus. In this review, we discuss the principal mechanisms that enable KSHV to interact with the host cell surface receptors as well as the mechanisms that are required to modulate cell signaling machinery for a successful entry.

p130Cas scaffolds the signalosome to direct adaptor-effector cross talk during Kaposi's sarcoma-associated herpesvirus trafficking in human microvascular dermal endothelial cells.

UNLABELLED: Kaposi's sarcoma-associated herpesvirus (KSHV) interacts with cell surface receptors, such as heparan sulfate, integrins (α3ß1, αVß3, and αVß5), and EphrinA2 (EphA2), and activates focal adhesion kinase (FAK), Src, phosphoinositol 3-kinase (PI3-K), c-Cbl, and RhoA GTPase signal molecules early during lipid raft (LR)-dependent productive macropinocytic entry into human dermal microvascular endothelial cells. Our recent studies have identified CIB1 as a signal amplifier facilitating EphA2 phosphorylation and subsequent cytoskeletal cross talk during KSHV macropinocytosis. Although CIB1 lacks an enzymatic activity and traditional adaptor domain or known interacting sequence, it associated with the KSHV entry signal complex and the CIB1-KSHV association was sustained over 30 min postinfection. To identify factors scaffolding the EphA2-CIB1 signal axis, the role of major cellular scaffold protein p130Cas (Crk-associated substrate of Src) was investigated. Inhibitor and small interfering RNA (siRNA) studies demonstrated that KSHV induced p130Cas in an EphA2-, CIB1-, and Src-dependent manner. p130Cas and Crk were associated with KSHV, LRs, EphA2, and CIB1 early during infection. Live-cell microscopy and biochemical studies demonstrated that p130Cas knockdown did not affect KSHV entry but significantly reduced productive nuclear trafficking of viral DNA and routed KSHV to lysosomal degradation. p130Cas aided in scaffolding adaptor Crk to downstream guanine nucleotide exchange factor phospho-C3G possibly to coordinate GTPase signaling during KSHV trafficking. Collectively, these studies demonstrate that p130Cas acts as a bridging molecule between the KSHV-induced entry signal complex and the downstream trafficking signalosome in endothelial cells and suggest that simultaneous targeting of KSHV entry receptors with p130Cas would be an attractive potential avenue for therapeutic intervention in KSHV infection. IMPORTANCE: Eukaryotic cell adaptor molecules, without any intrinsic enzymatic activity, are well known to allow a great diversity of specific and coordinated protein-protein interactions imparting signal amplification to different networks for physiological and pathological signaling. They are involved in integrating signals from growth factors, extracellular matrix molecules, bacterial pathogens, and apoptotic cells. The present study identifies human microvascular dermal endothelial (HMVEC-d) cellular scaffold protein p130Cas (Crk-associated substrate) as a platform to promote Kaposi's sarcoma-associated herpesvirus (KSHV) trafficking. Early during KSHV de novo infection, p130Cas associates with lipid rafts and scaffolds EphrinA2 (EphA2)-associated critical adaptor members to downstream effector molecules, promoting successful nuclear delivery of the KSHV genome. Hence, simultaneous targeting of the receptor EphA2 and scaffolding action of p130Cas can potentially uncouple the signal cross talk of the KSHV entry-associated upstream signal complex from the immediate downstream trafficking-associated signalosome, consequently routing KSHV toward lysosomal degradation and eventually blocking KSHV infection and associated malignancies.

The Kaposi's sarcoma-associated herpesvirus (KSHV)-induced 5-lipoxygenase-leukotriene B4 cascade plays key roles in KSHV latency, monocyte recruitment, and lipogenesis.

Kaposi's sarcoma-associated herpesvirus (KSHV) is etiologically associated with Kaposi's sarcoma (KS) and primary effusion lymphoma (PEL). KS lesions are characterized by endothelial cells with multiple copies of the latent KSHV episomal genome, lytic replication in a low percentage of infiltrating monocytes, and inflammatory cytokines plus growth factors. We demonstrated that KSHV utilizes inflammatory cyclooxygenase 2/prostaglandin E2 to establish and maintain latency (Sharma-Walia, N., A. G. Paul, V. Bottero, S. Sadagopan, M. V. Veettil, N. Kerur, and B. Chandran, PLoS Pathog 6:e1000777, 2010 [doi:10.1371/journal.ppat.1000777]). Here, we evaluated the role of 5-lipoxygenase (5LO) and its chemotactic metabolite leukotriene B4 (LTB4) in KSHV biology. Abundant staining of 5LO was detected in human KS tissue sections. We observed elevated levels of 5LO and high levels of secretion of LTB4 during primary KSHV infection of endothelial cells and in PEL B cells (BCBL-1 and BC-3 cells). Blocking the 5LO/LTB4 cascade inhibited viral latent ORF73, immunomodulatory K5, viral macrophage inflammatory protein 1 (MIP-1), and viral MIP-2 gene expression, without much effect on lytic switch ORF50, immediate early lytic K8, and viral interferon-regulatory factor 2 gene expression. 5LO inhibition significantly downregulated latent viral Cyclin and latency-associated nuclear antigen 2 levels in PEL cells. 5LO/LTB4 inhibition downregulated TH2-related cytokine secretion, elevated TH1-related cytokine secretion, and reduced human monocyte recruitment, adhesion, and transendothelial migration. 5LO/LTB4 inhibition reduced fatty acid synthase (FASN) promoter activity and its expression. Since FASN, a key enzyme required in lipogenesis, is important in KSHV latency, these findings collectively suggest that 5LO/LTB4 play important roles in KSHV biology and that effective inhibition of the 5LO/LTB4 pathway could potentially be used in treatment to control KS/PEL.

Kaposi's sarcoma-associated herpesvirus-positive primary effusion lymphoma tumor formation in NOD/SCID mice is inhibited by neomycin and neamine blocking angiogenin's nuclear translocation.

Angiogenin (ANG) is a 14-kDa multifunctional proangiogenic secreted protein whose expression level correlates with the aggressiveness of several tumors. We observed increased ANG expression and secretion in endothelial cells during de novo infection with Kaposi's sarcoma-associated herpesvirus (KSHV), in cells expressing only latency-associated nuclear antigen 1 (LANA-1) protein, and in KSHV latently infected primary effusion lymphoma (PEL) BCBL-1 and BC-3 cells. Inhibition of phospholipase Cγ (PLCγ) mediated ANG's nuclear translocation by neomycin, an aminoglycoside antibiotic (not G418-neomicin), resulted in reduced KSHV latent gene expression, increased lytic gene expression, and increased cell death of KSHV(+) PEL and endothelial cells. ANG detection in significant levels in KS and PEL lesions highlights its importance in KSHV pathogenesis. To assess the in vivo antitumor activity of neomycin and neamine (a nontoxic derivative of neomycin), BCBL-1 cells were injected intraperitoneally into NOD/SCID mice. We observed significant extended survival of mice treated with neomycin or neamine. Markers of lymphoma establishment, such as increases in animal body weight, spleen size, tumor cell spleen infiltration, and ascites volume, were observed in nontreated animals and were significantly diminished by neomycin or neamine treatments. A significant decrease in LANA-1 expression, an increase in lytic gene expression, and an increase in cleaved caspase-3 were also observed in neomycin- or neamine-treated animal ascitic cells. These studies demonstrated that ANG played an essential role in KSHV latency maintenance and BCBL-1 cell survival in vivo, and targeting ANG function by neomycin/neamine to induce the apoptosis of cells latently infected with KSHV is an attractive therapeutic strategy against KSHV-associated malignancies.