Properties and Functions of Feline Immunodeficiency Virus Gag Domains in Virion Assembly and Budding.

Feline immunodeficiency virus (FIV) is an important cat pathogen worldwide whose biological and pathophysiological properties resemble those of human immunodeficiency virus type 1 (HIV-1). Therefore, the study of FIV not only benefits its natural host but is also useful for the development of antiviral strategies directed against HIV-1 infections in humans. FIV assembly results from the multimerization of a single but complex viral polypeptide, the Gag precursor. In this review, we will first give an overview of the current knowledge of the proteins encoded by the FIV pol, env, rev, vif, and orf-A genes, and then we will describe and discuss in detail the critical roles that each of the FIV Gag domains plays in virion morphogenesis. Since retroviral assembly is an attractive target for therapeutic interventions, gaining a better understanding of this process is highly desirable.

Th17 cytokine profiling of colorectal cancer patients with or without enterovirus 71 antigen expression.

OBJECT: Th17 cytokines have been identified in several types of human cancers. In this pilot study, the expression of Th17 cytokines profiling in enteroviruses 71 (EV71) associated colorectal cancer (CRC) were explored. METHODS: 66 patients with CRC were enrolled in this study; immune- histochemical analyses were performed on cancerous tissues and adjacent non- cancerous tissues of the patients. Serum Th17 cytokines of CRC patients and healthy controls were measured using a Luminex 200 analyzer. RESULTS: Cancerous tissues had more positive EV71 antigen expression than adjacent non- cancerous tissues. In TNM II-III CRC, 59.9% of cancerous tissues were observed to be EV71 positive; on the contrary, 65.2% of the adjacent non- cancerous epithelium was EV71 negative. In TNM I CRC, all adjacent non- cancerous epithelium was virus negative, but in TNM IV, half of adjacent non- cancerous tissues were virus positive. Serum IL-10 were significantly higher in CRC patients than in healthy controls, and IL-10 concentrations in the EV71 positive group were higher than those of the EV71 negative group, with the highest IL-10 levels being observed in CRC patients with strong positive group (P < 0.05). Similar results were found for IL-21 and IL-23. IL-17 levels were higher in CRC patients than in healthy controls, there was no significant difference in IL-17 between the viral positive and viral negative groups (P > 0.05). CONCLUSION: Persistent existing EV71 viral antigens in intestinal tissues are positively associated with TNM III/IV CRC. EV71 latent infection recruits Th17 cells in the colorectal tumor site, stimulating Th17 cytokine production that closely associated with CRC carcinogenesis.

Kaposi's sarcoma-associated herpesvirus LANA recruits the DNA polymerase clamp loader to mediate efficient replication and virus persistence.

Kaposi's sarcoma-associated herpesvirus (KSHV) latently infects tumor cells and persists as a multiple-copy, extrachromosomal, circular episome. To persist, the viral genome must replicate with each cell cycle. The KSHV latency-associated nuclear antigen (LANA) mediates viral DNA replication and persistence, but little is known regarding the underlying mechanisms. We find that LANA recruits replication factor C (RFC), the DNA polymerase clamp [proliferating cell nuclear antigen (PCNA)] loader, to drive DNA replication efficiently. Mutated LANA lacking RFC interaction was deficient for LANA-mediated DNA replication and episome persistence. RFC depletion had a negative impact on LANA's ability to replicate and maintain viral DNA in cells containing artificial KSHV episomes or in infected cells, leading to loss of virus. LANA substantially increased PCNA loading onto DNA in vitro and recruited RFC and PCNA to KSHV DNA in cells. These findings suggest that PCNA loading is a rate-limiting step in DNA replication that is incompatible with viral survival. LANA enhancement of PCNA loading permits efficient virus replication and persistence, revealing a previously unidentified mechanism for KSHV latency.

Dengue viral protease interaction with NF-κB inhibitor α/ß results in endothelial cell apoptosis and hemorrhage development.

Hemorrhagic manifestations occur frequently accompanying a wide range of dengue disease syndromes. Much work has focused on the contribution of immune factors to the pathogenesis of hemorrhage, but how dengue virus (DENV) participates in the pathogenic process has never been explored. Although there is no consensus that apoptosis is the basis of vascular permeability in human dengue infections, we showed in dengue hemorrhage mouse model that endothelial cell apoptosis is important to hemorrhage development in mice. To explore the molecular basis of the contribution of DENV to endothelial cell death, we show in this study that DENV protease interacts with cellular IκBα and IκBß and cleaves them. By inducing IκBα and IκBß cleavage and IκB kinase activation, DENV protease activates NF-κB, which results in endothelial cell death. Intradermal inoculation of DENV protease packaged in adenovirus-associated virus-9 induces endothelial cell death and dermal hemorrhage in mice. Although the H51 activity site is not involved in the interaction between DENV protease and IκB-α/ß, the enzymatic activity is critical to the ability of DENV protease to induce IκBα and IκBß cleavage and trigger hemorrhage development. Moreover, overexpression of IκBα or IκBß protects endothelial cells from DENV-induced apoptosis. In this study, we show that DENV protease participates in the pathogenesis of dengue hemorrhage and discover IκBα and IκBß to be the new cellular targets that are cleaved by DENV protease.

Latency-associated nuclear antigen of Kaposi sarcoma-associated herpesvirus promotes angiogenesis through targeting notch signaling effector Hey1.

Notch signaling has been implicated in the pathogenesis of Kaposi sarcoma. Kaposi sarcoma is an angioproliferative neoplasm that originates from Kaposi sarcoma-associated herpesvirus (KSHV) infection. Previously, we showed that the KSHV LANA protein can stabilize intracellular Notch in KSHV-infected tumor cells and promote cell proliferation. However, whether Notch signaling functions in pathologic angiogenesis of Kaposi sarcoma remains largely unknown. Hey1, an essential downstream effector of the Notch signaling pathway, has been demonstrated to play a fundamental role in vascular development. In the present study, we performed whole transcriptome, paired-end sequencing on three patient-matched clinical Kaposi sarcoma specimens and their corresponding adjacent stroma samples, with an average depth of 42 million reads per sample. Dll4, Hey1, and HeyL displayed significant upregulation in Kaposi sarcoma. Further verification based on immunohistochemistry analysis demonstrated that Hey1 was indeed highly expressed in Kaposi sarcoma lesions. Using the Matrigel plug assay, we showed that downregulation of Hey1 and γ-secretase inhibitor treatment caused dramatic reduction in the formation of new blood vessels in mice. Interestingly, LANA was responsible for the elevated level of Hey1 through inhibition of its degradation. Importantly, Hey1 stabilized by LANA promoted the neoplastic vasculature. Taken together, our data suggest that hijacking of the proangiogenic property of Hey1 by LANA is an important strategy utilized by KSHV to achieve pathologic angiogenesis and that Hey1 is a potential therapeutic target in Kaposi sarcoma.

Recruitment of the tumour suppressor protein p73 by Kaposi's Sarcoma Herpesvirus latent nuclear antigen contributes to the survival of primary effusion lymphoma cells.

Kaposi's Sarcoma Herpesvirus (KSHV) is the causative agent of Kaposi's Sarcoma (KS) and two rare lymphoproliferative disorders, primary effusion lymphoma (PEL) and the plasmablastic variant of multicentric Castleman's disease (MCD). The KSHV latency-associated nuclear antigen-1 (LANA), required for the replication and maintenance of latent viral episomal DNA, is involved in the transcriptional regulation of viral and cellular genes and interacts with different cellular proteins, including the tumour suppressor p53. Here, we report that LANA also recruits the p53-related nuclear transcription factor p73, which influences cellular processes like DNA damage response, cell cycle progression and apoptosis. Both the full-length isoform TAp73α, as well as its dominant negative regulator ΔNp73α, interact with LANA. LANA affects TAp73α stability and sub-nuclear localisation, as well as TAp73α-mediated transcriptional activation of target genes. We observed that the small-molecule inhibitor Nutlin-3, which disrupts the interaction of p53 and p73 with MDM2, induces apoptotic cell death in p53 wild-type, as well as p53-mutant PEL cell lines, suggesting a possible involvement of p73. The small-molecule RETRA, which activates p73 in the context of mutant p53, leads to the induction of apoptosis in p53-mutant PEL cell lines. RNAi-mediated knockdown of p73 confirmed that these effects depend on the presence of the p73 protein. Furthermore, both Nutlin-3 and RETRA disrupt the LANA-p73 interaction in different PEL cell lines. These results suggest that LANA modulates p73 function and that the LANA-p73 interaction may represent a therapeutic target to interfere with the survival of latently KSHV-infected cells.

Unbiased mutagenesis of MHV68 LANA reveals a DNA-binding domain required for LANA function in vitro and in vivo.

The Latency-Associated Nuclear Antigen (LANA), encoded by ORF73, is a conserved gene among the γ2-herpesviruses (rhadinoviruses). The Kaposi's Sarcoma-Associated Herpesvirus (KSHV) LANA is consistently expressed in KSHV-associated malignancies. In the case of the rodent γ2-herpesvirus, murine gammaherpesvirus 68 (MHV68), the LANA homolog (mLANA) is required for efficient virus replication, reactivation from latency and immortalization of murine fetal liver-derived B cells. To gain insights into mLANA function(s), knowing that KSHV LANA binds DNA and can modulate transcription of a variety of promoters, we sought out and identified a mLANA-responsive promoter which maps to the terminal repeat (TR) of MHV68. Notably, mLANA strongly repressed activity from this promoter. We extended these analyses to demonstrate direct, sequence-specific binding of recombinant mLANA to TR DNA by DNase I footprinting. To assess whether the DNA-binding and/or transcription modulating function is important in the known mLANA phenotypes, we generated an unbiased library of mLANA point mutants using error-prone PCR, and screened a large panel of mutants for repression of the mLANA-responsive promoter to identify loss of function mutants. Notably, among the mutant mLANA proteins recovered, many of the mutations are in a predicted EBNA-1-like DNA-binding domain. Consistent with this prediction, those tested displayed loss of DNA binding activity. We engineered six of these mLANA mutants into the MHV68 genome and tested the resulting mutant viruses for: (i) replication fitness; (ii) efficiency of latency establishment; and (iii) reactivation from latency. Interestingly, each of these mLANA-mutant viruses exhibited phenotypes similar to the mLANA-null mutant virus, indicating that DNA-binding is critical for mLANA function.

Quantitative analysis of the bidirectional viral G-protein-coupled receptor and lytic latency-associated nuclear antigen promoter of Kaposi's sarcoma-associated herpesvirus.

Kaposi's sarcoma-associated herpesvirus (KSHV) establishes sustained latent persistence in susceptible cells. This is dependent on the latency-associated nuclear antigen (LANA). Understanding how LANA transcription is regulated thus aids our fundamental understanding of KSHV biology. Two hundred ninety-four base pairs are sufficient to regulate LANA transcription in response to the viral RTA protein and RBPjκ. The same region controls K14/viral G-protein-coupled receptor (vGPCR) transcription in the opposite direction. We used a quantitative analysis in conjunction with specific nucleotide substitutions and defined gain-of-function and loss-of-function RTA mutants to dissect this region. We used a bidirectional reporter driving red and green luciferase to study the LANApi and K14p promoters simultaneously. This established that LANApi/K14p functions as a canonical bidirectional promoter. Both were TATA dependent. K14p was favored by ∼50-fold in this context. Eliminating the distal LANApi TATA box increased maximal output and lowered the induction threshold (T) of K14p even further. Two RBPjκ binding sites were independently required; however, at high concentrations of RTA, direct interactions with an RTA-responsive element (RRE) could complement the loss of one RBPjκ binding site. Intracellular Notch (ICN) was no longer able to activate RBPjκ in the viral context. This suggests a model whereby KSHV alters ICN-RBPjκ gene regulation. When the architecture of this pair of head-to-head RBPjκ binding sites is changed, the sites now respond exclusively to the viral transactivator RTA and no longer to the host mediator ICN.

Tissue factor and glycoprotein C on herpes simplex virus type 1 are protease-activated receptor 2 cofactors that enhance infection.

The coagulation system provides physiologic host defense, but it can also be exploited by pathogens for infection. On the HSV1 surface, host-cell-derived tissue factor (TF) and virus-encoded glycoprotein C (gC) can stimulate protease activated receptor 1 (PAR1)-enhanced infection by triggering thrombin production. Using novel engineered HSV1 variants deficient in either TF and/or gC, in the present study, we show that activated coagulation factors X (FXa) or VII (FVIIa) directly affect HSV1 infection of human umbilical vein endothelial cells in a manner that is dependent on viral TF and gC. The combination of FXa and FVIIa maximally enhanced infection for TF(+)/gC(+) HSV1 and receptor desensitization and Ab inhibition demonstrated that both proteases act on PAR2. Inhibitory TF Abs showed that the required TF source was viral. Individually, TF or gC partly enhanced the effect of FXa, but not FVIIa, revealing gC as a novel PAR2 cofactor for FVIIa. In sharp contrast, thrombin enhanced infection via PAR1 independently of viral TF and gC. Thrombin combined with FXa/FVIIa enhanced infection, suggesting that PAR1 and PAR2 are independently involved in virus propagation. These results show that HSV1 surface cofactors promote cellular PAR2-mediated infection, indicating a novel mode by which pathogens exploit the initiation phase of the host hemostatic system.

Epstein-Barr virus-associated B-cell lymphomas: pathogenesis and clinical outcomes.

Epstein-Barr virus (EBV) is a ubiquitous human γ-herpesvirus that establishes a life-long asymptomatic infection in immunocompetent hosts. It is also found to be frequently associated with a broad spectrum of B-cell lymphomas predominantly seen in immunodeficient patients. Despite many resemblances, these EBV-linked lymphoproliferative disorders display heterogeneity at the clinical and the molecular level. Moreover, EBV-associated lymphoproliferative diseases differ in their differential expression patterns of the EBV-encoded latent antigens, which are directly related to their interactions with the host. EBV-driven primary B-cell immortalization is linked to the cooperative functions of these latent proteins, which are critical for perturbing many important cell-signaling pathways maintaining B-cell proliferation. Additionally, it is used as a surrogate model to explore the underlying mechanisms involved in the development of B-cell neoplasms. Recent discoveries have revealed that a number of sophisticated mechanisms are exploited by EBV during cancer progression. This finding will be instrumental in the design of novel approaches for therapeutic interventions against EBV-associated B-cell lymphomas. This review limits the discussion to the biology and pathogenesis of EBV-associated B-cell lymphomas and the related clinical implications.

Epstein-Barr virus nuclear antigen 3C facilitates G1-S transition by stabilizing and enhancing the function of cyclin D1.

EBNA3C, one of the Epstein-Barr virus (EBV)-encoded latent antigens, is essential for primary B-cell transformation. Cyclin D1, a key regulator of G1 to S phase progression, is tightly associated and aberrantly expressed in numerous human cancers. Previously, EBNA3C was shown to bind to Cyclin D1 in vitro along with Cyclin A and Cyclin E. In the present study, we provide evidence which demonstrates that EBNA3C forms a complex with Cyclin D1 in human cells. Detailed mapping experiments show that a small N-terminal region which lies between amino acids 130-160 of EBNA3C binds to two different sites of Cyclin D1- the N-terminal pRb binding domain (residues 1-50), and C-terminal domain (residues 171-240), known to regulate Cyclin D1 stability. Cyclin D1 is short-lived and ubiquitin-mediated proteasomal degradation has been targeted as a means of therapeutic intervention. Here, we show that EBNA3C stabilizes Cyclin D1 through inhibition of its poly-ubiquitination, and also increases its nuclear localization by blocking GSK3ß activity. We further show that EBNA3C enhances the kinase activity of Cyclin D1/CDK6 which enables subsequent ubiquitination and degradation of pRb. EBNA3C together with Cyclin D1-CDK6 complex also efficiently nullifies the inhibitory effect of pRb on cell growth. Moreover, an sh-RNA based strategy for knock-down of both cyclin D1 and EBNA3C genes in EBV transformed lymphoblastoid cell lines (LCLs) shows a significant reduction in cell-growth. Based on these results, we propose that EBNA3C can stabilize as well as enhance the functional activity of Cyclin D1 thereby facilitating the G1-S transition in EBV transformed lymphoblastoid cell lines.

Provision of continuous maturation signaling to dendritic cells by RIG-I-stimulating cytosolic RNA synthesis of Sendai virus.

Dendritic cell (DC)-based immunotherapy has potential for treating infections and malignant tumors, but the functional capacity of DC must be assessed in detail, especially maturation and Ag-specific CTL priming. Recent reports suggest that DC that are provided with continuous maturation signals in vivo after transfer into patients are required to elicit the full DC functions. We demonstrate in this study that the rSendai virus vector (SeV) is a novel and ideal stimulant, providing DC with a continuous maturation signal via viral RNA synthesis in the cytosol, resulting in full maturation of monocyte-derived DC(s). Both RIG-I-dependent cytokine production and CD4 T cell responses to SeV-derived helper Ags are indispensable for overcoming regulatory T cell suppression to prime melanoma Ag recognized by T cell-1-specific CTL in the regulatory T cell abundant setting. DC stimulated via cytokine receptors, or TLRs, do not show these functional features. Therefore, SeV-infected DC have the potential for DC-directed immunotherapy.

Extensive co-operation between the Epstein-Barr virus EBNA3 proteins in the manipulation of host gene expression and epigenetic chromatin modification.

Epstein-Barr virus (EBV) is able to drive the transformation of B-cells, resulting in the generation of lymphoblastoid cell lines (LCLs) in vitro. EBV nuclear proteins EBNA3A and EBNA3C are necessary for efficient transformation, while EBNA3B is dispensable. We describe a transcriptome analysis of BL31 cells infected with a series of EBNA3-knockout EBVs, including one deleted for all three EBNA3 genes. Using Affymetrix Exon 1.0 ST microarrays analysed with the MMBGX algorithm, we have identified over 1000 genes whose regulation by EBV requires one of the EBNA3s. Remarkably, a third of the genes identified require more than one EBNA3 for their regulation, predominantly EBNA3C co-operating with either EBNA3B, EBNA3A or both. The microarray was validated by real-time PCR, while ChIP analysis of a selection of co-operatively repressed promoters indicates a role for polycomb group complexes. Targets include genes involved in apoptosis, cell migration and B-cell differentiation, and show a highly significant but subtle alteration in genes involved in mitosis. In order to assess the relevance of the BL31 system to LCLs, we analysed the transcriptome of a set of EBNA3B knockout (3BKO) LCLs. Around a third of the genes whose expression level in LCLs was altered in the absence of EBNA3B were also altered in 3BKO-BL31 cell lines.Among these are TERT and TCL1A, implying that EBV-induced changes in the expression of these genes are not required for B-cell transformation. We also identify 26 genes that require both EBNA3A and EBNA3B for their regulation in LCLs. Together, this shows the complexity of the interaction between EBV and its host, whereby multiple EBNA3 proteins co-operate to modulate the behaviour of the host cell.

Role of BC loop residues in structure, function and antigenicity of the West Nile virus envelope protein receptor-binding domain III.

Site-directed mutagenesis of residues in the BC loop (residues 329-333) of the envelope (E) protein domain III in a West Nile virus (WNV) infectious clone and in plasmids encoding recombinant WNV and dengue type 2 virus domain III proteins demonstrated a critical role for residues in this loop in the function and antigenicity of the E protein. This included a strict requirement for the tyrosine at residue 329 of WNV for virus viability and E domain III folding. The absence of an equivalent residue in this region of yellow fever group viruses and most tick-borne flavivirus suggests there is an evolutionary divergence in the molecular mechanisms of domain III folding employed by different flaviviruses.

Immune evasion by gammaherpesvirus genome maintenance proteins.

Viruses that establish lifelong latent infections must ensure that the viral genome is maintained within the latently infected cell throughout the life of the host, yet at the same time must also be capable of avoiding elimination by the immune surveillance system. Gammaherpesviruses, which include the human viruses Epstein-Barr virus and Kaposi's sarcoma-associated herpesvirus, establish latent infections in lymphocytes. Infection of this dynamic host-cell population requires that the viruses have appropriate strategies for enabling the viral genome to persist while these cells go through rounds of mitosis, but at the same time must avoid detection by host CD8(+) cytotoxic T lymphocytes (CTLs). The majority of gammaherpesviruses studied have been found to encode a specific protein that is critical for maintenance of the viral genome within latently infected cells. This protein is termed the genome maintenance protein (GMP). Due to its vital role in long-term latency, this offers the immune system a crucial target for detection and elimination of virus-infected cells. GMPs from different gammaherpesviruses have evolved related strategies that allow the protein to be present within latently infected cells, but to remain effectively hidden from circulating CD8(+) CTLs. In this review, I will summarize the role of the GMPs and highlight the available data describing the immune-evasion properties of these proteins.

Kaposi's sarcoma-associated herpesvirus Lana-1 is a major activator of the serum response element and mitogen-activated protein kinase pathways via interactions with the Mediator complex.

In cells infected with Kaposi's sarcoma-associated herpesvirus (KSHV), the activation of mitogen-activated protein kinase (MAPK) pathways plays a crucial role early after virus infection as well as during reactivation. In order to systematically identify viral proteins activating MAPK pathways in KSHV-infected cells, a clone collection of KSHV open reading frames (ORFs) was screened for induction of the serum response element (SRE), as SRE is induced by MAPKs. The strongest induction of the SRE was found with ORF73 (latency-associated nuclear antigen 1, or Lana-1), although weaker activation was also found with the kaposin B isoform, ORF54 (dUTPase) and ORF74 (G-protein-coupled receptor). The bipartite SRE is bound by a ternary complex consisting of serum response factor (SRF) and ternary complex factor. Lana-1 bound directly to SRF, but also to the MED25 (ARC92/ACID-1), MED15 (PCQAP) and MED23 (Sur-2) subunits of the Mediator complex, a multi-subunit transcriptional co-activator complex for RNA polymerase II. Lana-1-induced SRE activation was inhibited by the dominant-negative N-terminal domain of the MED25 mediator subunit, suggesting that this subunit mediates Lana-1-induced SRE activation. In summary, these data suggest a model in which Lana-1 acts as an adaptor between the transcription factor SRF and the basal transcriptional machinery.

Epstein-Barr virus nuclear protein 3C domains necessary for lymphoblastoid cell growth: interaction with RBP-Jkappa regulates TCL1.

B lymphocytes converted into lymphoblastoid cell lines (LCLs) by an Epstein-Barr virus that expresses a conditional EBNA3C require complementation with EBNA3C for growth under nonpermissive conditions. Complementation with relatively large EBNA3C deletion mutants identified amino acids (aa) 1 to 506 (which includes the RBP-Jkappa/CSL [RBP-Jkappa] binding domain) and 733 to 909 to be essential for LCL growth, aa 728 to 732 and 910 to 992 to be important for full wild-type (wt) growth, and only aa 507 to 727 to be unimportant (S. Maruo, Y. Wu, T. Ito, T. Kanda, E. D. Kieff, and K. Takada, Proc. Natl. Acad. Sci. USA 106:4419-4424, 2009). When mutants with smaller deletions were used, only aa 51 to 400 and 851 to 900 were essential for LCL growth; aa 447 to 544, 701 to 750, 801 to 850, and 901 to 992 were important for full wt growth; and aa 4 to 50, 401 to 450, 550 to 707, and 751 to 800 were unimportant. These data reduce the EBNA3C essential residues from 68% to 40% of the open reading frame. Point mutations confirmed RBP-Jkappa binding to be essential for wt growth and indicated that SUMO and CtBP binding interactions were important only for full wt growth. EBNA3C aa 51 to 150, 249 to 311, and 851 to 900 were necessary for maintaining LCL growth, but not RBP-Jkappa interaction, and likely mediate interactions with other key cell proteins. Moreover, all mutants null for LCL growth had fewer S+G(2)/M-phase cells at 14 days, consistent with EBNA3C interaction with RBP-Jkappa as well as aa 51 to 150, 249 to 311, and 851 to 900 being required to suppress p16(INK4A) (S. Maruo, Y. Wu, S. Ishikawa, T. Kanda, D. Iwakiri, and K. Takada, Proc. Natl. Acad. Sci. USA 103:19500-19505, 2006). We have confirmed that EBNA3C upregulates TCL1 and discovered that EBNA3C upregulates TCL1 through RBP-Jkappa, indicating a central role for EBNA3C interaction with RBP-Jkappa in mediating cell gene transcription.

Inhibition of KSHV-infected primary effusion lymphomas in NOD/SCID mice by gamma-secretase inhibitor.

Primary effusion lymphoma (PEL) is a common cancer in AIDS patients closely associated with Kaposi's sarcoma-associated herpesvirus (KSHV). Previously, we showed that KSHV latency associated nuclear antigen (LANA) stabilizes intracellular activated Notch1 (ICN) involved in maintenance of the malignant phenotype of KSHV infected PEL cells in vitro. The gamma-secretase inhibitor (GSI) which specifically blocks the production of ICN slows down the proliferation of the KSHV infected PEL cell lines BCBL1, BC3 as well as JSC1 in vitro. In this study, we extended these studies to explore the possibility that manipulation of the Notch signaling by GSI would prevent the growth of the PEL tumors in vivo. We observed that the onset of tumorigenesis of KSHV infected PELs was significantly delayed in GSI treated SCID mice harboring the PEL cell lines. We also found that GSI treatment resulted in necrosis as well as apoptosis in tumors generated by the xenotransplanted KSHV positive PEL cell lines. In contrast, GSI had no effect on mice harboring BJAB cells, a KSHV negative Burkitt's lymphoma cell line where ICN levels were negligible. Our study provides further evidence to suggest that targeted downregulation of abnormal Notch signaling has therapeutic potential for KSHV related primary effusion lymphomas.

A function essential to viral entry underlies the hepatitis B virus "a" determinant.

The hepatitis B virus (HBV) particles bear a receptor-binding site located in the pre-S1 domain of the large HBV envelope protein. Using the hepatitis delta virus (HDV) as a surrogate of HBV, a second infectivity determinant was recently identified in the envelope proteins antigenic loop (AGL), and its activity was shown to depend upon cysteine residues that are essential for the structure of the HBV immunodominant "a" determinant. Here, an alanine-scanning mutagenesis approach was used to precisely map the AGL infectivity determinant to a set of conserved residues, which are predicted to cluster together with cysteines in the AGL disulfide bridges network. Several substitutions suppressed both infectivity and the "a" determinant, whereas others were infectivity deficient with only a partial impact on antigenicity. Interestingly, G145R, a substitution often arising under immune pressure selection and detrimental to the "a" determinant, had no effect on infectivity. Altogether, these findings indicate that the AGL infectivity determinant is closely related to, yet separable from, the "a" determinant. Finally, a selection of HDV entry-deficient mutations were introduced at the surface of HBV virions and shown to also abrogate infection in the HBV model. Therefore, a function can at last be assigned to the orphan "a" determinant, the first-discovered marker of HBV infection. The characterization of the AGL functions at viral entry may lead to novel approaches in the development of antivirals against HBV.

Disruption of LANA in rhesus rhadinovirus generates a highly lytic recombinant virus.

Rhesus monkey rhadinovirus (RRV) is a gammaherpesvirus that is closely related to human Kaposi's sarcoma-associated herpesvirus (KSHV/HHV-8). RRV is the closest relative to KSHV that has a fully sequenced genome and serves as an in vitro and an in vivo model system for KSHV. The latency-associated nuclear antigen (LANA) protein of both KSHV and RRV plays key roles in the establishment and maintenance of these herpesviruses. We have constructed a RRV recombinant virus (RRVDeltaLANA/GFP) in which the RRV LANA open reading frame has been disrupted with a green fluorescent protein (GFP) expression cassette generated by homologous recombination. The integrity of the recombinant virus was confirmed by diagnostic PCR, restriction digestion, Southern blot analysis, and whole-genome sequencing. We compared the single-step and multistep replication kinetics of RRVDeltaLANA/GFP, RRV-GFP, wild-type (WT) RRV H26-95, and a revertant virus using traditional plaque assays, as well as real-time quantitative PCR-based genome quantification assays. The RRVDeltaLANA/GFP recombinant virus exhibited significantly higher lytic replicative properties compared to RRV-GFP, WT RRV, or the revertant virus. This was observed upon de novo infection and in the absence of chemical inducers such as phorbol esters. In addition, by using a quantitative real-time PCR-based viral array, we are the first to report differences in global viral gene expression between WT and recombinant viruses. The RRVDeltaLANA/GFP virus displayed increased lytic gene transcription at all time points postinfection compared to RRV-GFP. Moreover, we also examined several cellular genes that are known to be repressed by KSHV LANA and report that these genes are derepressed during de novo lytic infection with the RRVDeltaLANA/GFP virus compared to RRV-GFP. Finally, we also demonstrate that the RRVDeltaLANA/GFP virus fails to establish latency in B cells, as measured by the loss of GFP-positive cells and intracellular viral genomes.