EBV can protect latently infected B cell lymphomas from death receptor-induced apoptosis

The relationship between EBV infection and sensitivity to death receptor (DR)-induced apoptosis is poorly understood. Using EBV- and EBV+ BJAB cells, we provide the first evidence that EBV can protect latently infected B cell lymphomas from apoptosis triggered through Fas or TRAIL receptors. Caspase 8 activation was impaired and cellular FLIP recruitment was enriched in death-inducing signaling complexes formed in EBV-infected BJAB cells relative to parent BJAB cells. Furthermore, latent membrane protein 1 expression alone could reduce caspase activation and confer partial resistance to DR apoptosis in BJAB cells. This protective effect was dependent on C-terminal activating region 2-driven NF-kappaB activation, which in turn up-regulated cellular FLIP expression in latent membrane protein 1+ BJAB cells. Thus, the ability of latent EBV to block DR apoptosis may help to ensure the survival of host cells during B cell differentiation, and contribute to the development of B cell lymphomas, especially in immunocompromised individuals.     

Long-term MHC class II presentation of the EBV lytic protein BHRF1 by EBV latently infected b cells following capture of BHRF1 antigen

Although T lymphocytes are considered essential for the control of EBV infection, it remains uncertain how this control occurs. We previously reported unexpected killing of EBV-transformed B-lymphoblastoid cells (LCLs) that did not express BHRF1 by CD4+ T cells specific for BHRF1, an EBV lytic cycle protein. Using LCLs transformed with an EBV mutant, in which the BHRF1 gene was deleted, we showed that killing of latently infected cells through the recognition of a protein produced during the lytic cycle is due to transfer of BHRF1 from lytically infected to latently infected cells, which occurs in culture. Accordingly, LCLs efficiently presented exogenous BHRF1 protein. Furthermore, we present evidence for persistence of captured BHRF1 Ag for several days. Due to this long-term persistence, repeated loading of suboptimal amounts of BHRF1 led to accumulation of BHRF1 Ags in LCLs and, ultimately, to their optimal recognition by BHRF1-specific CD4+ T cells. These results unveil an MHC class II-dependent pathway that could be important for the control of EBV latent infection through recognition of lytic cycle Ags.     

Ex vivo stimulation of B cells latently infected with gammaherpesvirus 68 triggers reactivation from latency

Murine gammaherpesvirus 68 (gammaHV68) infection of mice results in the establishment of a chronic infection, which is largely maintained through latent infection of B lymphocytes. Acute virus replication is almost entirely cleared by 2 weeks postinfection. Spontaneous reactivation of gammaHV68 from latently infected splenocytes upon ex vivo culture can readily be detected at the early stages of infection (e.g., day 16). However, by 6 weeks postinfection, very little spontaneous reactivation is detected upon explant into tissue culture. Here we report that stimulation of latently infected splenic B cells harvested at late times postinfection with cross-linking surface immunoglobulin (Ig), in conjunction with anti-CD40 antibody treatment, triggers virus reactivation. As expected, this treatment resulted in B-cell activation, as assessed by upregulation of CD69 on B cells, and ultimately B-cell proliferation. Since anti-Ig/anti-CD40 stimulation resulted in splenic B-cell proliferation, we assessed whether this reactivation stimulus could overcome the previously characterized defect in virus reactivation of a v-cyclin null gammaHV68 mutant. This analysis demonstrated that anti-Ig/anti-CD40 stimulation could drive reactivation of the v-cyclin null mutant virus in latently infected splenocytes, but not to the levels observed with wild-type gammaHV68. Thus, there appears to be a role for the v-cyclin in B cells following anti-Ig/anti-CD40 stimulation independent of the induction of the cell cycle. Finally, to assess signals that are not mediated through the B-cell receptor, we demonstrate that addition of lipopolysaccharide to explanted splenocyte cultures also enhanced virus reactivation. These studies complement and extend previous analyses of Epstein-Barr virus and Kaposi's sarcoma-associated virus reactivation from latently infected cell lines by investigating reactivation of gammaHV68 from latently infected primary B cells recovered from infected hosts.     

Identifying the membrane proteome of HIV-1 latently infected cells

Profiling integral plasma membrane proteins is of particular importance for the identification of new biomarkers for diagnosis and for drug development. We report in this study the identification of surface markers by performing comparative proteomics of established human immunodeficiency virus-1 (HIV-1) latent cell models and parental cell lines. To this end we isolated integral membrane proteins using a biotin-directed affinity purification method. Isolated proteins were separated by two-dimensional gel electrophoresis and identified by matrix-assisted laser desorption/ionization-time-of-flight (MALDI-TOF) after in gel digestion. Seventeen different proteins were found to vary on the surface of T-cells due to HIV-1 infection. Of these proteins, 47% were integral membrane proteins, and 18% were membrane-associated. Through the use of complementary techniques such as Western blotting and fluorescent staining, we confirmed the differential expression of some of the proteins identified by MALDI-TOF including Bruton's tyrosine kinase and X-linked inhibitor of apoptosis. Finally, using phosphatidylinositol 3-kinase inhibitors and flavopiridol to inhibit Bruton's tyrosine kinase localization at the membrane and X-linked inhibitor of apoptosis protein expression, respectively, we showed that HIV-1 latently infected cells are more sensitive to these drugs than uninfected cells. This suggests that HIV-1 latently infected cells may be targeted with drugs that alter several pathways that are essential for the establishment and maintenance of latency.     

The murine gammaherpesvirus 68 M2 gene is required for efficient reactivation from latently infected B cells

Murine gammaherpesvirus 68 (gammaHV68) infection of mice provides a tractable small-animal model system for assessing the requirements for the establishment and maintenance of gammaherpesvirus latency within the lymphoid compartment. The M2 gene product of gammaHV68 is a latency-associated antigen with no discernible homology to any known proteins. Here we focus on the requirement for the M2 gene in splenic B-cell latency. Our analyses showed the following. (i) Low-dose (100 PFU) inoculation administered via the intranasal route resulted in a failure to establish splenic B-cell latency at day 16 postinfection. (ii) Increasing the inoculation dose to 4 x 10(5) PFU administered via the intranasal route partially restored the establishment of B-cell latency at day 16, but no virus reactivation was detected upon explant into tissue cultures. (iii) Although previous data failed to detect a phenotype of the M2 mutant upon high-dose intraperitoneal inoculation, decreasing the inoculation dose to 100 PFU administered intraperitoneally revealed a splenic B-cell latency phenotype at day 16 that was very similar to the phenotype observed upon high-dose intranasal inoculation. (iv) After low-dose intraperitoneal inoculation, fractionated B-cell populations showed that the M2 mutant virus was able to establish latency in surface immunoglobulin D-negative (sIgD(-)) B cells; by 6 months postinfection, equivalent frequencies of M2 mutant and marker rescue viral genome-positive sIgD(-) B cells were detected. (v) Like the marker rescue virus, the M2 mutant virus also established latency in splenic naive B cells upon low-dose intraperitoneal inoculation, but there was a significant lag in the decay of this latently infected reservoir compared to that seen with the marker rescue virus. (vi) After low-dose intranasal inoculation, by day 42 postinfection, latency was observed in the spleen, although at a frequency significantly lower than that in the marker rescue virus-infected mice; by 3 months postinfection, nearly equivalent levels of viral genome-positive cells were observed in the spleens of marker rescue virus- and M2 mutant virus-infected mice, and these cells were exclusively sIgD(-) B cells. Taken together, these data convincingly demonstrate a role for the M2 gene product in reactivation from splenic B cells and also suggest that disruption of the M2 gene leads to dose- and route-specific defects in the efficient establishment of splenic B-cell latency.     

CpG oligodeoxynucleotides activate HIV replication in latently infected human T cells

CpG oligodeoxynucleotides (CpG ODNs) stimulate immune cells via the Toll-like receptor 9 (TLR9). In this study, we have investigated the effects of CpG ODNs on latent human immunodeficiency virus (HIV) infection in human T cells. Treatment of the latently infected T cell line ACH-2 with CpG ODNs 2006 or 2040 stimulated HIV replication, whereas no effects were evident when ODNs without the CpG motif were used. CpG-induced virus reactivation was blocked by chloroquine, indicating the involvement of TLR9. In contrast to the responsiveness of ACH-2 cells, CpG ODNs failed to activate HIV provirus in the latently infected Jurkat clone J1.1. We also studied the effects of CpG ODNs on productive HIV infection and found enhancement of viral replication in A3.01 T cells, whereas again no stimulating effects were observed in Jurkat T cells. CpG ODN treatment activated NF-kappaB in ACH-2 cells, which was similarly triggered in uninfected A3.01 T cells following exposure to CpG ODNs, indicating that TLR9-induced signal transduction was not dependent on proviral infection. Our study demonstrates that CpG ODNs directly trigger the activation of NF-kappaB and reactivation of latent HIV in human T cells. Our results point to a novel role for CpG ODNs as stimulators of HIV replication and open new avenues to eradicate the latent viral reservoirs in HIV-infected patients treated with antiretroviral therapy.     

Epstein-barr virus latently infected cells are selectively deleted in simulated-microgravity cultures

Summary Rotating-wall vessels (RWVs) allow for the cultivation of cells in simulated microgravity. Previously, we showed that the cultivation of lymphoblastoid cells in simulated microgravity results in the suppression of Epstein—Barr virus (EBV) reactivation. To determine if the suppression generated by simulated microgravity could be reversed by changing to static culture conditions, cells were cultured in an RWV for 5 d, and then switched to static conditions. Following the switch to static conditions, viral reactivation remained suppressed (significantly lower) relative to static control cultures over a 4-d period. Additionally, experiments were conducted to determine if chemical treatment could induce viral reactivation in cells from simulated-microgravity cultures. Cells were cultured in static flask cultures and in simulated microgravity in RWVs for 4–7 d. The cells were then transferred to 50-cm³ tubes, and treated with 3 mM n-butyrate for 48 h, or 18 ng/ml of phorbol ester, viz., 12-0-tetradecanoylphorbol-13 acetate (TPA) for either 2 or 48 h, under static conditions. Although EBV was inducible, the cells from simulated-microgravity cultures treated withn-butyrate displayed significantly lower levels of viral-antigen expression compared with the treated cells from static cultures. Also, incubation with TPA for 2–3 h, but not for 48 h, reactivated EBV in cells from RWV cultures. In contrast, EBV was inducible in cells from static cultures treated for either 2–3 or 48 h with TPA. TPA reactivation of EBV following a 2–3-h period of treatment indicates that the protein kinase C signal-transduction pathway is not impaired in lymphoblastoid cells cultured in simulated microgravity. However, the exposure of B-lymphoblastoid cells from simulated-microgravity cultures to TPA for more than 3–4 h triggered a lytic event (apoptosis or necrosis), which prevented replication of the virus. Thus, EBV-infected cells in simulated microgravity were negatively selected in the absence of any cytotoxic cells.     

Rapid expression of human immunodeficiency virus following activation of latently infected cells

The host cell activation state impacts the nature of human immunodeficiency virus infection. Activated cells facilitate productive infections; quiescent cells enable the virus to enter a latent state, the major obstacle to viral clearance. We wanted to understand how these differences affected viral gene expression. In quiescent cells activated prior to infection, viral RNA was seen 12 h postinfection; when cells were stimulated postinfection, viral RNA was not seen until 36 h postinfection. Up-regulation of viral RNA in latently infected cells occurred within 2 h poststimulation. This hierarchy also held true for viral protein production. These results may explain the rapid reemergence of viremia following termination of therapy.     

Identification of glycoproteins associated with HIV latently infected cells using quantitative glycoproteomics

HIV infection is not curable due to viral latency. Compelling reports suggest that there is a distinct profile of surface proteins that can be used for targeting latently infected cells. We have recently reported that glycoproteins were differentially secreted from HIV latently infected ACH‐2 cells compared to the parental A3.01 cells. This finding suggests that glyco‐phenotype might be different in these two cell lines. To determine the difference, the ACH‐2 and A3.01 cell lines were subjected to a glycoproteomic analysis. A total number of 940 unique N‐linked glycosite‐containing peptides from 515 glycoproteins were identified. Among the glycoproteins, 365 and 104 were annotated as cell surface and membrane‐associated proteins, respectively. Quantitative LC‐MS/MS analysis revealed a change of 236 glycosite‐containing peptides from 172 glycoproteins between the two cell lines without reactivation. Bioinformatic analysis suggests that cell adhesion, immune response, glycoprotein metabolic process, cell motion, and cell activation were associated with the changed proteins. After reactivation of latency, changes in glycosite‐containing peptides were observed in both cell lines. The changed proteins suggest that cell migration, response to wounding and immune response might be impaired in reactivated latently infected cells. Glycoproteomics merits future application using primary cells to discover reveal mechanisms in HIV pathogenesis.     

Cytotoxic CD4+ T-cells specific for EBV capsid antigen BORF1 are maintained in long-term latently infected healthy donors

Epstein Barr Virus (EBV) infects more than 95% of the population whereupon it establishes a latent infection of B-cells that persists for life under immune control. Primary EBV infection can cause infectious mononucleosis (IM) and long-term viral carriage is associated with several malignancies and certain autoimmune diseases. Current efforts developing EBV prophylactic vaccination have focussed on neutralising antibodies. An alternative strategy, that could enhance the efficacy of such vaccines or be used alone, is to generate T-cell responses capable of recognising and eliminating newly EBV-infected cells before the virus initiates its growth transformation program. T-cell responses against the EBV structural proteins, brought into the newly infected cell by the incoming virion, are prime candidates for such responses. Here we show the structural EBV capsid proteins BcLF1, BDLF1 and BORF1 are frequent targets of T-cell responses in EBV infected people, identify new CD8+ and CD4+ T-cell epitopes and map their HLA restricting alleles. Using T-cell clones we demonstrate that CD4+ but not CD8+ T-cell clones specific for the capsid proteins can recognise newly EBV-infected B-cells and control B-cell outgrowth via cytotoxicity. Using MHC-II tetramers we show a CD4+ T-cell response to an epitope within the BORF1 capsid protein epitope is present during acute EBV infection and in long-term viral carriage. In common with other EBV-specific CD4+ T-cell responses the BORF1-specific CD4+ T-cells in IM patients expressed perforin and granzyme-B. Unexpectedly, perforin and granzyme-B expression was sustained over time even when the donor had entered the long-term infected state. These data further our understanding of EBV structural proteins as targets of T-cell responses and how CD4+ T-cell responses to EBV change from acute disease into convalescence. They also identify new targets for prophylactic EBV vaccine development.     

Identification of Marek''s disease virus nuclear antigen in latently infected lymphoblastoid cells.

A new Marek's disease virus (MDV) nuclear antigen (MDNA) was identified in two MDV-transformed T-lymphoblastoid cell lines, MKT-1 and MSB-1, derived from chickens bearing tumors induced by MDV. This MDNA was not detected in MSB-1 cells maintained in iododeoxyuridine, which activates the latent MDV genome. Moreover, it was not found in chicken embryo fibroblasts undergoing productive and cytolytic infection with MDV. Expression of MDNA is not related to strain pathogenicity in chickens, because chicken embryo fibroblasts productively infected with the pathogenic RBIB strain or the nonpathogenic CV-1 strain of MDV did not express this antigen. DNA-protein immunoprecipitation studies revealed that MDNA bound to two sites in the 190,00-base-pair (bp) MDV genome. One of these loci identified by MDNA obtained from MKT-1 and MSB-1 cells corresponded to a 476-bp segment within the short unique region of BamHI-A MDV DNA. A second locus located in a 280-bp segment within the short inverted repeat region of BamHI-A was also identified by MDNA from MSB-1 cells but not by MDNA obtained from MKT-1 cells. Analyses of the nucleotide sequence by DNase digestion showed that MDNA protected a 60-bp segment spanning a 22-bp palindromic sequence of the short unique region and a 103-bp sequence encompassing a 32-bp palindrome in the short inverted repeat region of BamHI-A MDV DNA.     

Signaling through Toll-like receptors triggers HIV-1 replication in latently infected mast cells

Evidence that human progenitor mast cells are susceptible to infection with CCR5-tropic strains of HIV-1 and that circulating HIV-1-infected FcepsilonRIalpha(+) cells with a similar progenitor phenotype have been isolated from AIDS patients has led to speculation that mast cells may serve as a potential reservoir for infectious HIV-1. In this study, progenitor mast cells, developed in vitro from CD34(+) cord blood stem cells, were experimentally infected with the CCR5-tropic strain HIV-1Bal after 28 days in culture as they reached their HIV-1-susceptible progenitor stage. HIV-1 p24 Ag levels were readily detectable by day 7 postinfection (PI), peaked at 2-3 wk PI as mature (tryptase/chymase-positive) HIV-1 infection-resistant mast cells emerged, and then steadily declined to below detectable limits by 10 wk PI, at which point integrated HIV-1 proviral DNA was confirmed by PCR quantitation in ( approximately 34% of) latently infected mast cells. Stimulation by ligands for Toll-like receptor (TLR) 2, TLR4, or TLR9 significantly enhanced viral replication in a dose- and time-dependent manner in both HIV-1-infected progenitor and latently infected mature mast cells, without promoting degranulation, apoptosis, cellular proliferation, or dysregulation of TLR agonist-induced cytokine production in infected mast cells. Limiting dilution analysis of TLR activated, latently infected mature mast cells indicated that one in four was capable of establishing productive infections in A301 sentinel cells. Taken together, these results indicate that mast cells may serve both as a viral reservoir and as a model for studying mechanisms of postintegration latency in HIV infection.     

Leukotriene B4 protects latently infected mice against murine cytomegalovirus reactivation following allogeneic transplantation

Human CMV is often associated with transplant rejection and opportunistic infections such as pneumonia in immunosuppressed patients. Current anti-CMV therapies, although effective, show relatively high toxicity, which seriously limits their long-term use. In this study, we provide evidence that leukotriene B(4) (LTB(4)) plays an important role in the fight against murine CMV (MCMV) infection in vivo. Intravenous administration of 50 and 500 ng/kg/day of LTB(4) to mice infected with a lethal dose of MCMV significantly increases their survival (50 and 70%, respectively), compared with the placebo-treated group (10% of survival). In mice infected with a sublethal dose of MCMV and treated daily with 50 ng/kg/day of LTB(4), the salivary gland viral loads were found to be reduced by 66% compared with the control group. Furthermore, using an allogeneic bone marrow transplantation mouse model, the frequency of MCMV reactivation from latently infected mice was much lower (38%) in LTB(4) (500 ng/kg)-treated mice than in the placebo-treated group (78%). Finally, in experiments using 5-lipoxygenase-deficient mice, MCMV viral loads in salivary glands were found to be higher in animals unable to produce leukotrienes than in the control groups, supporting a role of endogenous 5-lipoxygenase products, possibly LTB(4), in host defense against CMV infection.     

Zinc-finger-nucleases mediate specific and efficient excision of HIV-1 proviral DNA from infected and latently infected human T cells

HIV-infected individuals currently cannot be completely cured because existing antiviral therapy regimens do not address HIV provirus DNA, flanked by long terminal repeats (LTRs), already integrated into host genome. Here, we present a possible alternative therapeutic approach to specifically and directly mediate deletion of the integrated full-length HIV provirus from infected and latently infected human T cell genomes by using specially designed zinc-finger nucleases (ZFNs) to target a sequence within the LTR that is well conserved across all clades. We designed and screened one pair of ZFN to target the highly conserved HIV-1 5′-LTR and 3′-LTR DNA sequences, named ZFN-LTR. We found that ZFN-LTR can specifically target and cleave the full-length HIV-1 proviral DNA in several infected and latently infected cell types and also HIV-1 infected human primary cells in vitro. We observed that the frequency of excision was 45.9% in infected human cell lines after treatment with ZFN-LTR, without significant host-cell genotoxicity. Taken together, our data demonstrate that a single ZFN-LTR pair can specifically and effectively cleave integrated full-length HIV-1 proviral DNA and mediate antiretroviral activity in infected and latently infected cells, suggesting that this strategy could offer a novel approach to eradicate the HIV-1 virus from the infected host in the future.     

Calpain is involved in the HIV replication from the latently infected OM10.1 cells

Treatment of OM10.1 cells latently infected with human immunodeficiency virus type 1 (HIV-1) with phorbol ester and calcium ionophore (A23187) induced virus replication which was blocked by N-Ac-Leu-Leu-norleucinal (ALLnL), a calpain inhibitor I, and not by lactacystin, a specific proteasome inhibitor. When the purified NF-kappa B/I kappa B complex was treated with mu-calpain, the specific DNA-binding activity was demonstrated by using electrophoretic mobility shift assay in vitro. This effect of mu-calpain was inhibited by ALLnL and calpastatin and not by lactacystin. In fact, we found that mu-calpain efficiently degraded I kappa B alpha. Furthermore, our Western blotting analysis has revealed that mu-calpain cleaves I kappa B alpha at its N-terminal and C-terminal regions that were previously reported to be involved in the interaction with NF-kappa B p65. These observations indicate that in monocyte/macrophage cells calcium signaling is involved in NF-kappa B activation through activation of calpain and thus calpain inhibitors may be effective in inhibiting the activation of latently infected HIV.     

Immune control of the number and reactivation phenotype of cells latently infected with a gammaherpesvirus

Despite active immune responses, gammaherpesviruses establish latency. In a related process, these viruses also persistently replicate by using a mechanism that requires different viral genes than acute-phase replication. Many questions remain about the role of immunity in chronic gammaherpesvirus infection, including whether the immune system controls latency by regulating latent cell numbers and/or other properties and what specific immune mediators control latency and persistent replication. We show here that CD8(+) T cells regulate both latency and persistent replication and demonstrate for the first time that CD8(+) T cells regulate both the number of latently infected cells and the efficiency with which infected cells reactivate from latency. Furthermore, we show that gamma interferon (IFN-gamma) and perforin, which play no significant role during acute infection, are essential for immune control of latency and persistent replication. Surprisingly, the effects of perforin and IFN-gamma are site specific, with IFN-gamma being important in peritoneal cells while perforin is important in the spleen. Studies of the mechanisms of action of IFN-gamma and perforin revealed that perforin acts primarily by controlling the number of latently infected cells while IFN-gamma acts primarily by controlling reactivation efficiency. The immune system therefore controls chronic gammaherpesvirus infection by site-specific mechanisms that regulate both the number and reactivation phenotype of latently infected cells.     

Oocyte development in Hydra involves selection from competent precursor cells

We have investigated oocyte development in Hydra vulgaris, a member of one of the oldest metazoan phyla. We show that oocyte determination involves a mechanism that establishes a subset of precursor interstitial cells competent to differentiate into oocytes. The oocyte is singled out from this subset and the competence of the remaining cells to become oocytes dramatically decreases as they adopt the alternative nurse cell fate. Progression through the nurse cell differentiation program requires the presence of the oocyte. When the oocyte is removed from the egg field, nurse cells abort their differentiation program, undergo apoptosis, and are phagocytosed and degraded by somatic epithelial cells. However, in the presence of the oocyte, nurse cells differentiate and enter an unusual apoptosis program where they are phagocytosed by the oocyte, but are not degraded. We show that the oocyte is able to induce this unusual apoptosis program in immature nurse cells that have not completed differentiation. A new model for oocyte development in Hydra is discussed.