Selective Gene Expression of Latent Murine Gammaherpesvirus 68 in B Lymphocytes

Selective Gene Expression of Latent Murine Gammaherpesvirus 68 in B Lymphocytes

Summary:  Nearly all human beings, by the time they reach adolescence, are infected with multiple herpesviruses. Viral latent-protein expression is critical in inducing and maintaining KSHV latency. 1981. Each assay found HHV-8 seroprevalence to be high (49–87%) in the African populations and generally lower (9–54%) in Malta. This project also has the potential to examine the impact of latent infection on host cell gene expression, to address the hypothesis that CMV orchestrates an environment within the host cell that enhances the capacity of this virus to successfully remain within the host in a latent state (from which it can reactivate years later under conditions of immunosuppression to cause life-threatening disease). KSHV-infected fibroblasts expressed normal levels of IFN-γR1 and responded to exogenous IFN-γ by upregulating MHC class I, ICAM-1 and HLA-E and resisting activated NK-cell killing. Furthermore, NK cells armed by latent infection protected the host against a lethal lymphoma challenge.

Current strategies for inducing durable allograft survival involve long-term use of immunosuppressive drugs following transplantation. Src family kinases are required for the activation of HIF and the downstream gene VEGFR1 by KSHV. Proliferation and associated expansion of latently infected cells culminates with the generation of a persistently infected memory B-cell pool (2). & Opp, M. At 70 days PI, gB expression was detected only in the spleen, and at 105 days PI, gB expression was not detected in any of the lymphoid tissue (thymus, spleen, or bursa). The initial infection may be followed by latency with subsequent reactivation. Idiopathic pulmonary fibrosis (IPF) is characterized by progressive and relentless lung scarring and is the most lethal interstitial lung disease (1).

Spread of infection to the spleen generates an antigen-nonspecific, T-cell-dependent B-cell activation (32, 38) and an infectious mononucleosis-like disease similar to that observed in adolescent EBV infection (13, 43). Previous studies have shown that MHV-68 infection of the spleen results in the establishment of latency in B lymphocytes, macrophages, and dendritic cells (14, 42, 56). K1 is a positional homologue of LMP-1 of EBV and contains the two most variable regions (VR1 and VR2) across the entire KSHV genome which are used to classify KSHV into four clades (A, B, C, and D) (Zong et al., 1999). In uninfected mice, LPS administration induces modest and relatively transient (that is, less than 18 h) hypothermia, reduced running-wheel activity, and alterations in sleep.28 In contrast, mice with latent MuGHV infection develop prolonged hypothermia, hypoactivity, hypersomnolence, and fragmented sleep, all of which persist for as long as 5 d after LPS administration.28 This duration is consistent with the time reported for viral reactivation from latency in MuGHV-infected mice treated with LPS.14 Therefore, as compared with uninfected mice, mice with latent infections appear to show a greater magnitude and duration of LPS-induced behavioral perturbations that may reflect fatigue, perhaps in association with virus reactivation. Since MCMV can cause aortic inflammation in weanling mice (16), we focused on the great vessels as possible targets for chronic MCMV disease. Thus, the identification of viral genes expressed during the establishment of infection in GC B cells is central for an understanding of MHV-68 pathogenesis. During the establishment of latent infection in the spleen a restricted number of transcribed viral genes have been identified.

These include M2, M3, K3, M8, and M9 (20, 30, 34, 44, 52). Of these putative latency-associated genes, only K3 and M2 have been formally shown to be necessary for the establishment of a normal latent load (21, 39). K3 downregulates major histocompatibility complex I surface expression via a proteosome-dependent mechanism (4, 18). No function has as yet been attributed to M2. With the exception of K3 (39), whether M2, M3, or any of the other putative latency-associated genes are expressed in GC B cells or in any of the other proposed latent sites, i.e., macrophages and dendritic cells, is still unknown. This suggests that a γ-herpesvirus is able to manipulate innate immunity and influence both the skewing and the strength of T cell responses upon a second pro-inflammatory stimulus. For each cell type, cell-specific patterns of virus transcription were quantified at 14 days p.i.

The virus open reading frames (ORFs) analyzed included unique genes and cellular homologues that are predicted to be involved in activities, such as immune evasion and latency. Similar to EBV-associated lymphomas, cell lines with B-cell characteristics established from BCBL carry KSHV in a latent form, and a lytic cycle can be induced by 12-O-tetradecanoylphorbol-13-acetate (TPA) (14, 18, 29, 40, 46, 47). S11 cells (45) were cultured in RPMI medium plus 10% FCS. As obligate pathogens, viruses have evolved with strategies to evade and exploit host innate immune signaling events. Virus working stocks were prepared by a low multiplicity of infection (MOI; 0.001 PFU/cell) of BHK-21 cells (33). Female BALB/c mice (Gulbenkian Institute of Science, Oeiras, Portugal), 6 to 8 weeks of age, were inoculated intranasally under the effect of light halothane anesthesia with 104 PFU of virus in 20 μl of phosphate-buffered saline (PBS). At different time points after infection, mice were killed by inhalation of CO2.

Lungs or spleens were removed and kept at −80°C or immersed in PBS-2% FCS at 4°C for subsequent use, respectively. Virus assays.Infectious virus was plaque assayed on BHK-21 cells, and latent virus was assayed by explant coculture of fluorescence-activated cell-sorted (FACS) splenocytes with BHK-21 cells, incubated for 5 days, fixed with 10% formal saline, and counterstained with toluidine blue, and plaques were counted with a plate microscope (33). FACS-purified populations of splenocytes were also freeze-thawed and sonicated prior to plaque assay in order to determine the level of preformed infectious virus. Purification of different splenocyte populations.Single-cell suspensions of at least five pooled spleens were prepared per time point and passed trough a 100-μm (pore-size) filter to remove stromal debris. Cell suspensions were washed in PBS-2% FCS and red blood cells lysed in 154 mM ammonium chloride, 14 mM sodium hydrogen carbonate, and 1 mM EDTA (pH 7.3). For surface staining, single-cell suspensions were preincubated with 2.4G2 [anti-CD16/CD32 (FcγIII/II receptor), rat immunoglobulin G2b(κ) culture supernatant; Pharmingen] before staining with the following monoclonal antibodies (Pharmingen) and lectins (Vector Laboratories): anti-CD19, anti-CD11b, anti-CD11c, anti-CD21, anti-CD23, anti-B220 (CD45R), and peanut agglutinin (PNA). Using a MoFlo cytometer (Cytomation, Fort Collins, Colo.), the following enriched cell subpopulations were obtained: CD19+ for total B cells, B220+ CD21int CD23hi for Fo cells, B220+ CD21− CD23− for NF B cells, B220+ CD21hi CD23+ for MZ B cells, B220+ PNAhi for GC cells B cells, B220− CD11b+ CD11c− for macrophages, and B220− CD11c+ for dendritic cells.

The inoculated groups were kept in separate cages in an animal isolation facility of the Federal University of Santa Maria (UFSM, building #20, room 4006). The purities of sorted populations were usually >95% and always >90%. Real-time PCR.Real-time PCR was performed by using a LightCycler from Roche Molecular Biochemicals (Mannheim, Germany) according to the manufacturer’s instructions by using sequence-specific fluorescence detection oligonucleotide hybridization probes coupled to suitable fluorophores (see Table 1 for probe genome coordinates). Labeled probes were designed and supplied by TIB Biomol. This expanded T-cell compartment appears to be highly functional. Treatment with IL-4 after MHV68 infection increased viral replication (fig. It then passes on part of its excitation energy to the adjacent LC Red-640, which is directly conjugated to the 5′ end of another oligonucleotide.
Selective Gene Expression of Latent Murine Gammaherpesvirus 68 in B Lymphocytes

The excited LC Red-640 then emits measurable light. PCR amplification was done at 94°C for 6 min (1 cycle); 94°C for 1 min, 58°C for 1 min, and 72°C for 1 min (35 cycles); and 72°C for 5 min (1 cycle). KSHV infection of cultured endothelial cells yields similar levels of latent and lytic infection [17]. Of importance, the target sample had kinetics of amplification identical to those of the standard amplification reactions, i.e., the slope of the target sample at each cross point, over a large range, was the same as for the standard samples. Restriction enzyme sites NruI, AgeI, andStuI used to construct the fusion protein clones are indicated. When used, PAA was added to 0.5 mM. Proteinase K was then inactivated (5 min at 95°C), and the samples were analyzed by real-time PCR (as described above), with primer-probe sets specific for K3 in a final volume of 10 μl per PCR (2 mM MgCl2, 4 ng of each primer/μl, a 0.02 mM concentration of each internal probe, a 1× DNA mix [Roche], and 1 μl of cell lysate).

Although different primer sets used in the present study had slightly different amplification kinetics, they all amplified viral genomes with equivalent sensitivity, i.e., S11 cells (harboring ca. Since all genes have one copy in each KSHV genome, similar levels of signals in both assays should be observed after calibration of amplification efficiencies if only full-length KSHV genome is present. Therefore, any primer-probe combination could have been used for the detection of viral genomic DNA. Real-time DNA-PCR.Real-time PCR was performed to quantify the internalized viral DNA in the infected cells. Interestingly, cells transfected with KSHV-LANAΔ329-931 also failed to establish stable clones in the presence of all of the hygromycin concentrations tested (Table 1). Fifty micrograms of nuclear extracts or 10 μg of whole-cell lysates were resolved on a 4 to 15% gradient gel (Bio-Rad), transferred to Immobilon (Millipore), blocked with 5% milk in phosphate-buffered saline with 0.05% Tween 20, and probed with the following specific antibodies in the same solution: anti-HIF1α, anti-HIF1β, and anti-transcription factor IIβ (TFIIβ) (BD Biosciences); anti-HIF2α (U.S. Additionally, we show that KSHV infection induces protein expression of c-Myc, its dimerization partner Max, MondoA, and its dimerization partner, Mlx.

Estimation of the cell subset frequency of MHV-68 infection consisted of computation by maximal-likelihood estimation as follows: let f be the estimate of the cell frequency; the maximum likelihood of f is the value of f that maximizes where log(L) is the natural logarithm of the likelihood function L and Pi is given by Pi = exp(−f xi) according to the SHPM. Cycle threshold (CT) values were determined by automated analysis. The 95% confidence interval (CI) for f was calculated as 95% CI (f) = f ± 1.96SE (f). (A) A diagram of KSHV genome with mapped viral pA sites (red triangles for plus strand and blue triangles for minus strand). Reverse transcription-PCR (RT-PCR) analysis of virus transcription.RNA was isolated from 106 to 3 × 106 splenocytes purified by sorting from pools of five spleens and NIH 3T3 cells by using the RNeasy Minikit with the RNase-free DNase set protocol (Qiagen) according to the manufacturer’s instructions. RNA was also extracted from lung tissue by using Trizol as specified by the manufacturer (Gibco-BRL). IFN-α/βR−/− mice are hypersensitive to γHV68 infection but clear low-dose intranasal infection.Following intranasal infection, 100% of IFN-α/βR−/− mice receiving 1,000 PFU or more succumbed within 12 days postinfection (dpi), whereas no deaths occurred in wt mice receiving up to 106 PFU (data not shown).

Samples were then reverse transcribed in a total reaction volume of 40 μl containing 0.5 mM concentrations of each of deoxynucleoside triphosphate, 1× first-strand buffer (Gibco-BRL), 1 U of RNase OUT RNase inhibitor (Gibco-BRL), and 400 U of Superscript II reverse transcriptase (Gibco-BRL). Reactions were performed for 50 min at 37°C, followed by 5 min at 90°C. Viral cDNAs were quantified by using real-time PCR, as described above. KSHV de novo infection.Approximately 8 × 107 human PBMCs were infected with KSHV purified from the induced TRExBCBL1-RTA cells. Primer and probe genome coordinates are listed in Table 1. Our analytic models depended on a conditional independence assumption, which considered the errors of the various tests to be uncorrelated, conditional on individuals’ true infection status. This gave the relative quantity of each viral transcript in each sample.

The signal in RT-negative controls, indicative of residual viral DNA, was subtracted from the total to give a cDNA-specific signal. In the present study, only RT-PCRs yielding 10 or more copies of viral transcript per RT-PCR were considered. Chimerism analysis by flow cytometry using H-2d, CD45.1, and B220 staining showed that while latently infected B6 mice resisted chimerism of allogeneic BALB/c BM, they readily accepted congeneic CD45.1B6 BM and skin grafts (data not shown). 1 and 2 and Table 2). During the establishment of latency at 14 days p.i., virus DNA was detected in all populations of cells analyzed. L. In MZ B cells and GC B cells, the estimated frequency of virus genome-positive cells (1 infected cell per 63 cells and 1 infected cell per 89 cells, respectively) was at least 2 to 3 orders of magnitude higher than in Fo B cells, dendritic cells, and macrophages.

This result showed that B cells constitute the principal target for virus infection during the establishment of latency. However, FITC-induced reactivation of lytic viral RNA transcription was two orders of magnitude less than that of peak viral gene expression on Day 5 after primary infection ( and data not shown). Splenocyte populations analyzed in this study. After intranasal infection of BALB/c mice with 10,000 PFU. However, the first case of a kidney transplant recipient who developed iatrogenic Kaposi’s sarcoma was reported soon afterwards. Data are expressed as differences from values obtained for each mouse during the comparable baseline … Reactivation was detected by transfer of 100 μl culture medium to fresh MEF monolayers which were observed for cytopathic effect for 12 d (31).

Graphical representation of limiting-dilution data estimating the frequency of MHV-68 genome-positive cells in different splenocyte populations. Real-time PCR was performed on limiting dilutions of purified splenocytes and frequencies of infection were determined by limiting-dilution analysis. (A to C) Total B cells at 14, 21, and 153 days p.i., respectively; (D to H) GC B cells at 14, 21, 80, 122, and 253 days p.i., respectively; (I to K) Fo B cells at 14, 21, and 112 days p.i., respectively; (L to N) NF B cells at 14, 21, and 112 days p.i., respectively; (O to Q) MZ B cells at 14, 21, and 112 days p.i., respectively; (R to T) dendritic cells at 14, 21, and 70 days p.i., respectively; (U to X) macrophages at 14, 21, 70, and 147 days p.i., respectively. Data are plotted as the log of the fraction of negative PCRs as a function of cells per reaction (○). The prediction equation of the regression line (plain line) is −log(μi) = f xi, where f is the cell frequency estimated by the maximum-likelihood method according to the SHPM hypothesis. Upper and lower dotted lines are plotted by using upper and lower values of the 95% CI of f. When all PCRs were negative to extrapolate the maximum value of the cell frequency f in the assay, the log of (ni − 1/ni, where ni is the number of replicate reactions) is plotted as a function of the maximum number of cells analyzed (cross) and the prediction inequation is −log(μi) > f xi (shaded area).

Finally, the frequency of Tregs was decreased in γHV-68 C57Bl/6 wt mice before EAE induction (50 days post γHV-68 infection), but it was rescued in γHV-68 C57Bl/6 CD40KO mice that showed the same Treg frequency as naïve CD40 KO mice (). the frequency of virus genome-positive cells decreased sharply in all cell populations analyzed. In GC B cells the frequency of infection fell to 1 infected cell per 880 cells at 21 days p.i. The RNA transcripts from the infected and uninfected samples were converted into cDNA using Superscript II (Invitrogen) in the presence of the fluorescent dyes Cy-3 and Cy-5, respectively. In NF B cells stable levels of virus persistence were also reached but at a higher frequency than GC B cells of 1 infected cell per 11,000 cells. Although IKKβ and IKKε were shown to phosphorylate the inhibitor of κB (IκB, e.g., IκBα), these kinases can directly phosphorylate NF-κB subunits such as RelA, representing a regulatory step likely functioning at post-nuclear translocation. and in Fo B cells, MZ B cells, total B cells, and macrophages beyond 21 days p.i.

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