A Role for the Internal Repeat of the Kaposi’s Sarcoma-Associated Herpesvirus Latent Nuclear Antigen in the Persistence of an Episomal Viral Genome

A Role for the Internal Repeat of the Kaposi's Sarcoma-Associated Herpesvirus Latent Nuclear Antigen in the Persistence of an Episomal Viral Genome

Kaposi’s sarcoma-associated herpesvirus (KSHV or HHV-8) is the etiological agent of Kaposi’s sarcoma, a highly vascularized, endothelial-derived tumor. KSHV establishes a predominantly latent infection in the main KS tumor cell type, the spindle cell, which is of endothelial cell origin. (B) Immunofluorescence staining of KSHV LANA expression in KSHV-THP-1 cells compared to that in uninfected THP-1 cells. Thus, KSHV minimizes its immunological signature by suppressing key immune response factors, enabling persistent infection and evasion from host detection. The vast majority of people carry a combination of latent herpesviruses which may cause severe disease and death if they reactivate upon immune suppression (1–4). The usage of each mapped pA site correlates to its peak size, the larger (broad and wide) peak size, the more usage and thus, the higher expression of the pA site-associated gene(s). Three months after primary infection, experimental reactivation showed that BHV-5 was able to establish latency in the trigeminal ganglia but also the CNS of surviving calves.

Conditioned medium from KSHV-infected endothelial cells is able to transiently activate STAT3, indicating the involvement of a secreted factor and that a latency-associated factor in KSHV-infected cells is necessary for sustained activation. These data demonstrate that an IFN-α/β-induced pathway regulates γHV68 gene expression patterns during latent viral infection in vivo and that IFN-α/β plays a critical role in inhibiting viral reactivation during latency. The IR consists of three segments, a segment (amino acids [aa] 340 to 431) containing multiple repeats of the amino acids DEED or DEEED; a segment (aa 440 to 756) containing multiple repeats of the motifs QQQEP, QQREP, and QQQDE; and a segment (aa 760 to 931) containing multiple repeats of the motifs QEQELEE and QELEVEE with L, V, and Q spaced in a leucine zipper-like pattern (19). In contrast to EBV, comparatively little is known about the natural sequence of events during KSHV infection or the phenotype of B cells during long-term latent infection. The function of this IR region is not completely resolved. IMPORTANCE Kaposi’s sarcoma-associated herpesvirus (KSHV) is the causative agent of multiple human malignancies in immunocompromised individuals. 1981.

© 2000 Wiley-Liss, Inc. This results in shedding of virus in saliva, urine or other body secretions. Both of these downregulate surface expression of HLA-A and HLA-B but K3 also downregulates the NK-cell inhibitory ligands HLA-C and HLA-E 16. To delete the coding region for LANA, we inserted a kanamycin resistance gene instead of most of the LANA gene using an ET cloning strategy (Gene Bridges) (Fig. Many γ-herpesviruses, including the human pathogens EBV and Kaposi’s sarcoma-associated herpesvirus (KSHV) and the murine γHV68 (γ-herpesvirus 68), establish life-long latent infections in hemopoietic cells (3, 20, 21). As a result of the cassette insertion, KSHV-ΔLANA lacks the whole N-terminal domain, the IR, and a small part of the C-terminal domain of orf73, corresponding to nucleotide position 127379 upstream of the LANA ATG and nucleotide position 124438 downstream of the IR region (equivalent to aa 1 to 954 of LANA) (Fig. 1A).

Exacerbation of experimental autoimmune encephalomyelitis in rodents infected with murine gammaherpesvirus-68. Se tomaron muestras de timo, bazo, bolsa de Fabricio, plexo braquial, plexo ciático y extremos de las plumas a los 21, 28, 35, 70 y 105 días después de la inoculación. Acute HHV-6 infection is rare in immunocompetent adults but may manifest as a mononucleosislike illness characterized by fever, lymphadenopathy, and hepatitis or encephalitis, with negative test results for CMV or Epstein-Barr virus (EBV). A role for latent herpesviruses in fibrosis has not been studied. To delete the IR region only, we used the shuttle mutagenesis strategy (Fig. 1B) (4) and generated KSHV-LANAΔ329-931. Of note, CTL epitopes were also identified in vMIR1 and vMIR2 (Ribechini et al., 2006), two lytic KSHV genes that downregulate expression of MHC-I and other co-stimulatory proteins (Chapters 31 and 62), whereas vFLIP is able to upregulate expression of MHC-I and ICAM-1 (Lagos et al., unpublihsed data).

All surgery was conducted by using standard aseptic techniques. After a period of persistent productive infection in the salivary gland, MCMV establishes a predominantly latent infection in immunocompetent mice (31, 32). Immunoblot assays of lysates from HEK293 cells transiently transfected with the KSHV genome containing wild-type or mutant LANA and sorted with a fluorescence-activated cell sorter (FACS) for GFP expression showed the presence of the expected 150- to 250-kDa LANA protein in the case of KSHV-LANAwt and a band of approximately 90 kDa for KSHV-LANAΔ329-931, while no specific band was seen in KSHV-ΔLANA-transfected cells (Fig. 1D). An immunofluorescence assay of HeLa cells transiently transfected with wild-type KSHV (KSHV-wt), KSHV-ΔLANA, or KSHV-LANAΔ329-931 confirmed the expression of the LANAΔ329-931 protein, which localizes to the nucleus and shows a speckled pattern similar to that of full-length LANA in the context of the entire genome (Fig. 1E). Construction and characterization of KSHV-ΔLANA and KSHV-LANAΔ329-931.

(A) KSHV-ΔLANA was generated by ET cloning. A DNA fragment carrying the kanamycin resistance cassette (rpsl-neo) flanked by 60 bp homologous to the area to be knocked out was generated by PCR and inserted instead of aa 1 to 954 of LANA-1. Essentially, the ability to skew the adaptive immune response towards a Th1 phenotype and retain low frequencies of Treg cells through CD40 expression and co-stimulation represents a novel mechanism in which latent γ-herpesviruses like EBV can act to facilitate and induce autoimmune disease. The transcript encoding vcyc and vFLIP (left side) starts from a reported latent promoter in the 3′ end of orf73 (see text). term, terminus. Studies have shown that RTA can activate the KSHV immediate-early/early or primary (K8, K5, K2, K12, Nut1, and ORF 6, 57, and 74), early or secondary (K9, ORF 59, ORF 65, and K3), and late or tertiary (K1, K8.1A, and ORF 21) lytic cycle genes, either alone or in synergy with other viral regulatory genes (14, 18, 29, 46, 50, 55). LANA lacking aa 329 to 931 flanked by extra homologous regions (in gray) was cloned into the shuttle vector (pST76-NSR), electroporated into Escherichia coli harboring the KSHV-wt genome, and then passed through several steps of selection and counterselection.

Posttranslational modifications of the RelA subunit, e.g., phosphorylation and acetylation, are important means to regulate NF-κB-dependent gene expression (31,–34). NC_009333. (C) Restriction digestion analysis of the KSHV LANA mutants with the KpnI enzyme. The 9.6-kb band in the KSHV-wt is shifted to around 8.0 kb in the case of KSHV-ΔLANA (lanes 1 and 2). The same band was shifted to 7.8 kb in the case of KSHV-LANAΔ329-931 (lanes 5 and 6). To confirm the identities of these bands, Southern blot analysis was performed with the rpsl-neo cassette in the case of KSHV-ΔLANA (lanes 3 and 4) and the amino-terminal domain of LANA (aa 1 to 328) as a probe in the case of KSHV-LANAΔ329-931 (lanes 7 and 8). (D) Expression of LANA in the context of the whole KSHV-BAC genome.

HEK293 cells were transfected with KSHV-wt, KSHV-ΔLANA, or KSHV-LANAΔ329-931 and then sorted with a FACS for GFP expression and lysed with lysis buffer for Western blot analysis. (E) Immunofluorescence assay of HeLa cells transfected with KSHV-wt (I), KSHV-LANAΔ329-931 (II), or KSHV-ΔLANA (III) DNA showing the intracellular localization of LANA and LANAΔ329-931. (F) Immunofluorescence assay showing the expression and localization of LANA and LANAΔ329-931 from the pGTR4 plasmid constructs. I and II, pGTR4:LANA; III and IV, pGTR4:LANAΔ329-931; V and VI, PGTR4. Once the clinical signs of neurological disease appeared, clinical monitoring was performed twice a day. Human serum from a patient with Kaposi’s sarcoma was used at a dilution of 1:500 to detect the expression of LANA and LANAΔ329-931. We first evaluated the ability of KSHV-wt and the two KSHV LANA mutants to establish stable clones under selection.
A Role for the Internal Repeat of the Kaposi's Sarcoma-Associated Herpesvirus Latent Nuclear Antigen in the Persistence of an Episomal Viral Genome

HEK293 cells were transfected with KSHV-wt or the KSHV-LANA mutants. In these subjects, who were acutely infected with HBV, nearly a quarter of the CD8+ T-cell pool was activated and this population included CD8+ T cells specific for herpesviruses. Etomoxir blocks IL-4-induced changes in fatty–acid oxidation(21) and upregulation of CD206 (fig. As expected, the KSHV-ΔLANA genome failed to produce stable clones under selection, supporting an important role for LANA in KSHV episomal persistence (Table 1). 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). The apparent molecular mass of LNA ranged from 204/212 kDa for cell line PK-1 to 226/234 kDa for cell line BC-1. 1D and E).

Furthermore, LANAΔ329-931 does not appear to be degraded more quickly than wild-type LANA in the presence of cycloheximide (see Fig. The recombinant proteins represent a nonoverlapping set and contain no apparent common epitopes, which suggests that LN20 is a mixed antibody population, requiring further rounds of monocloning. The membrane was washed and exposed to Kodak XAR5 film for 5 days. 1G) and selecting the transfectants with 150 μg hygromycin. This result excludes the possibility of an inadvertent alteration in the remainder of the KSHV genome or the bacterial artificial chromosome backbone being responsible for the observed phenotype. Isolation of a defective KSHV, KV-1.Further virologic and phenotypic characterizations of the defective KSHV detected by PCR-DSG were not possible because of the limited amount of the paraffin-embedded specimens. Early after transfection, wells transfected with KSHV-wt and KSHV-LANAΔ329-931 consistently showed a higher number of GFP-expressing cells than wells transfected with KSHV-ΔLANA (Fig.

The lower limit of ORF 73 gene detection was 10 to 100 copies, and the most accurate detection was from 100 to 106 copies. This may reflect the transient replication of the KSHV episome in cells transfected with KSHV-wt and KSHV-LANAΔ329-931 but not in KSHV-ΔLANA. HRE-luciferase assays.TIME cells were transfected in a six-well dish using the TransIt Jurkat reagent (Mirus) with 2 μg of a firefly luciferase expression vector containing either a wild-type or a mutant triple repeat of the erythropoietin 3′ HRE in the enhancer region (31) as well as 1 ng of control Renilla luciferase expression vector pRL-SV40 (Promega) for transfection efficiency normalization. (A) Intracellular glutamine levels are elevated following KSHV infection. As shown in Fig. Cell sorting was performed by staining cells as described above for CD86, and results were analyzed by the UNC Flow Cytometry Core facility using iCyt/Sony Reflection under sterile conditions. We performed the same experiment with the 293LANA cell line.

The remaining 12 pA sites unable to assign would indicate the presence of transcripts from unknown KSHV genes for further validation. 2C), indicating that the presence of LANA in trans enhanced the replication of transfected KSHV genomes and thereby the expression of the GFP marker in the backbone of the bacterial artificial chromosome. As in the absence of LANA provided in trans, the number of GFP-positive cells (as a proportion of the total number of cells in the culture) was consistently higher in cells transfected with the KSHV-wt and KSHV-LANAΔ329-931 genomes than in those transfected with KSHV-ΔLANA (Fig. (B) Age-matched 129SvPas or IFN-α/βR−/− mice were infected with 100 PFU γHV68 intranasally, and spleen, liver, and lung were harvested 7 to 9 or 21 to 28 dpi. The fact that the cultures transfected with KSHV-wt and KSHV-LANAΔ329-931 showed a proportion of GFP-positive cells higher than that of KSHV-ΔLANA-transfected cells suggests an additional role for LANA provided in cis and that LANAΔ329-931 behaves like wild-type LANA-1 in this context. For all cell populations, levels of virus genome positive cells were maximal at 14 days p.i., with MZ B cells and GC B cells harboring the highest frequency of infection. 2D, the presence or absence of LANA-1 or its IR in cis, however, did not have an impact on the rate of loss of viral episomes, even in the presence of excess LANA-1 provided in trans.

The data were acquired on a FACSCalibur flow cytometer equipped with CellQuest Pro software and analyzed using FlowJo software. HEK293 cells were shown to retain KSHV episomes in a small percentage of originally infected cells, slightly more than several other cell lines (10). In these analyses, prevalence estimates were 81–92% for Congo and 1–11% for Malta. KSHV genomes with mutant forms of LANA are lost from HEK293 and 293LANA cells at the same rate as KSHV genomes with wild-type LANA. All three KSHV constructs were transfected into HEK293 cells (A and B) or the 293LANA cell line (C and D), and the loss of GFP-positive cells was monitored over time by FACS. (A and C) Fractions of GFP-positive cells as percentages of all of the cells in the culture plotted over time. Latent infection-associated resistance to allograft tolerance is not mediated by NK1.1 cells.

We employed the minireplicon system to study the ability of LANAΔ329-931 to replicate viral DNA (10). The minireplicon (pGTR4:LANA) replicates spontaneously because it carries four copies of the KSHV TR, which contains the latent origin of replication, in addition to LANA. Regulatory T cell activity in primary and persistent Epstein-Barr virus infection. A short-term replication assay (9, 20) was employed to study the ability of LANAΔ329-931 to replicate this plasmid. In five independent experiments, pGTR4:LANAΔ329-931 replicated the minireplicon either at a level comparable to that of pGTR4:LANA or at slightly reduced levels, which were, however, comparable to those of pGTR4:LANA when the different levels of protein expression of LANA and LANAΔ329-931 in minireplicon experiments were taken into account (Fig. Reactivation of γherpesvirus-68 (γHV-68) is not necessary to augment fibrosis. Replication competence of the LANAΔ329-931 protein.

(A) KSHV minireplicons carrying four copies of the KSHV TR region (which contains the LANA binding sites) and LANA (pGTR4:LANA), LANAΔ329-931 (pGTR4:LANAΔ329-931), or no LANA (pGTR4) were transfected into HEK293 cells. Extrachromosomal DNA was extracted from transfected cells by Hirt extraction on day 3 after transfection and digested with MfeI (input DNA) or MfeI and DpnI (unmethylated, replicated DNA). Within-subjects analysis showed significant effects of time on both percentage time in SWS (P = 0.008) and SWS bout length (P < 0.001), with no significant interactions. Statistical analysis was carried out through the Division of Biostatistics at Washington University School of Medicine. (B) Immunoblot assay showing the expression of the LANA and LANAΔ329-931 proteins in these cultures. (C) Quantification of the replication efficiency of LANA and LANAΔ329-931 from panel A. After the bands were measured with the ImageJ program, the quantity of the replicated DNA (represented by the band after digestion with MfeI and DpnI) was divided by the quantity of the input DNA (represented by the band after digestion with MfeI only) for each construct separately and multiplied by 100 to calculate the percentage of replicated DNA. The average percentage of four experiments, without adjustment for LANA protein levels, is shown on the left side of the panel. The right side of the panel presents replication efficiency results adjusted for LANA protein expression levels. (D) Stability of LANA and LANAΔ329-931 in the presence of cycloheximide. At 24 h after the transfection of 293 cells with pGTR4 expressing LANA or LANAΔ329-931, cells were treated with 100 μg/ml cycloheximide or left untreated. Our results show for the first time, to our knowledge, that γHV-68 mice have decreased splenic percentages of Tregs and that this reduction is long lasting, still evident more than 50 days after infection. A recent study (6) found that KSHV with extensive internal deletions of LANA removing most of the N-terminal domain and the entire IR region (e.g., LANAΔ33-929) retained the ability to replicate viral DNA in the transient replication assay shown in Fig. 3 but was not capable of mediating the persistence of viral episomes in BJAB B cells under selection. After hybridization, slides were washed in low-stringency buffer containing 1% SSC and 0.2% SDS for 5 min three times at 42°C. We conclude that the LANA IR region plays an important role in supporting the persistence of latent viral genomes.

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