Rachel Bagni

Conservation of Virally Encoded MicroRNAs in Kaposi Sarcoma-Associated Herpesvirus in Primary Effusion Lymphoma Cell Lines and in Patients with Kaposi Sarcoma or Multicentric Castleman Disease

BACKGROUND MicroRNAs are small noncoding RNAs that posttranscriptionally regulate gene expression. Kaposi sarcoma (KS)-associated herpesvirus (KSHV) encodes 12 distinct microRNA genes, all of which are located within the latency-associated region that is highly expressed in all KSHV-associated malignancies. METHODS We amplified, cloned, and sequenced a 2.8-kbp-long region containing a cluster of 10 microRNAs plus a 646-bp fragment of K12/T0.7 containing the remaining 2 microRNAs from 5 primary effusion lymphoma-derived cell lines and from 17 patient samples. The patients included 2 with classic KS, 12 with AIDS-KS (8 from the United States, 1 from Europe, 3 from Africa, and 4 from Central/South America), and 2 with multicentric Castleman disease (MCD). Additionally, we analyzed the K1, open reading frame 75, and K15 genes to determine KSHV subtypes, and we performed a phylogenetic analysis. RESULTS Phylogenetic analysis of the 2.8-kbp microRNA region revealed 2 distinct clusters of sequences: a major (A/C) and a variant (B/Q) cluster. The variant cluster included sequences from 3 patients of African origin and both patients with MCD. Some microRNAs were highly conserved, whereas others had changes that could affect processing and, therefore, biological activity. CONCLUSIONS These data demonstrate that KSHV microRNA genes are under tight selection in vivo and suggest that they contribute to the biological activity and possibly the pathogenesis of KSHV-associated malignancies.

Infection of Lymphoid Cells by Integration-Defective Human Immunodeficiency Virus Type 1 Increases De Novo Methylation

ABSTRACT DNA methylation, by regulating the transcription of genes, is a major modifier of the eukaryotic genome. DNA methyltransferases (DNMTs) are responsible for both maintenance and de novo methylation. We have reported that human immunodeficiency virus type 1 (HIV-1) infection increases DNMT1 expression and de novo methylation of genes such as the gamma interferon gene in CD4+ cells. Here, we examined the mechanism(s) by which HIV-1 infection increases the cellular capacity to methylate genes. While the RNAs and proteins of all three DNMTs (1, 3a, and 3b) were detected in Hut 78 lymphoid cells, only the expression of DNMT1 was significantly increased 3 to 5 days postinfection. This increase was observed with either wild-type HIV-1 or an integrase (IN) mutant, which renders HIV replication defective, due to the inability of the provirus to integrate into the host genome. Unintegrated viral DNA is a common feature of many retroviral infections and is thought to play a role in pathogenesis. These results indicate another mechanism by which unintegrated viral DNA affects the host. In addition to the increase in overall genomic methylation, hypermethylation and reduced expression of thep16 INK4A gene, one of the most commonly altered genes in human cancer, were seen in cells infected with both wild-type and IN-defective HIV-1. Thus, infection of lymphoid cells with integration-defective HIV-1 can increase the methylation of CpG islands in the promoters of genes such as thep16 INK4A gene, silencing their expression.

Reactivation of Kaposi’s sarcoma-associated herpesvirus by natural products from Kaposi’s sarcoma endemic regions

Kaposis sarcoma (KS) and its causative agent, Kaposis sarcoma associated herpesvirus (KSHV/HHV‐8), a gamma2 herpesvirus, have distinctive geographical distributions that are largely unexplained. We propose the “oncoweed” hypothesis to explain these differences, namely that environmental cofactors present in KS endemic regions cause frequent reactivation of KSHV in infected subjects, leading to increased viral shedding and transmission leading to increased prevalence of KSHV infection as well as high viral load levels and antibody titers. Reactivation also plays a role in the pathogenesis of KSHV‐associated malignancies. To test this hypothesis, we employed an in vitro KSHV reactivation assay that measured increases in KSHV viral load in KSHV infected primary effusion lymphoma (PEL) cells and screened aqueous natural product extracts from KS endemic regions. Of 4,842 extracts from 38 countries, 184 (5%) caused KSHV reactivation. Extracts that caused reactivation came from a wide variety of plant families, and extracts from Africa, where KSHV is highly prevalent, caused the greatest level of reactivation. Time course experiments were performed using 28 extracts that caused the highest levels of reactivation. The specificity of the effects on viral replication was examined using transcriptional profiling of all viral mRNAs. The array data indicated that the natural extracts caused an ordered cascade of lytic replication similar to that seen after induction with synthetic activators. These in vitro data provide support for the “oncoweed” hypothesis by demonstrating basic biological plausibility.

Molecular Evidence for Mother-to-Child Transmission of Kaposi Sarcoma–Associated Herpesvirus in Uganda and K1 Gene Evolution within the Host

BACKGROUND Epidemiological studies of Kaposi sarcoma (KS)-related herpesvirus (KSHV) indicate that having a KSHV-seropositive mother is a risk factor for KSHV infection in children. METHODS We determined the KSHV K1 sequences in concordantly polymerase chain reaction-positive Ugandan mother-child pairs, to ascertain whether they shared the same viral strain. We also examined sequences amplified from saliva and buffy coat samples from the same subjects, to investigate potential intrasubject sequence differences. RESULTS We obtained K1 sequences from 6 of 10 mother-child pairs. In 1 pair, the subtypes differed between mother and child. The mother and child in 2 other pairs shared the same subtype, but the sequences differed. The mother and child in 2 pairs shared KSHV strains with exact (100%) nucleotide homology. The last pair showed evidence of viral strain concordance between mother and child but also showed evidence of evolution of the viral sequence within the child. Of 26 study subjects, 19 showed no evidence of intrasubject K1 sequence variability, but, in 7 subjects, all of whom were children, amino acid variation of 1%-4% was observed. CONCLUSIONS Our findings are consistent with KSHV transmission from maternal and nonmaternal sources in KS-endemic regions. Our results also provide evidence for ongoing evolution of the K1 gene in KSHV-infected children.

Prediagnostic Serum Levels of Cytokines and Other Immune Markers and Risk of Non-Hodgkin Lymphoma

Although severe immune dysregulation is an established risk factor for non-Hodgkin lymphoma (NHL), it is unclear whether subclinical immune system function influences lymphomagenesis. To address this question, we conducted a nested case-control study within the Prostate, Lung, Colorectal, and Ovarian Cancer Screening Trial to investigate whether circulating levels of cytokines and other immune markers are associated with future risk of NHL. Selected cytokines [interleukin (IL)-4, IL-6, IL-10, and TNF-α] and other immune markers [soluble TNF receptor 1 (sTNF-R1), sTNF-R2, C-reactive protein, and sCD27] were measured in prediagnostic serum specimens from 297 incident NHL cases and 297 individually matched controls. ORs and 95% confidence intervals (CI) relating quartiles of analyte concentration to NHL risk were calculated by using conditional logistic regression. Statistically significant associations with increased NHL risk were observed for elevated serum levels of sTNF-R1 (quartile 4 vs. quartile 1: OR = 1.7, 95% CI: 1.1-2.8; P(trend) = 0.02) and sCD27 (OR = 5.3, 95% CI: 2.9-9.4; P(trend) < 0.0001). These associations remained in analyses of cases diagnosed longer than 6 years following blood collection (sTNF-R1: OR = 2.1, 95% CI: 1.0-4.0, P(trend) = 0.01; sCD27: OR = 4.1, 95% CI: 1.9-8.5, P(trend) = 0.0001). Elevated levels of IL-10, TNF-α and sTNF-R2 were also significantly associated with increased risk of NHL overall; however, these associations weakened with increasing time from blood collection to case diagnosis and were null for cases diagnosed longer than 6 years postcollection. Our findings for sTNF-R1 and sCD27, possible markers for inflammatory and B-cell stimulatory states, respectively, support a role for subclinical inflammation and chronic B-cell stimulation in lymphomagenesis.

Kaposi's sarcoma-associated herpesvirus transmission and primary infection.

Purpose of reviewKaposis sarcoma-associated herpesvirus (KSHV), also known as human herpesvirus 8, is the causal agent of Kaposis sarcoma, one of the commonest cancers in HIV-infected individuals. Transmission and risk factors for infection by KSHV are not fully understood. The purpose of this review is to highlight recent advances in our understanding of KSHV transmission in various settings. Recent findingsKSHV and HIV are both common in southern Africa where KSHV infection occurs during childhood via saliva. HIV infection is a major risk factor for KSHV infection. In developed countries, KSHV transmission among men who have sex with men is related to sexual risk factors such as number of sexual partners and to sexual practices involving saliva. KSHV can be transmitted by transfusion of infected blood, but storage of blood products diminishes the risk. SummaryRecent reports have provided much additional insight into KSHV transmission in different populations, but have also provided a number of important questions for the research and public health communities. Most critically, the role of HIV in increasing risk for KSHV infection and the possible effects on KSHV prevalence, and consequently the incidence of Kaposis sarcoma warrants urgent further study.

Intra-individual variability over time in serum cytokine levels among participants in the Prostate, Lung, Colorectal, and Ovarian Cancer Screening Trial

BACKGROUND Serum measurements of cytokines, mediators of various B and T cell activities, are important markers of inflammation and immune dysregulation. We assessed the reproducibility of serum cytokine measurements over a five-year period among participants in the Prostate, Lung, Colorectal, and Ovarian Cancer Screening Trial (PLCO). METHODS Levels of 13 cytokines [interleukin (IL) 1β, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-12p70, IL-13, interferon-gamma (IFNγ), granulocyte macrophage colony-stimulating factor (GM-CSF), and tumor necrosis factor-α (TNFα)] in stored sera from three collections (study baseline,+1 year, and+5 years) among 28 randomly selected PLCO participants were measured using a high-sensitivity Luminex xMap-based multiplex panel. Within- and between-subject components of variance were estimated from random effects models and were used to calculate the coefficient of variation (CV) and intraclass correlation coefficient (ICC) for analytes with <30% of samples below the limit of detection (LOD). Spearman correlation coefficients between measurements of the same analyte over time and between analytes were also calculated. RESULTS Among the six cytokines with <30% of samples below the LOD, we observed excellent reproducibility for IL-6, IL-7, IL-13, and TNFα (ICC≥0.73), and fair to good reproducibility for IL-8 (ICC=0.55) and IL-10 (ICC=0.60). Spearman correlation coefficients comparing paired measurements of each cytokine at baseline and at +5 years were high (ρ≥0.74) with the exception of IL-10 (ρ=0.44). CONCLUSIONS These results suggest that measurements of most of the cytokines evaluated in this study were highly reproducible over five-year periods.

Kaposi Sarcoma (KS)-Associated Herpesvirus MicroRNA Sequence Analysis and KS Risk in a European AIDS-KS Case Control Study

BACKGROUND We recently identified polymorphisms in Kaposi sarcoma-associated herpesvirus (KSHV)-encoded microRNA (miRNA) sequences from clinical subjects. Here, we examine whether any of these may contribute to KS risk in a European AIDS-KS case-control study. METHODS KSHV load in peripheral blood was determined by real-time quantitative polymerase chain reaction. Samples that had detectable viral loads were used to amplify the 2.8-kb miRNA encoding region plus a 646-bp fragment of the K12/T0.7 gene. Additionally, we characterized an 840-bp fragment of the K1 gene to determine KSHV subtypes. RESULTS KSHV DNA was detected in peripheral blood mononuclear cells of 49.6% of case patients and 6.8% of controls, and viral loads tended to be higher in case patients. Sequences from the miRNA-encoding regions were conserved overall, but distinct polymorphisms were detected, some of which occurred in primary miRNAs, pre-miRNAs, or mature miRNAs. CONCLUSIONS Patients with KS were more likely to have detectable viral loads than were controls without disease. Despite high conservation in KSHV miRNA-encoded sequences, polymorphisms were observed, including some that have been reported elsewhere. Some polymorphisms could affect mature miRNA processing and appear to be associated with KS risk.

Heterogeneity and breadth of host antibody response to KSHV infection demonstrated by systematic analysis of the KSHV proteome.

The Kaposi sarcoma associated herpesvirus (KSHV) genome encodes more than 85 open reading frames (ORFs). Serological evaluation of KSHV infection now generally relies on reactivity to just one latent and/or one lytic protein (commonly ORF73 and K8.1). Most of the other polypeptides encoded by the virus have unknown antigenic profiles. We have systematically expressed and purified products from 72 KSHV ORFs in recombinant systems and analyzed seroreactivity in US patients with KSHV-associated malignancies, and US blood donors (low KSHV seroprevalence population). We identified several KSHV proteins (ORF38, ORF61, ORF59 and K5) that elicited significant responses in individuals with KSHV-associated diseases. In these patients, patterns of reactivity were heterogeneous; however, HIV infection appeared to be associated with breadth and intensity of serological responses. Improved antigenic characterization of additional ORFs may increase the sensitivity of serologic assays, lead to more rapid progresses in understanding immune responses to KSHV, and allow for better comprehension of the natural history of KSHV infection. To this end, we have developed a bead-based multiplex assay detecting antibodies to six KSHV antigens.

Purify First: rapid expression and purification of proteins from XMRV.

Purifying proteins from recombinant sources is often difficult, time-consuming, and costly. We have recently instituted a series of improvements in our protein purification pipeline that allows much more accurate choice of expression host and conditions and purification protocols. The key elements are parallel cloning, small scale parallel expression and lysate preparation, and small scale parallel protein purification. Compared to analyzing expression data only, results from multiple small scale protein purifications predict success at scale-up with greatly improved reliability. Using these new procedures we purified eight of nine proteins from xenotropic murine leukemia virus-related virus (XMRV) on the first attempt at large scale.