Epstein-Barr Virus Infection of Mammary Epithelial Cells Promotes Malignant Transformation.

Whether the human tumor virus, Epstein-Barr Virus (EBV), promotes breast cancer remains controversial and a potential mechanism has remained elusive. Here we show that EBV can infect primary mammary epithelial cells (MECs) that express the receptor CD21. EBV infection leads to the expansion of early MEC progenitor cells with a stem cell phenotype, activates MET signaling and enforces a differentiation block. When MECs were implanted as xenografts, EBV infection cooperated with activated Ras and accelerated the formation of breast cancer. Infection in EBV-related tumors was of a latency type II pattern, similar to nasopharyngeal carcinoma (NPC). A human gene expression signature for MECs infected with EBV, termed EBVness, was associated with high grade, estrogen-receptor-negative status, p53 mutation and poor survival. In 11/33 EBVness-positive tumors, EBV-DNA was detected by fluorescent in situ hybridization for the viral LMP1 and BXLF2 genes. In an analysis of the TCGA breast cancer data EBVness correlated with the presence of the APOBEC mutational signature. We conclude that a contribution of EBV to breast cancer etiology is plausible, through a mechanism in which EBV infection predisposes mammary epithelial cells to malignant transformation, but is no longer required once malignant transformation has occurred.

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

Kaposi's sarcoma (KS) and its causative agent, Kaposi's 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.

Baboon model for West Nile virus infection and vaccine evaluation.

Animal models that closely mimic the human condition are of paramount significance to study pathogenic mechanisms, vaccine and therapy scenarios. This is particularly true for investigations that involve emerging infectious diseases. Nonhuman primate species represent an alternative to the more intensively investigated rodent animal models and in a number of instances have been shown to represent a more reliable predictor of the human response to infection. West Nile virus (WNV) has emerged as a new pathogen in the Americas. It has a 5% fatality rate, predominantly in the elderly and immune compromised. Typically, infections are cleared by neutralizing antibodies, which suggests that a vaccine would be efficacious. Previously, only macaques had been evaluated as a primate model for WNV vaccine design. The macaques did not develop WNV disease nor express the full complement of IgG subclasses that is found in humans. We therefore explored baboons, which exhibit the similar four IgG subclasses observed in humans as a new model for WNV infection and vaccine evaluation. In this present report, we describe the experimental infection of baboons with WNV and test the efficacy of an inactivated WNV vaccination strategy. All experimentally infected animals developed transient viremia and subsequent neutralizing antibodies. Anti-WNV IgM antibodies peaked at 20 days post-infection. Anti-WNV IgG antibodies appeared later and persisted past 60 days. Prior vaccination with chemically inactivated virus induced neutralizing titers and a fast, high titer IgG recall response, which resulted in lower viremia upon challenge. This report is the first to describe the development of the baboon model for WNV experimental infection and the utility of this model to characterize the immunologic response against WNV and a candidate WNV vaccine.

Whole-genome transcription profiling of rhesus monkey rhadinovirus.

Rhesus monkey rhadinovirus (RRV) and Kaposi's sarcoma-associated herpesvirus (KSHV; also called human herpesvirus 8) belong to the gamma-2 grouping of herpesviruses. RRV and KSHV share a high degree of sequence similarity, and their genomes are organized in a similar fashion. RRV serves as an excellent animal model system to study the gamma herpesvirus life cycle both in vitro and in vivo. We have developed a high-sensitivity, high-throughput, high-specificity real-time quantitative reverse transcriptase-based PCR assay for RRV and have used this assay to profile transcription from the whole RRV genome during de novo productive infection of rhesus fibroblasts. Using this assay, we demonstrate that the genome-wide transcription profile for RRV closely parallels the genome-wide transcription profile for KSHV.

Real-time quantitative PCR analysis of viral transcription.

Whole-genome profiling using DNA arrays has led to tremendous advances in our understanding of cell biology. It has had similar success when applied to large viral genomes, such as the herpesviruses. Unfortunately, most DNA arrays still require specialized and expensive resources and, generally, large amounts of input RNA. An alternative approach is to query entire viral genomes using real-time quantitative PCR. We have designed such PCR-based arrays for every open reading frame of human herpesvirus 8 and describe here the general design criteria, validation procedures, and detailed application to quantify viral mRNAs. This should provide a useful resource either for whole-genome arrays or just to measure transcription of any one particular mRNA of interest. Because these arrays are RT-PCR-based, they are inherently more sensitive and robust than current hybridization-based approaches and are ideally suited to query viral gene expression in models of pathogenesis.

The tumor microenvironment controls primary effusion lymphoma growth in vivo.

Certain lymphomas in AIDS patients, such as primary effusion lymphoma (PEL), are closely associated with the lymphotropic gamma herpes virus Kaposi's sarcoma-associated herpes virus (KSHV), also called human herpesvirus 8. The virus is thought to be essential for tumorigenesis, yet systems to investigate PEL in vivo are rare. Here we describe PEL tumorigenesis in a new xenograft model. Embedded in Matrigel, PEL cells formed rapid, well-organized, and angiogenic tumors after s.c. implantation of C.B.17 SCID mice. Without Matrigel we did not observe comparable tumors, which implies that extracellular support and/or signaling aids PEL. All of the tumors maintained the KSHV genome, and the KSHV latent protein LANA/orf73 was uniformly expressed. However, the expression profile for key lytic mRNAs, as well as LANA-2/vIRF3, differed between tissue culture and sites of implantation. We did not observe a net effect of ganciclovir on PEL growth in culture or as xenograft. These findings underscore the importance of the microenvironment for PEL tumorigenesis and simplify the preclinical evaluation of potential anticancer agents.

Real-time Quantitative PCR Arrays for Virally-associated Cancers.

Virally-associated cancers are unique in that their origin is typically well defined and suitable to genomic analysis on a smaller scale. We recently reported the transcription profile of Kaposi's sarcoma-associated herpesvirus (KSHV/HHV-8) in Kaposi's sarcoma (KS) using a real-time quantitative PCR (QPCR) array. This review explores the advantages and limitations of such an approach as well as the possibilities of extending PCR-based profiling to human cancers. Since real-time QPCR records a truly quantitative transcription profile, this technology will improve statistical analysis and solidify clinical decision-making.

Tissue specificity of the Kaposi's sarcoma-associated herpesvirus latent nuclear antigen (LANA/orf73) promoter in transgenic mice.

Kaposi's sarcoma-associated herpesvirus (KSHV/HHV-8) is a human-oncogenic herpesvirus. Cells from KSHV-associated tumors, such as Kaposi's sarcoma (KS) and primary effusion lymphoma (PEL), are of endothelial and B-cell origin, respectively. KSHV persists indefinitely in these cell lineages during latent infection. Indeed, cellular latency is a hallmark of all herpesviruses that is intimately linked to their pathogenesis. We previously characterized the promoter for the KSHV latency-associated nuclear antigen LANA/orf73. LANA is required for latent episome maintenance and has also been implicated in oncogenesis. Hence, regulation of LANA expression is critical to KSHV persistence. We find that a region extending to bp -1299 upstream of the LANA transcription start site is able to drive lacZ-reporter gene expression in several lines of transgenic mice. In agreement with KSHV's natural tropism, we detected reporter gene expression in CD19-positive B cells but not in CD3-positive T cells. We also detected expression in the kidney and, at a lower level, in the liver. In contrast to KS tumors, transgene expression was localized to kidney tubular epithelium rather than vascular endothelial cells. This suggests that our promoter fragment contains all cis-regulatory elements sufficient for B-cell specificity but not those required for endothelial specificity. Alternatively, while the trans-acting factors required for LANA expression in B cells are evolutionarily conserved, those that regulate endothelial cell-specific expression are unique to humans. Our in vivo studies address a conundrum in KSHV biology: in culture, KSHV is able to infect a variety of cell types indiscriminately, while in healthy latent carriers KSHV is found in B lymphocytes. The transgenic-mouse experiments reported here suggest that tissue-restricted LANA gene expression could explain B-cell-specific viral persistence.