Prashant Desai

Reconstitution of the Kaposi's Sarcoma-Associated Herpesvirus Nuclear Egress Complex and Formation of Nuclear Membrane Vesicles by Coexpression of ORF67 and ORF69 Gene Products

ABSTRACT The Kaposis sarcoma-associated herpesvirus nuclear egress complex is composed of two proteins, ORF67 and ORF69. In this study, we have recapitulated the KSHV complex by coexpression of these two proteins in insect cells using expression from recombinant baculoviruses. The proteins form a complex at the nuclear membrane as judged by live-cell analysis of protein fusions tagged with green fluorescent protein (GFP) and mCherry. Ultrastructural analysis of infected cells showed that ORF67 expression results in reduplication of the nuclear membrane. When the two proteins are expressed together, numerous virion-size nuclear membrane-derived vesicles were evident at the nuclear margins.

KSHV Regulation of Fibulin-2 in Kaposi's Sarcoma: Implications for Tumorigenesis

Kaposis sarcoma is an angioproliferative tumor caused by Kaposis sarcoma-associated herpesvirus (KSHV) infection of vascular endothelial cells. Fibulins, proteins that associate with extracellular matrix (ECM) proteins, may have both tumor-suppressive and oncogenic activities. We found that the expression of fibulin-2 protein and mRNA were decreased 50-fold and 26-fold, respectively, in 10-day KSHV-infected dermal microvascular endothelial cells (DMVEC). Using quantitative RT-PCR, we found a fivefold and 25-fold decrease of fibulin-2 extracellular matrix binding partners, fibronectin and tropoelastin, respectively. Time-course transcriptional analyses over 10 days showed that in addition to that of fibulin-2, expression of fibulins 3 and 5 was decreased in KSHV-infected DMVEC, fibulins 1C/1D were increased, and fibulins 4, 6, and 7 were unchanged. KSHV latency-associated nuclear antigen (LANA) transcription levels rose consistently over the same period. Addition of recombinant fibulin-3 or -5 for 48 hours to 10-day KSHV-infected cells caused a suppression of KSHV-induced vascular endothelial growth factor (VEGF) protein and mRNA levels. Recombinant fibulin-3 also significantly reduced VEGF receptor 3 expression. In pleural effusion lymphoma cell lines that express variable levels of KSHV lytic replication, we observed no detectable fibulin-2 or -5 expression. Finally, fibulin-2 expression was decreased in tissue microarrays from KSHV-infected, LANA-positive patient cells as compared to that in patient nontumor controls. Understanding the interactions between KSHV and the fibulins may lead to the development of novel therapies for treatment of Kaposis sarcoma.

KSHV downregulation of galectin-3 in Kaposi’s sarcoma

Galectins are a family of proteins that share an affinity for beta-galactoside containing glycoconjugates. In prostate, ovarian and breast cancer, downregulation of galectin-3 is associated with malignancy and tumor progression. Kaposis sarcoma (KS) is characterized as an angioproliferative tumor of vascular endothelial cells and produces rare B cell lymphoproliferative diseases in the form of primary effusion lymphomas and some forms of multicentric Castlemans disease. Kaposis sarcoma-associated herpesvirus (KSHV) is the etiological agent of KS. We found reduced levels of galectin-3 expression in a significant fraction of latency-associated nuclear antigen (LANA)-positive spindle cell regions in human archival KS tissue and as measured in KS tissue microarrays. Here we demonstrate that galectin-3 protein expression is downregulated 10-fold in 10-day KSHV-infected dermal microvascular endothelial cells (DMVEC) accompanied by downregulation of message. There is loss of galectin-3 staining in KSHV-infected DMVEC by dual labeled immunohistochemistry in LANA-positive spindle cells. We observed a consistent downregulation of galectin-3 by time-course transcriptional analysis. Of the galectins assayed, only galectin-1 was also downregulated in KSHV-infected DMVEC. We examined 86 KS tumors; 19 were LANA positive (22%) and 67 LANA negative (78%). All 86 tumors were found to be galectin-3 positive; 11 of 19 showed reduced expression of galectin-3 in LANA-positive spindle cell regions. Our data suggest that KSHV vFLIP and LANA are the viral genes targeting galectin-3 downregulation. The contribution of host factors to the pathogenesis of KS is essential for early detection and development of innovative therapies for treatment.

Development and Application of a High-Content Virion Display Human GPCR Array

G protein-coupled receptors (GPCRs) comprise the largest membrane protein family in humans and can respond to a wide variety of ligands and stimuli. Like other multi-pass membrane proteins, the biochemical properties of GPCRs are notoriously difficult to study because they must be embedded in lipid bilayers to maintain their native conformation and function. To enable an unbiased, high-throughput platform to profile biochemical activities of GPCRs in native conformation, we individually displayed 315 human non-odorant GPCRs (>85% coverage) in the envelope of human herpes simplex virus-1 and immobilized on glass to form a high-content Virion Display (VirD) array. Using this array, we found that 50% of the tested commercial anti-GPCR antibodies (mAbs) is ultra-specific, and that the vast majority of those VirD-GPCRs, which failed to be recognized by the commercial mAbs, could bind to their canonical ligands, indicating that they were folded correctly. Next, we used the VirD-GPCR arrays to examine binding specificity of two known peptide ligands and recovered expected interactions, as well as new off-target interactions, three of which were confirmed with real-time kinetics measurements. Finally, we explored the possibility of discovering novel pathogen targets by probing VirD-GPCR arrays with live group B Streptococcus (GBS), a common Gram-positive bacterium causing neonatal meningitis. Using cell invasion assays and a mouse model of hematogenous meningitis, we showed that inhibition of one of the five newly identified GPCRs, CysLTR1, greatly reduced GBS penetration in brain-derived endothelial cells and in mouse brains. Therefore, our work demonstrated that the VirD-GPCR array holds great potential for high-throughput, unbiased screening for small molecule drugs, affinity reagents, and deorphanization.

Rapid and efficient in vitro excision of BAC sequences from herpesvirus genomes using Cre-mediated recombination

Cre-mediated recombination is a widely used technique for the re-arrangement of DNA sequences that are bracketed by loxP recognition sites. This bacteriophage P1 enzyme is commonly used to excise the bacterial artificial chromosome (BAC) sequence, a vector sequence used for large herpesvirus genomes for the purposes of propagation and manipulation in Escherichia coli. Most methods utilize cell lines that can be induced for the expression of Cre enzyme to facilitate this excision. In addition, methods have been developed that express Cre from the virus genome and enable auto-excision of the BAC plasmid. We report a versatile and rapid in vitro method based on purified Cre enzyme to carry out the same process in a test tube and does not require cell line generation or cloning into the virus genome. This method greatly increases the repertoire of methods available to modify the genome prior to reconstitution of virus infectivity in a mammalian host.

Visualization of herpes simplex virus type 1 virions using fluorescent colors

Our laboratory was one of the first to engineer a live fluorescent tag, enhanced green fluorescent protein (eGFP), that marked the capsid of herpes simplex virus type 1 (HSV-1) and subsequently maturing virus as the particle made its way to the cell surface. In the present study we sought to increase the repertoire of colors available as fusion to the small capsid protein, VP26, so that they can be used alone or in conjunction with other fluorescent tags (fused to other HSV proteins) to follow the virus as it enters and replicates within the cell. We have now generated viruses expressing VP26 fusions with Cerulean, Venus, mOrange, tdTomato, mCherry, and Dronpa3 fluorescent proteins. These fusions were made in a repaired UL35 gene (VP26) background. These fusions do not affect the replication properties of the virus expressing the fusion polypeptide and the fusion tag was stably associated with intranuclear capsids and mature virions. Of note we could not isolate viruses expressing fusions with fluorescent proteins that have a tendency to dimerize.

KSHV regulation of fibulins in Kaposi’s sarcoma: implications for tumorigenesis

Kaposi’s sarcoma (KS) is an angioproliferative tumor of vascular endothelial cells and produces rare B cell lymphoproliferative diseases in the form of primary effusion lymphomas (PELs) and some forms of Multicentric Castleman’s Disease (MCD). Kaposi’s sarcoma-associated herpesvirus, also known as KSHV or human herpesvirus type 8 (HHV8), is the etiological agent of KS. Fibulins are extra-cellular matrix (ECM) proteins involved in cell adhesion, proliferation, migration, invasion, and angiogenesis, and have been linked to progression of several cancer types. However, to our knowledge, they have not been studied in KS. We examined fibulin-2 because we found it to be significantly downregulated by microarray analysis in KSHV-infected DMVEC cells. Here we demonstrate that fibulin-2 protein expression is downregulated 50-fold in 10 day KSHV-infected dermal microvascular endothelial cells (DMVEC) with a 26-fold reduction in fibulin-2 message. By time-course transcriptional analysis there was consistent reduction of fibulin-2 message accompanied by an increase in KSHV latency associated nuclear antigen (LANA) transcription. Of the fibulins assayed, fibulins -2, -5, and -3 were downregulated over time in KSHV infected DMVEC, while fibulins 1C and 1D were upregulated, with no change in fibulins 4, 6, and 7. In pleural effusion lymphoma cell lines that express different levels of KSHV lytic replication, we observed no detectable fibulin-2 expression. Tissue microarrays representing patch/plaque and nodular forms of KS from 86 different patients were shown to be statistically significant for downregulation of fibulin-2 in virus-infected LANA positive cells by dual labeled immunohistochemical staining. This represents the first study that examines fibulin-2 expression in KSHV-infected DMVEC and KS to determine whether suppression of this ECM protein plays a role in KS tumor progression. Understanding the interactions between KSHV and the fibulin family of extra-cellular matrix proteins that modulate angiogenesis cancer cell proliferation, migration and invasion could lead to development of novel therapies for treatment of KS.

Suppression of galectin-3 in KSHV infected DMVEC cells and Kaposi's sarcoma: implications for tumorigenesis

The Galectins are a family of proteins that share an affinity for Beta-galactoside containing glycoconjugates. Galectin-3 has been implicated in tumor progression and metastasis. It is also known that in prostate, ovarian and breast cancer, down regulation of Galectin-3 is associated with malignancy. Kaposis sarcoma (KS) is characterized as an angioproliferative tumor of vascular endothelial cells and produces rare B cell lymphoproliferative diseases in the form of primary effusion lymphomas (PEL) and some forms of Multicentric Castlemans Disease (MDC). Kaposis Sarcoma-Associated Herpesvirus also known as KSHV or Human Herpesvirus type 8 (HHV8) is the etiological agent of KS. We have observed suppression of galectin-3 expression in Kaposis sarcoma (KS). Here we demonstrate that galectin-3 protein expression is down-regulated 20-fold in KSHV infected dermal microvascular endothelial cells (DMVEC) cells. We show loss of Galectin-3 staining by dual labeled immunofluorescence in latency associated nuclear antigen (LANA) positive spindle cells. We also find reduced levels of galectin-3 expression in LANA positive spindle cell regions in archival KS tissue. There is also transcriptional suppression of Galectin-3 message in KSHV infected DMVEC cells compared to mock infected controls. We demonstrate that KSHV vFLIP is the likely viral gene that targets galectin-3 downregulation in HeLa cells. In pleural effusion lymphoma cell lines (PEL), we observe different levels of galectin-3 expression associated with varying levels of KSHV replication. The search for novel host cell factors that may contribute to the overall pathogenesis of KS is essential for early detection of KS and development of innovative therapies for treatment.