miRNA-36 inhibits KSHV, EBV, HSV-2 infection of cells via stifling expression of interferon induced transmembrane protein 1 (IFITM1)

Kaposi’s sarcoma-associated herpesvirus (KSHV) is etiologically associated with all forms of Kaposi’s sarcoma worldwide. Little is currently known about the role of microRNAs (miRNAs) in KSHV entry. We recently demonstrated that KSHV induces a plethora of host cell miRNAs during the early stages of infection. In this study, we show the ability of host cell novel miR-36 to specifically inhibit KSHV-induced expression of interferon induced transmembrane protein 1 (IFITM1) to limit virus infection of cells. Transfecting cells with miR-36 mimic specifically lowered IFITM1 expression and thereby significantly dampening KSHV infection. In contrast, inhibition of miR-36 using miR-36 inhibitor had the direct opposite effect on KSHV infection of cells, allowing enhanced viral infection of cells. The effect of miR- 36 on KSHV infection of cells was at a post-binding stage of virus entry. The highlight of this work was in deciphering a common theme in the ability of miR-36 to regulate infection of closely related DNA viruses: KSHV, Epstein-Barr virus (EBV), and herpes simplexvirus-2 (HSV-2). Taken together, we report for the first time the ability of host cell miRNA to regulate internalization of KSHV, EBV, and HSV-2 in hematopoietic and endothelial cells

Raman spectroscopy: the gateway into tomorrow's virology

In the molecular world, researchers act as detectives working hard to unravel the mysteries surrounding cells. One of the researchers' greatest tools in this endeavor has been Raman spectroscopy. Raman spectroscopy is a spectroscopic technique that measures the unique Raman spectra for every type of biological molecule. As such, Raman spectroscopy has the potential to provide scientists with a library of spectra that can be used to unravel the makeup of an unknown molecule. However, this technique is limited in that it is not able to manipulate particular structures without disturbing their unique environment. Recently, a novel technology that combines Raman spectroscopy with optical tweezers, termed Raman tweezers, evades this problem due to its ability to manipulate a sample without physical contact. As such, Raman tweezers has the potential to become an incredibly effective diagnostic tool for differentially distinguishing tissue, and therefore holds great promise in the field of virology for distinguishing between various virally infected cells. This review provides an introduction for a virologist into the world of spectroscopy and explores many of the potential applications of Raman tweezers in virology. Originally published in Virology Journal, 2006 Vol. 3, No. 51

Raman tweezers provide the fingerprint of cells supporting the late stages of KSHV reactivation

Kaposi's sarcoma-associated herpesvirus (KSHV) has both latent and lytic phases of replication. The molecular switch that triggers a reactivation is still unclear. Cells from S phase of cell cycle provide apt conditions for an active reactivation. In order to specifically delineate the Raman spectra of cells supporting KSHV reactivation, we followed a novel approach where cells were sorted based on the state of infection (latent Vs lytic) by a flow cytometer and then analyzed by the Raman tweezers. The Raman bands at 785, 813, 830, 1095, and 1128 cm-1 are specifically altered in cells supporting KSHV reactivation. These 5 peaks make up the Raman fingerprint of cells supporting KSHV reactivation. The physiological relevance of the changes in these peaks with respect to KSHV reactivation is discussed in the following report. Originally published Journal of Cellular and Molecular Medicine, Vol. 13, No. 8b, Aug 200

Resveratrol Inhibits KSHV Reactivation by Lowering the Levels of Cellular EGR-1

In the field of herpesvirus research, the exact molecular mechanism by which such viruses reactivate from latency remains elusive. Kaposi's sarcoma-associated herpesvirus (KSHV) primarily exists in a latent state, while only 1–3% of cells support lytic infection at any specific time. KSHV reactivation from latency is an exceedingly intricate process mediated by the integration of viral and cellular factors. Previously, our lab has described early growth response-1 (Egr-1) as an essential component for the KSHV reactivation process via its ability to mediate transcription of KSHV ORF50, the gene encoding for replication and transcription activator (RTA), a viral component known to control the switch from latent to lytic infection. In here, electrophoretic mobility shift assays (EMSA) and chromatin immunoprecipitation (ChIP) experiments revealed that Egr-1 binds KSHV ORF50 promoter (ORF50P) in at least two different GC-rich binding domains. Expression profiles of cellular egr-1 and KSHV-encoded ORF50 follow a similar pattern during de novo KSHV infection. Over-expressing Egr-1, a signaling component downstream of Raf>MEK>ERK1/2, in KSHV-infected cells activates KSHV lytic replication. Through performing more physiologically relevant experiments, we analyzed the effect of a dietary supplement containing resveratrol on KSHV-infected cells. Our results, for the first time, demonstrate resveratrol to act in lowering ERK1/2 activity and expression of Egr-1 in KSHV-infected cells, resulting in the suppression of virus reactivation from latency. Taken together, these findings will undoubtedly contribute to future studies on not only combating KSHV related disease conditions, but also on other herpesviruses-induced pathogenesis

KSHV gB associated RGD interactions promote attachment of cells by inhibiting the potential migratory signals induced by the disintegrin-like domain

Background: Kaposi's sarcoma-associated herpesvirus (KSHV) glycoprotein B (gB) is not only expressed on the envelope of mature virions but also on the surfaces of cells undergoing lytic replication. Among herpesviruses, KSHV gB is the only glycoprotein known to possess the RGD (Arg-Gly-Asp) binding integrin domain critical to mediating cell attachment. Recent studies described gB to also possess a disintegrin-like domain (DLD) said to interact with non-RGD binding integrins. We wanted to decipher the roles of two individually distinct integrin binding domains (RGD versus DLD) within KSHV gB in regulating attachment of cells over cell migration

Abilities of berberine and chemically modified berberines to interact with metformin and inhibit proliferation of pancreatic cancer cells

Pancreatic cancer is devastating cancer worldwide with few if any truly effective therapies. Pancreatic cancer has an increasing incidence and may become the second leading cause of death from cancer. Novel, more effective therapeutic approaches are needed as pancreatic cancer patients usually survive for less than a year after being diagnosed. Control of blood sugar levels by the prescription drug metformin in diseases such as diabetes mellitus has been examined in association with pancreatic cancer. While the clinical trials remain inconclusive, there is hope that certain diets and medications may affect positively the outcomes of patients with pancreatic and other cancers. Other natural compounds may share some of the effects of metformin. One "medicinal" fruit consumed by millions worldwide is berberine (BBR). Metformin and BBR both activate AMP-activated protein kinase (AMPK) which is a key mediator of glucose metabolism. Glucose metabolism has been shown to be very important in cancer and its significance is increasing. In the following studies, we have examined the effects of metformin, BBR and a panel of modified BBRs (NAX compounds) and chemotherapeutic drugs on the growth of four different human pancreatic adenocarcinoma cell lines (PDAC). Interestingly, the effects of metformin could be enhanced by BBR and certain modified BBRs. Upon restoration of WT-TP53 activity in MIA-PaCa-2 cells, an altered sensitivity to the combination of certain NAX compounds and metformin was observed compared to the parental cells which normally lack WT-TP53. Certain NAX compounds may interact with WT-TP53 and metformin treatment to alter the expression of key molecules involved in cell growth. These results suggest a therapeutic approach by combining certain pharmaceutical drugs and nutraceuticals to suppress the growth of cancer cells

Effects of the MDM-2 inhibitor Nutlin-3a on PDAC cells containing and lacking WT-TP53 on sensitivity to chemotherapy, signal transduction inhibitors and nutraceuticals

Mutations at the TP53 gene are readily detected (approximately 50-75%) in pancreatic ductal adenocarcinoma (PDAC) patients. TP53 was previously thought to be a difficult target as it is often mutated, deleted or inactivated on both chromosomes in certain cancers. In the following study, the effects of restoration of wild-type (WT) TP53 activity on the sensitivities of MIA-PaCa-2 pancreatic cancer cells to the MDM2 inhibitor nutlin-3a in combination with chemotherapy, targeted therapy, as well as, nutraceuticals were examined. Upon introduction of the WT-TP53 gene into MIA-PaCa-2 cells, which contain a TP53 gain of function (GOF) mutation, the sensitivity to the MDM2 inhibitor increased. However, effects of nutlin-3a were also observed in MIA-PaCa-2 cells lacking WT-TP53, as upon co-treatment with nutlin-3a, the sensitivity to certain inhibitors, chemotherapeutic drugs and nutraceuticals increased. Interestingly, co-treatment with nutlin-3a and certain chemotherapeutic drug such as irinotecan and oxaliplatin resulted in antagonistic effects in cells both lacking and containing WT-TP53 activity. These studies indicate the sensitizing abilities that WT-TP53 activity can have in PDAC cells which normally lack WT-TP53, as well as, the effects that the MDM2 inhibitor nutlin-3a can have in both cells containing and lacking WT-TP53 to various therapeutic agents

Wild Type and Gain of Function Mutant TP53 can Regulate the Sensitivity of Pancreatic Cancer Cells to Chemotherapeutic Drugs, EGFR/Ras/Raf/MEK, and PI3K/mTORC1/GSK-3 Pathway Inhibitors, Nutraceuticals and Alter Metabolic Properties

TP53 is a master regulator of many signaling and apoptotic pathways involved in: aging, cell cycle progression, metastasis, and metabolism. Most pancreatic cancers are classified as pancreatic ductal adenocarcinomas (PDAC). The tumor suppressor gene TP53 is mutated frequently (50-75%) in PDAC. Different types of TP53 mutations have been observed including gain of function (GOF) point mutations and various deletions of the TP53 gene resulting in lack of the protein expression. Most PDACs have point mutations at the KRAS gene which result in constitutive activation of KRas and multiple downstream signaling pathways. It has been difficult to develop specific KRas inhibitors and/or methods that result in recovery of functional TP53 activity. To further elucidate the roles of TP53 in drug-resistance of pancreatic cancer cells, we introduced wild-type (WT) TP53 or a control vector into two different PDAC cell lines. Introduction of WT-TP53 increased the sensitivity of the cells to multiple chemotherapeutic drugs, signal transduction inhibitors, drugs and nutraceuticals and influenced key metabolic properties of the cells. Therefore, TP53 is a key molecule which is critical in drug sensitivity and metabolism of PDAC