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

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

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

Regulation of KSHV replication by glycoprotein B: chronicles of a virus and its host

Kaposi's sarcoma-associated herpesvirus (KSHV), the causative agent of three cancers: Kaposi's sarcoma (KS), primary effusion lymphoma (PEL), and the plasmablastic form of multicentric Castleman disease (MCD), shares many characteristics with other herpesviruses including the unique ability to maneuver between latent and lytic modes of infection. During a latent stage of infection, viruses remain dormant with very few genes being expressed. Alternatively, lytic infection is distinguished by the production of infectious virions and virus dissemination. In KSHV pathogenesis, the majority of infected cells support latent infections while less than 3% of them support spontaneous lytic replication. Regulation of latent and lytic infections involves interactions between virus-encoded and cellular factors. However, the exact mechanism(s) supporting it is poorly understood. In the following study, we provide evidence supporting the role for KSHV-encoded glycoprotein B (gB), a structural protein, to inhibit KSHV reactivation from latency via altering the expression of the cellular transcription factor, Early growth response-1 (Egr-1). We observed Egr-1 to specifically bind to two separate locations on the KSHV-encoded ORF50 promoter which plays a significant role in KSHV reactivation. Furthermore, we provide evidence supporting the potential mechanism for KSHV gB to regulate KSHV reactivation. These studies provide new insights and appreciation for the role of virus-encoded structural proteins to support virus latency and may contribute to the development of future therapies.  Ph.D

Regulation of KSHV replication by glycoprotein B: chronicles of a virus and its host

Kaposi's sarcoma-associated herpesvirus (KSHV) the causative agent of three cancers: Kaposi's sarcoma (KS) primary effusion lymphoma (PEL) and the plasmablastic form of multicentric Castleman disease (MCD) shares many characteristics with other herpesviruses including the unique ability to maneuver between latent and lytic modes of infection. During a latent stage of infection viruses remain dormant with very few genes being expressed. Alternatively lytic infection is distinguished by the production of infectious virions and virus dissemination. In KSHV pathogenesis the majority of infected cells support latent infections while less than 3% of them support spontaneous lytic replication. Regulation of latent and lytic infections involves interactions between virus-encoded and cellular factors. However the exact mechanism(s) supporting it is poorly understood. In the following study we provide evidence supporting the role for KSHV-encoded glycoprotein B (gB) a structural protein to inhibit KSHV reactivation from latency via altering the expression of the cellular transcription factor Early growth response-1 (Egr-1). We observed Egr-1 to specifically bind to two separate locations on the KSHV-encoded ORF50 promoter which plays a significant role in KSHV reactivation. Furthermore we provide evidence supporting the potential mechanism for KSHV gB to regulate KSHV reactivation. These studies provide new insights and appreciation for the role of virus-encoded structural proteins to support virus latency and may contribute to the development of future therapies.

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