Norbert K. Herzog

Identification of Differentially Activated Cell-Signaling Networks Associated with Pichinde Virus Pathogenesis by Using Systems Kinomics

ABSTRACT Phosphorylation plays a key role in regulating many signaling pathways. Although studies investigating the phosphorylated forms of signaling pathways are now commonplace, global analysis of protein phosphorylation and kinase activity has lagged behind genomics and proteomics. We have used a kinomics approach to study the effect of virus infection on host cell signaling in infected guinea pigs. Delineating the host responses which lead to clearance of a pathogen requires the use of a matched, comparative model system. We have used two passage variants of the arenavirus Pichinde, used as a biosafety level 2 model of Lassa fever virus as it produces similar pathologies in guinea pigs and humans, to compare the host cell responses between infections which lead to either a mild, self-limiting infection or lethal disease. Using this model, we can begin to understand the differences in signaling events which give rise to these markedly different outcomes. By contextualizing these data using pathway analysis, we have identified key differences in cellular signaling matrices. By comparing these differentially involved networks, we have identified a number of key signaling “nodes” which show differential phosphorylations between mild and lethal infections. We believe that these nodes provide potential targets for the development of antiviral therapies by acting at the level of the host response rather than by directly targeting viral proteins.

Alterations in NF-κB and RBP-Jκ by Arenavirus Infection of Macrophages In Vitro and In Vivo

ABSTRACT Pichinde virus is an arenavirus that infects guinea pigs and serves as an animal model for human Lassa fever. An attenuated Pichinde virus variant (P2) and a virulent variant (P18) are being used to delineate pathogenic mechanisms that culminate in shock. In guinea pigs, the infection has been shown to begin in peritoneal macrophages following intraperitoneal inoculation and then spreads to the spleen and other reticuloendothelial organs. We show here that infection of the murine monocytic cell line P388D1 with either Pichinde virus variant resulted in the induction of inflammatory cytokines and effectors, including interleukin-6 and tumor necrosis factor alpha. Since these genes are regulated in part by the cellular transcription factors NF-κB and RBP-Jκ, we compared the activities of NF-κB and RBP-Jκ in P388D1 cells following infection with Pichinde virus. The attenuated P2 virus inhibited NF-κB activation and caused a shift in the size of the RBP-Jκ complex. The virulent P18 virus showed less inhibition of NF-κB and failed to alter the size of the RBP-Jκ complex. Peritoneal cells from P2-infected guinea pigs showed induction of NF-κB RelA/p50 heterodimer and p50/p50 homodimer and manifested an increase in the size of RBP-Jκ. By contrast, P18 induced large amounts of the NF-κB p50/p50 dimer but failed to induce RelA/p50 or to cause an increase in the RBP-Jκ size. Taken together, these changes suggest that the attenuated viral strain induces an “activation” of macrophages, while the virulent form of the virus does not.

Induction of granulysin in CD8+ T cells by IL-21 and IL-15 is suppressed by human immunodeficiency virus-1

Immunosuppression following infection with HIV‐1 predisposes patients to a myriad of opportunistic pathogens, one of the most important of which is Mtb. Granulysin, expressed by NK cells and CTL, exhibits potent antimicrobial activity against Mtb and several other opportunistic pathogens associated with HIV‐1 infection. The immune signals that promote granulysin expression in human CTL are not fully understood. Using primary human CD8+ T cells, in this study, we identify IL‐21 as a strong inducer of granulysin, demonstrate that IL‐21 and IL‐15 activate granulysin expression within CD8+ CD45RO+ T cells, and establish a role for Jak/STAT signaling in the regulation of granulysin within CD8+ T cells. We show that infection of PBMC from healthy donors in vitro with HIV‐1 suppresses granulysin expression by CD8+ T cells, concomitant with reduced p‐STAT3 and p‐STAT5, following activation with IL‐15 and IL‐21. Of note, simultaneous signaling through IL‐15 and IL‐21 could partially overcome the immunosuppressive effects of HIV‐1 on granulysin expression by CD8+ T cells. These results suggest that HIV‐1 infection of PBMC may reduce the antimicrobial profile of activated CD8+ T cells by disrupting signaling events that are critical for the induction of granulysin. Understanding the effects of HIV‐1 on CD8+ T cell activation is essential to understanding the physiological basis for inadequate cytotoxic lymphocyte activity in HIV+ patients and for informed guidance of cytokine‐based therapy to restore T cell function.

Differential Signaling Networks Induced by Mild and Lethal Hemorrhagic Fever Virus Infections

ABSTRACT The family Arenaviridae includes several National Institutes of Allergy and Infections Diseases category A select agents which cause hemorrhagic fever. There are few vaccines available, and treatment is limited to ribavirin, which varies in efficacy. Development of new antiviral compounds has been hindered by a lack of understanding of the molecular basis of pathogenesis. We used two variants of Pichinde virus, one attenuated and one virulent in the guinea pig model, to delineate the host determinants which lead to either viral clearance or lethal disease. By analyzing protein level changes using pathway analysis, we have identified key intermediates which may be targets for therapeutic intervention.

Selection of Thioaptamers for Diagnostics and Therapeutics

Abstract:  Thioaptamers offer advantages over normal phosphate ester backbone aptamers due to their enhanced affinity, specificity, and higher stability, largely due to the properties of the sulfur backbone modifications. Over the past several years, in vitro thioaptamer selection and bead‐based thioaptamer selection techniques have been developed in our laboratory. Furthermore, several thioaptamers targeting specific proteins such as transcription factor NF‐κB and AP‐1 proteins have been identified. Selected thioaptamers have been shown diagnostic promise in proteome screens. Moreover, some promising thioaptamers have been shown in preliminary animal therapeutic dosing to increase survival in animal models of infection with West Nile virus.

Solution structure and design of dithiophosphate backbone aptamers targeting transcription factor NF-κB☆

Abstract A variety of monothio- and dithiosubstituted duplex aptamers targeting NF-κB have been synthesized and designed. The specificity and affinity of the dithioate aptamers of p50 and RelA(p65) NF-κB homodimers was determined by gel shift experiments. The NMR solution structures for several unmodified and dithioate backbone modified 14-base paired duplex aptamers have been determined by a hybrid, complete matrix (MORASS)/restrained molecular dynamics method. Structural perturbations of the dithioate substitutions support our hypothesis that the dithioate binds cations less tightly than phosphoryl groups. This increases the electrostatic repulsion across the B-form narrow minor groove and enlarges the minor groove, similar to that found in A-form duplexes. Structural analysis of modeled aptamer complexes with NF-κB homo- and heterodimers suggests that the dithioate backbone substitution can increase the aptamer’s relative affinity to basic groups in proteins such as NF-κB by helping to “strip” the cations from the aptamer backbone.

Attenuated and lethal variants of pichindé virus induce differential patterns of NF-κB activation suggesting a potential target for novel therapeutics

Lassa virus pathogenesis is believed to involve dysregulation of cytokines. We have previously shown nuclear factor-kappaB (NF-kappaB) inhibition using a BSL-2 model for Lassa fever. Here we further define the potential mechanism for NF-kappaB inhibition as involving increased levels of repressive p50/p50 homodimers, and suggest a novel therapeutic strategy that acts via modulation of host signaling.

Analysis of the Differential Host Cell Nuclear Proteome Induced by Attenuated and Virulent Hemorrhagic Arenavirus Infection

ABSTRACT Arenaviruses are important emerging pathogens and include a number of hemorrhagic fever viruses classified as NIAID category A priority pathogens and CDC potential biothreat agents. Infection of guinea pigs with the New World arenavirus Pichindé virus (PICV) has been used as a biosafety level 2 model for the Lassa virus. Despite continuing research, little is known about the molecular basis of pathogenesis, and this has hindered the design of novel antiviral therapeutics. Modulation of the host response is a potential strategy for the treatment of infectious diseases. We have previously investigated the global host response to attenuated and lethal arenavirus infections by using high-throughput immunoblotting and kinomics approaches. In this report, we describe the differential nuclear proteomes of a murine cell line induced by mock infection and infection with attenuated and lethal variants of PICV, investigated by using two-dimensional gel electrophoresis. Spot identification using tandem mass spectrometry revealed the involvement of a number of proteins that regulate inflammation via potential modulation of NF-κB activity and of several heterogeneous nuclear ribonuclear proteins. Pathway analysis revealed a potential role for transcription factor XBP-1, a transcription factor involved in major histocompatibility complex II (MHC-II) expression; differential DNA-binding activity was revealed by electrophoretic mobility shift assay, and differences in surface MHC-II expression were seen following PICV infection. These data are consistent with the results of several previous studies and highlight potential differences between transcriptional and translational regulation. This study provides a number of differentially expressed targets for further research and suggests that key events in pathogenesis may be established early in infection.

Proteomic analysis of Pichindé virus infection identifies differential expression of prothymosin-alpha.

The arenaviruses include a number of important pathogens including Lassa virus and Junin virus. Presently, the only treatment is supportive care and the antiviral Ribavirin. In the event of an epidemic, patient triage may be required to more effectively manage resources; the development of prognostic biomarker signatures, correlating with disease severity, would allow rational triage. Using a pair of arenaviruses, which cause mild or severe disease, we analyzed extracts from infected cells using SELDI mass spectrometry to characterize potential biomarker profiles. EDGE analysis was used to analyze longitudinal expression differences. Extracts from infected guinea pigs revealed protein peaks which could discriminate between mild or severe infection, and between times post-infection. Tandem mass-spectrometry identified several peaks, including the transcriptional regulator prothymosin-α. Further investigation revealed differences in secretion of this peptide. These data show proof of concept that proteomic profiling of host markers could be used as prognostic markers of infectious disease.

Gender-related differences in expression of murine glutathione S-transferases and their induction by butylated hydroxyanisole

The basal levels of mu and pi class glutathione S-transferases RNA were 18-fold higher in the male mouse liver as compared with the female. When 0.75% (w/w) BHA was included in the diet it altered the RNA levels of alpha, mu, pi GST classes and mGSTA4-4 in a tissue and sex specific manner. The most marked induction of RNA was seen for the mu class GSTs of female liver, lung and kidney (52, 10 and 8-fold, respectively), and of male liver and kidney (25 and 3.5-fold, respectively), the pi class GSTs of female liver, lung, and kidney (11, 10, and 5-fold, respectively), and mGSTA4-4 of female liver (4-fold). The effect of BHA on the induction of the mu and pi class GST RNA was 2-9 fold greater in female as compared with male tissues. The degree of induction of GST RNA did not correlate directly with changes in GST protein indicating that post-transcriptional events regulating GST expression may be affected by BHA particularly for GST mu and mGSTA4-4.