David Davido, Ph.D., University of Kansas
HSV-1-mediated proteolysis of cellular targets
Herpes simplex virus (HSV) infections, typically characterized by physiologically-distinct lytic and latent phases, result in intermittent mouth (cold) and genital sores and are the primary cause of infectious blindness in western industrialized countries. Virus-host interactions dictate whether HSV initiates a lytic infection, establishes latent infection, or reactivates from latency by a variety of stress stimuli (e.g., heat shock). A key player in determining whether an infection will be lytic or latent is the immediate-early (IE), infected cell protein 0 (ICP0), which is an E3 ubiquitin (Ub) ligase activity that transactivates the expression of viral genes. E3 Ub ligases are components of pathways that typically attach and polymerize Ub (a 76 amino acid protein) to target proteins, marking them for proteolysis. Current data suggests that ICP0 directs the degradation of several cellular proteins that are, in turn, required for its transactivating activity. Efforts to identify specific targets of ICP0-mediated degradation by proteomic approaches have been challenging given the limitations in the purity, solubility, and detection of proteins. While several functions of ICP0 during viral infection have been characterized, the identities of cellular proteins marked for degradation by ICP0 are largely unknown. Until these new targets have been identified, it is unclear the exact role ICP0's E3 Ub ligase activity plays in its transactivating activity.
The long-term objective of our research is to determine how virus-host interactions, at the molecular level, control the HSV-1 life cycle. The objective of this proposal is to identify cellular proteins whose degradation is directed by ICP0. The central hypothesis of this pilot grant is that ICP0 mediates the degradation of specific cellular proteins. While unable to perform additional experiments in the time frame of this application (an 8-month time frame), we propose in future studies that degradation of these targets by ICP0 is essential for efficient HSV replication. Our approach is to identify functional targets of ICP0- mediated degradation in a novel screen. Our rationale for carrying out these studies is that by understanding how ICP0 facilitates productive infection via proteolysis, targets of ICP0 degradation could be used to develop new therapies to impair HSV replication and its associated diseases. Given the time frame of this application, we propose the following specific aim:
Specific Aim #1: Isolate novel targets of the HSV-1 E3 Ub ligase, ICP0. Our working hypothesis is that ICP0 directs the proteolysis of specific cellular proteins.
The contribution of this proposal is that we expect to identify cellular proteins involved in the biology of ICP0 using an innovative approach, which is a significant contribution that can applied to isolate and identify genes and pathways that play important roles in viral infections and proteolysis.
Revathi Govind, Ph.D., Kansas State University
Role of TcdR, the alternate sigma factor in Clostridium difficile virulence
Clostridium difficile is the leading cause of hospital-acquired diarrhea. Antibiotic use is the primary risk factor for the development of C. difficile-associated disease (CDAD) because it disrupts normal protective gut flora and enables C. difficile to colonize the colon. Toxigenic C. difficile strains produce two toxins, toxin A and toxin B that are considered to be the major virulence factors. The toxins encoding genes, tcdA and tcdB are part of a pathogenicity locus, which also carry the gene encodes for the toxin genes positive regulator tcdR. TcdR is an alternate sigma factor that binds with RNA polymerase core enzyme to make the holoenzyme that initiate transcription at tcdA and tcdB promoters. Alternate sigma factors are known to regulate virulence and virulence associated genes in many pathogenic bacteria. Including toxin genes, TcdR may regulate other virulence-associated genes in C. difficile. We have created and characterized, tcdR mutant in two different C. difficile strains. Mutation in tcdR affected both toxin production and sporulation in C. difficile. Microarray analysis revealed many differentially expressed sporulation-associated genes in tcdR mutant. In this project in our first aim, we propose to test the role of TcdR in C. difficile sporulation. In our second aim, we are proposing to monitor TcdR dependent promoter expression at cellular level, using a novel reporter system. During the current decade there has been a dramatic increase in the incidence and severity of C. difficile infections due to the emergence of hypertoxinogenic C. difficile strains. Our long- term goal is to unravel pathogenic mechanisms of C. difficile, thus new strategies to prevent, treat and manage C. difficile infection can be developed.
Jianming Qiu, Ph.D., University of Kansas Medical Center
Mechanisms of the cell cycle arrest induced during parvovirus B19 infection
Human parvovirus B19 (B19V) infection causes severe hematological disorders that in some cases can be fatal, including hydrops fetalis in pregnant women, transient aplastic crisis in patients with a high rate of red blood cell turnover, and chronic anemia in immunodeficient and immunocompromised patients. Currently, there are no specific antiviral drugs available to treat patients with B19V infection, and an effective vaccine to prevent B19V infection in high-risk individuals has yet to be developed. B19V replication is highly restricted to human erythroid progenitor cells (EPCs) in bone marrow and fetal liver. B19V-casued hematological disorders are largely due to direct killing of the EPCs, in which B19V replicates. B19V infection induces a DNA damage response (DDR) that is mainly mediated by activation of ATR. The B19V large non-structural protein NS1 is essential for B19V DNA replication and induces infected cells arrested at a phase with a 4N DNA content (4N phase). Replication of the B19V linear single-stranded (ss)DNA genome occurs in host cells arrested at the 4N phase, and is facilitated by the activation of ATR. Thus, B19V does not use the host double-stranded DNA replication machinery for replication of its ssDNA; rather, it appears to induce a DDR and subsequently to co-opt the host mechanism of DNA repair for its own replication. We hypothesize that during early infection, the ATR-mediated DDR induces intra-S phase arrest that facilitates viral DNA replication (repair) through inhibiting cellular DNA replication, and that in contrast, during late infection, the G2/M arrest induced by B19V NS1 promotes cell death.
We have established two experimental cell systems that will allow us to dissect the mechanism(s) of the cell cycle arrest during B19V infection: an efficient system of productive B19V infection involving the ex vivo-expansion of EPCs under conditions of hypoxia, which mimics the microenvironment of EPCs; a reverse genetics approach that involves transfection of a replicative form of B19V DNA into megakaryoblastoid UT7/Epo-S1 cells cultured under hypoxia. We will first explore a role of the ATR-Chk1 activation in inducing intra-S phase arrest during B19V infection and understand how the intra-S phase arrest facilitates B19V DNA replication. Second, we will characterize the B19V NS1-indcued cell cycle arrest at G2/M phase and understand the mechanism of how NS1 induces the G2/M arrest. Our studies will delineate the key molecular mechanisms of B19V replication and pathogenesis, which can be applied to develop anti-virus strategies for treating patients with B19V-casued hematological disorders.
Wolfram Zueckert, Ph.D., University of Kansas Medical Center
Mechanism of Borrelia Surface Lipoprotein Secretion
Borrelia spirochetes, the causative agents of arthropod-borne Lyme borreliosis and relapsing fever, are often described as gram-negative bacteria due to their Gram stain properties and diderm, i.e. double-membrane envelopes. Yet, a closer examination reveals significant differences in cell envelope composition and architecture. Of particular importance for transmission and human disease is the unique Borrelia-vector/host interface, which is dominated by surface lipoproteins. Despite the emergence of these peripherally membrane-associated proteins as major virulence factors, targets of the immune response and premier vaccine candidates, the processing and targeting pathways that guide them to their sites of biological activity have been only partially defined. The overall objective of our research is to gain an understanding of spirochetal envelope biogenesis, with a focus on determining the secretion and sorting mechanisms of spirochetal lipoproteins. Our seminal studies using Borrelia burgdorferi as a model spirochete have shown that (i) surface lipoprotein localization determinants commonly localize to N-terminal tether peptides, (ii) translocation through the outer membrane (OM) requires an at least partially unfolded lipopeptide, (iii) accordingly, dimeric lipoproteins assemble into their final quarternary fold after reaching the bacterial surface, and (iv) translocation through the OM can be initiated by an unfolded C terminus. Preliminary studies also suggested that at least one of the predicted inner membrane Lol pathway orthologs is not directly involved in surface lipoprotein localization. We therefore hypothesize that surface localization requires maintenance of a translocation-competent intermediate, likely by interaction with a periplasmic holding chaperone, which may work in concert with a so far unidentified OM lipoprotein translocon. To test these hypotheses, we have formulated the following independent but synergistic specific aims:
1. To identify and define the periplasmic and OM pathway components governing Borrelia surface lipoprotein secretion by reverse and forward genetics approaches, using conditional knockouts of candidate periplasmic chaperones and a powerful combination of established FACS-based lipoprotein localization and novel suppressor screens.
2. To define the Borrelia OM lipoprotein translocation mechanism using in vivo site-specific photocrosslinking and pulse-chase experiments.
These studies will (i) achieve further milestones in our investigation of Borrelia lipoprotein secretion, (ii) shed more light on the evolution of bacterial protein export mechanisms, (iii) significantly increase our understanding of spirochetal virulence, and may (iv) translate into the design of future intervention strategies.
This grant was made possible by NIH Grant Number P20 RR016443 from the COBRE program of the National Center for Research Resources and NIH Grant Number P30 GM103326 from the COBRE Program of the National Institute of General Medical Sciences.