Distinct domains in ORF52 tegument protein mediate essential functions in murine gammaherpesvirus 68 virion tegumentation and secondary envelopment.

Epstein-Barr virus and Kaposi's sarcoma-associated herpesvirus are etiologically associated with several types of human malignancies. However, as these two human gammaherpesviruses do not replicate efficiently in cultured cells, the morphogenesis of gammaherpesvirus virions is poorly understood. Murine gammaherpesvirus 68 (MHV-68) provides a tractable model to define common, conserved features of gammaherpesvirus biology. ORF52 of MHV-68 is conserved among gammaherpesviruses. We have previously shown that this tegument protein is essential for the envelopment and egress of viral particles and solved the crystal structure of ORF52 dimers. To more closely examine its role in virion maturation, we performed immunoelectron microscopy of MHV-68-infected cells and found that ORF52 localized to both mature, extracellular virions and immature viral particles in the cytoplasm. ORF52 consists of three α-helices followed by one ß-strand. To understand the structural requirements for ORF52 function, we constructed mutants of ORF52 and examined their ability to complement an ORF52-null MHV-68 virus. Mutations in conserved residues in the N-terminal α1-helix and C terminus, or deletion of the α2-helix, resulted in a loss-of-function phenotype. Furthermore, the α1-helix was crucial for the predominantly punctate cytoplasmic localization of ORF52, while the α2-helix was a key domain for ORF52 dimerization. Immunoprecipitation experiments demonstrated that ORF52 interacts with another MHV-68 tegument protein, ORF42; however, a single point mutation in R95 in the C terminus of ORF52 led to the loss of this interaction. Moreover, the homologues of MHV-68 ORF52 in Kaposi's sarcoma-associated herpesvirus and Epstein-Barr virus complement the defect in ORF52-null MHV-68 and interact with MHV-68 ORF52. Taken together, these data uncover the relationship between the α-helical structure and the molecular basis for ORF52 function. This is the first structure-based functional domain mapping study for an essential gammaherpesvirus tegument protein.

The virion-associated open reading frame 49 of murine gammaherpesvirus 68 promotes viral replication both in vitro and in vivo as a derepressor of RTA.

Replication and transcription activator (RTA), an immediate-early gene, is a key molecular switch to evoke lytic replication of gammaherpesviruses. Open reading frame 49 (ORF49) is conserved among gammaherpesviruses and shown to cooperate with RTA in regulating virus lytic replication. Here we show a molecular mechanism and in vivo functions of murine gammaherpesvirus 68 (MHV-68 or γHV-68) ORF49. MHV-68 ORF49 was transcribed and translated as a late gene. The ORF49 protein was associated with a virion, interacting with the ORF64 large tegument protein and the ORF25 capsid protein. Moreover, ORF49 directly bound to RTA and its negative cellular regulator, poly(ADP-ribose) polymerase-1 (PARP-1), and disrupted the interactions of RTA and PARP-1. Productive replication of an ORF49-deficient mutant virus (49S) was attenuated in vivo as well as in vitro. Likewise, latent infection was also impaired in the spleen of 49S-infected mice. Taken together, our results suggest that the virion-associated ORF49 protein may promote virus replication both in vitro and in vivo by providing an optimal environment in the early phase of virus infection as a derepressor of RTA.

An integrated approach to elucidate the intra-viral and viral-cellular protein interaction networks of a gamma-herpesvirus.

Genome-wide yeast two-hybrid (Y2H) screens were conducted to elucidate the molecular functions of open reading frames (ORFs) encoded by murine γ-herpesvirus 68 (MHV-68). A library of 84 MHV-68 genes and gene fragments was generated in a Gateway entry plasmid and transferred to Y2H vectors. All possible pair-wise interactions between viral proteins were tested in the Y2H assay, resulting in the identification of 23 intra-viral protein-protein interactions (PPIs). Seventy percent of the interactions between viral proteins were confirmed by co-immunoprecipitation experiments. To systematically investigate virus-cellular protein interactions, the MHV-68 Y2H constructs were screened against a cellular cDNA library, yielding 243 viral-cellular PPIs involving 197 distinct cellar proteins. Network analyses indicated that cellular proteins targeted by MHV-68 had more partners in the cellular PPI network and were located closer to each other than expected by chance. Taking advantage of this observation, we scored the cellular proteins based on their network distances from other MHV-68-interacting proteins and segregated them into high (Y2H-HP) and low priority/not-scored (Y2H-LP/NS) groups. Significantly more genes from Y2H-HP altered MHV-68 replication when their expression was inhibited with siRNAs (53% of genes from Y2H-HP, 21% of genes from Y2H-LP/NS, and 16% of genes randomly chosen from the human PPI network; p<0.05). Enriched Gene Ontology (GO) terms in the Y2H-HP group included regulation of apoptosis, protein kinase cascade, post-translational protein modification, transcription from RNA polymerase II promoter, and IκB kinase/NFκB cascade. Functional validation assays indicated that PCBP1, which interacted with MHV-68 ORF34, may be involved in regulating late virus gene expression in a manner consistent with the effects of its viral interacting partner. Our study integrated Y2H screening with multiple functional validation approaches to create γ-herpes viral-viral and viral-cellular protein interaction networks.

Current Trends in IBD-Development of Mucosal-Based Biomarkers and a Novel Minimally Invasive Recoverable Sampling System.

Despite recent developments in therapy for inflammatory bowel diseases (IBDs), there have been limited advances in diagnostic tools available to aid in disease management. A growing body of evidence suggests that there are important host-microbe interactions at the mucosal interface that modulate the inflammatory response in patients with IBD. Additionally, the importance of mucosal integrity and its disruption appears to be important in the pathophysiology and perpetuation of the disease. The ability to characterize this interface may provide valuable information for both disease monitoring and identification of new treatment targets. Endoscopy remains the primary tool for disease monitoring, and mucosal healing is the primary therapeutic target in IBD treatment. However, establishing mucosal healing requires repetitive endoscopic procedures, and endoscopy is limited by factors such as invasiveness, cost, and risk of adverse events. Moreover, the use of a bowel preparation for colonoscopies alters the mucus layer and thus perturbs evaluation of the host-microbe interaction. Stool sampling may also be inaccurate because it reflects the end state of metabolites and proteins, failing to take into account the degradation or alteration of substrates of interest by bacterial proteases and other enzymes during passage through the colon. A novel sampling capsule, called the Recoverable Sampling System (RSS), is being developed as a complementary tool to colonoscopy. The RSS is intended to be a platform for noninvasive autonomous sampling, preservation, handling, and storage of analytes of interest found in the gastrointestinal fluids. A proprietary preservative contained within the chambers of the capsule has been developed to stabilize DNA and proteins for ex vivo microbiome and metabolomics analyses. Surrogate markers such as SPP24 and GUCA2a have been identified to correlate with gut health, intestinal permeability, and inflammation and could be locally sampled by the RSS. The potential clinical utility of an RSS device is broad and would likely be able to guide therapy by allowing for more frequent disease monitoring, aiding in disease characterization, and facilitating in the identification of novel therapeutic targets.

A new technology is being developed, Recoverable Sampling System (RSS), that may allow sampling without a colonoscopy. This may lead to new biomarkers and even drug targets which may ultimately improve the care of these patients.

Regulation of Kaposi's sarcoma-associated herpesvirus reactivation by dopamine receptor-mediated signaling pathways.

BACKGROUND: Kaposi's sarcoma-associated herpesvirus (KSHV) possesses two distinct life cycles, lytic replication and latency. An immediate early viral protein, Replication and transcription activator (RTA), is responsible for the virus switch from latency to active replication. METHODS: To identify cellular pathways that reactivate KSHV replication, an RTA-responsive viral early promoter, PAN, coupled with an enhanced green fluorescent protein (EGFP) reporter was delivered into a KSHV latently infected B cell line. Five different chemical libraries with defined cellular targets were screened for their ability to induce the PAN promoter as an indication of lytic replication. RESULTS: We identified seven chemicals that disrupted latency in KSHV latently infected B cells, five being N-acyl-dopamine derivatives. We showed that these chemicals reactivate KSHV through interacting with dopamine receptors, and that KSHV utilizes dopamine receptors and the associated PKA and MAP kinase pathways to detect and transmit stress signals for reactivation. CONCLUSION: Our study identified two cellular signaling pathways that mediate KSHV reactivation and provided a chemical genetics approach to identify new endogenous activators with therapeutic potential against herpesvirus associated malignancies.

Structural and functional studies of the abundant tegument protein ORF52 from murine gammaherpesvirus 68.

The tegument is a layer of proteins between the nucleocapsid and the envelope of herpesviruses. The functions of most tegument proteins are still poorly understood. In murine gammaherpesvirus 68, ORF52 is an abundant tegument protein of 135 residues that is required for the assembly and release of infectious virus particles. To help understand the molecular basis for the function of this protein, we have determined its crystal structure at 2.1 A resolution. The structure reveals a dimeric association of this protein. Interestingly, an N-terminal alpha-helix that assumes different conformation in the two monomers of the dimer mediates the formation of an asymmetrical tetramer and contains many highly conserved residues. Structural and sequence analyses suggest that this helix is more likely involved in interactions with other components of the tegument or nucleocapsid of the virus and that ORF52 functions as a symmetrical dimer. The asymmetrical tetramer of ORF52 may be a "latent" form of the protein, when it is not involved in virion assembly. The self-association of ORF52 has been confirmed by co-immunoprecipitation and fluorescence resonance energy transfer experiments. Deletion of the N-terminal alpha-helix, as well as mutation of the conserved Arg(95) residue, abolished the function of ORF52. The results of the functional studies are fully consistent with the structural observations and indicate that the N-terminal alpha-helix is a crucial site of interaction for ORF52.