Ca2+ Regulates the Drosophila Stoned-A and Stoned-B Proteins Interaction with the C2B Domain of Synaptotagmin-1

The dicistronic Drosophila stoned gene is involved in exocytosis and/or endocytosis of synaptic vesicles. Mutations in either stonedA or stonedB cause a severe disruption of neurotransmission in fruit flies. Previous studies have shown that the coiled-coil domain of the Stoned-A and the µ-homology domain of the Stoned-B protein can interact with the C2B domain of Synaptotagmin-1. However, very little is known about the mechanism of interaction between the Stoned proteins and the C2B domain of Synaptotagmin-1. Here we report that these interactions are increased in the presence of Ca2+. The Ca2+-dependent interaction between the µ-homology domain of Stoned-B and C2B domain of Synaptotagmin-1 is affected by phospholipids. The C-terminal region of the C2B domain, including the tryptophan-containing motif, and the Ca2+ binding loop region that modulate the Ca2+-dependent oligomerization, regulates the binding of the Stoned-A and Stoned-B proteins to the C2B domain. Stoned-B, but not Stoned-A, interacts with the Ca2+-binding loop region of C2B domain. The results indicate that Ca2+-induced self-association of the C2B domain regulates the binding of both Stoned-A and Stoned-B proteins to Synaptotagmin-1. The Stoned proteins may regulate sustainable neurotransmission in vivo by binding to Ca2+-bound Synaptotagmin-1 associated synaptic vesicles

Heparan sulfate as a regulator of inflammation and immunity

Heparan sulfate is found on the surface of most cell types, as well as in basement membranes and extracellular matrices. Its strong anionic properties and highly variable structure enable this glycosaminoglycan to provide binding sites for numerous protein ligands, including many soluble mediators of the immune system, and may promote or inhibit their activity. The formation of ligand binding sites on heparan sulfate (HS) occurs in a tissue- and context-specific fashion through the action of several families of enzymes, most of which have multiple isoforms with subtly different specificities. Changes in the expression levels of these biosynthetic enzymes occur in response to inflammatory stimuli, resulting in structurally different HS and acquisition or loss of binding sites for immune mediators. In this review, we discuss the multiple roles for HS in regulating immune responses, and the evidence for inflammation-associated changes to HS structure

An In Vitro System for Studying Murid Herpesvirus-4 Latency and Reactivation

The narrow species tropisms of Epstein-Barr Virus (EBV) and the Kaposi's Sarcoma -associated Herpesvirus (KSHV) have made Murid Herpesvirus-4 (MuHV-4) an important tool for understanding how gammaherpesviruses colonize their hosts. However, while MuHV-4 pathogenesis studies can assign a quantitative importance to individual genes, the complexity of in vivo infection can make the underlying mechanisms hard to discern. Furthermore, the lack of good in vitro MuHV-4 latency/reactivation systems with which to dissect mechanisms at the cellular level has made some parallels with EBV and KSHV hard to draw. Here we achieved control of the MuHV-4 lytic/latent switch in vitro by modifying the 5′ untranslated region of its major lytic transactivator gene, ORF50. We terminated normal ORF50 transcripts by inserting a polyadenylation signal and transcribed ORF50 instead from a down-stream, doxycycline-inducible promoter. In this way we could establish fibroblast clones that maintained latent MuHV-4 episomes without detectable lytic replication. Productive virus reactivation was then induced with doxycycline. We used this system to show that the MuHV-4 K3 gene plays a significant role in protecting reactivating cells against CD8+ T cell recognition

Murine Gamma-herpesvirus Immortalization of Fetal Liver-Derived B Cells Requires both the Viral Cyclin D Homolog and Latency-Associated Nuclear Antigen

Human gammaherpesviruses are associated with the development of lymphoproliferative diseases and B cell lymphomas, particularly in immunosuppressed hosts. Understanding the molecular mechanisms by which human gammaherpesviruses cause disease is hampered by the lack of convenient small animal models to study them. However, infection of laboratory strains of mice with the rodent virus murine gammaherpesvirus 68 (MHV68) has been useful in gaining insights into how gammaherpesviruses contribute to the genesis and progression of lymphoproliferative lesions. In this report we make the novel observation that MHV68 infection of murine day 15 fetal liver cells results in their immortalization and differentiation into B plasmablasts that can be propagated indefinitely in vitro, and can establish metastasizing lymphomas in mice lacking normal immune competence. The phenotype of the MHV68 immortalized B cell lines is similar to that observed in lymphomas caused by KSHV and resembles the favored phenotype observed during MHV68 infection in vivo. All established cell lines maintained the MHV68 genome, with limited viral gene expression and little or no detectable virus production - although virus reactivation could be induced upon crosslinking surface Ig. Notably, transcription of the genes encoding the MHV68 viral cyclin D homolog (v-cyclin) and the homolog of the KSHV latency-associated nuclear antigen (LANA), both of which are conserved among characterized γ2-herpesviruses, could consistently be detected in the established B cell lines. Furthermore, we show that the v-cyclin and LANA homologs are required for MHV68 immortalization of murine B cells. In contrast the M2 gene, which is unique to MHV68 and plays a role in latency and virus reactivation in vivo, was dispensable for B cell immortalization. This new model of gammaherpesvirus-driven B cell immortalization and differentiation in a small animal model establishes an experimental system for detailed investigation of the role of gammaherpesvirus gene products and host responses in the genesis and progression of gammaherpesvirus-associated lymphomas, and presents a convenient system to evaluate therapeutic modalities

Analysis of the leader and capsid coding regions of persistent and neurovirulent strains of Theiler's virus.

Most strains of Theiler's virus (TMEV) cause a persistent infection of the central nervous system of the mouse and a chronic demyelinating disease considered a model for multiple sclerosis. Two strains, on the contrary, cause an acute encephalitis and kill mice in a matter of days. We sequenced the leader and capsid coding region of three persistent (TO4, WW, and Yale) isolates and one neurovirulent (FA) isolate of TMEV. We compared these sequences and those already published for other isolates (DA, BeAn, GDVII, and Vilyuisk). The results suggest that virulent and persistent strains did not evolve as two separate groups, but rather that neurovirulent strains arose from a subgroup of persistent strains. The sequences of viruses isolated in different geographic areas and at different times were highly homologous, a surprising finding for an RNA virus. This suggests that severe constraints are imposed on the genome during the viral life cycle. The sequences of the TO4 and WW strains were identical, suggesting that the latter came from a laboratory contamination. The genomes of all the persistent strains sequenced so far contain an alternate open reading frame in the L region, which has been shown, in the case of the DA strain, to code for an 18-kDa protein called "I"

A single amino acid change determines persistence of a chimeric Theiler's virus.

The DA strain of Theiler's virus persists in the central nervous system of mice and causes chronic inflammation and demyelination. On the other hand, the GDVII strain causes an acute encephalitis and does not persist in surviving animals. Series of recombinants between infectious cDNA clones of the genomes of DA and GDVII viruses have been constructed. The analysis of the phenotypes of the recombinant viruses has shown that determinants of persistence and demyelination are present in the capsid proteins of DA virus. Chimeric viruses constructed by the different research groups gave consistent results, with one exception. Chimeras GD1B-2A/DAFL3 and GD1B-2C/DAFL3, which contain part of capsid protein VP2, capsid proteins VP3 and VP1, and different portions of P2 of GDVII in a DA background, were able to persist and cause demyelination. Chimera R4, whose genetic map is identical to that of GD1B-2A/DAFL3, was not. After exchanging the viral chimeras between laboratories and verifying each other's observations, new chimeras were generated in order to explain this difference. Here we report that the discrepancy can be attributed to a single amino acid difference in the sequence of the capsid protein VP2 of the two parental DA strains. DAFL3 (University of Chicago) and the chimeras derived from it, GD1B-2A/DAFL3 and GD1B-2C/DAFL3, contain a Lys at position 141, while TMDA (Institut Pasteur) and R4, the chimera derived from it, contain an Asn in that position. This amino acid is located at the tip of the EF loop, on the rim of the depression spanning the twofold axis of the capsid. These results show that a single amino acid change can confer the ability to persist and demyelinate to a chimeric Theiler's virus, and they pinpoint a region of the viral capsid that is important for this phenotype

Chimeric Theiler's virus with altered tropism for the central nervous system.

Theiler's virus is a neurotropic murine picornavirus which, depending on the strain, causes either an acute encephalitis or a persistent demyelinating disease. Following intracranial inoculation, the demyelinating strains infect sequentially the grey matter of the brain, the grey matter of the spinal cord, and finally the white matter of the spinal cord, where they persist and cause chronic demyelination. The neurovirulent strains cause a generally fatal encephalitis with lytic infection of neurons. The study of chimeric Theiler's viruses, obtained by recombining the genomes of demyelinating and neurovirulent strains, has shown that the viral capsid contains determinants for persistence and demyelination. In this article we describe the recombinant virus R5, in which the capsid protein VP1 and a small portion of protein 2A come from the neurovirulent GDVII strain and the rest of the genome comes from the persistent DA strain. The capsid of virus R5 also contains one mutation at amino acid 34 of VP3 (Asn-->His). Virus R5 does not persist in the central nervous system (CNS) of immunocompetent SJL/J or BALB/c mice. However, it replicates efficiently and persists in the CNS of BALB/c nu/nu mice, showing that its growth in the CNS is not impaired. In BALB/c nu/nu mice, whereas virus DA causes mortality with large amounts of viral antigens in the white matter of the spinal cord, virus R5 does not kill the animals, persists in the neurons of the grey matter of the brain, and never reaches the white matter of the spinal cord. This phenotype is due to the chimerism of the capsid and/or to the mutation in VP3. These results indicate that the capsid plays an important role in the characteristic migration of Theiler's virus within the CNS