Kaposi’s sarcoma associated herpesvirus (KSHV), like other human herpes viruses, establishes a biphasic life cycle referred to as dormant or latent, and productive or lytic phases. The latent phase is characterized by the persistence of viral episomes in a highly ordered chromatin structure and with the expression of a limited number of viral genes. Latency Associated Nuclear Antigen (LANA) is among the most abundantly expressed proteins during latency and is required for various nuclear functions including the recruitment of cellular machineries for viral DNA replication and segregation of the replicated genomes to daughter cells. LANA achieves these functions by recruiting cellular proteins including replication factors, chromatin modifying enzymes and cellular mitotic apparatus assembly. LANA directly binds to the terminal repeat region of the viral genome and associates with nucleosomal proteins to tether to the host chromosome. Binding of LANA to TR recruits the replication machinery, thereby initiating DNA replication within the TR. However, other regions of the viral genome can also initiate replication as determined by Single Molecule Analysis of the Replicated DNA (SMARD) approach. Recent, next generation sequence analysis of the viral transcriptome shows the expression of additional genes during latent phase. Here, we discuss the newly annotated latent genes and the role of major latent proteins in KSHV biology.

RIG-I is an intracellularRIG-I is a recently identified RNA helicase containing two caspase activation and recruitment domains (CARDs). RNA binding and signaling by RIG-I are implicated in host defense against pathogens via stimulation of alpha/beta interferon (IFN-␣/␤) production and downstream transcription of various antiviral genes (26). In resting cells, RIG-I is maintained as a monomer in an autoinhibited state. Viral infection induces a conformational change in RIG-I that promotes selfassociation and CARD interactions with the VISA/IPS-1/ Cardif/MAVS adaptor protein and triggers IFN regulatory factor 3-and NF-B-responsive gene expression (15). The internal repressor domain (RD) controls RIG-I multimerization and interaction with VISA/IPS-1/Cardif/MAVS. Deletion of the RD results in constitutive signaling to the IFN-␤ promoter, whereas RD expression alone prevents signaling and increases cellular permissiveness to hepatitis C virus (HCV) (16). Further study reveals that the C-terminal regulatory domain RD of RIG-I binds viral RNA in a 5Ј-triphosphatedependent manner and activates the RIG-I ATPase by RNA-dependent dimerization. The RD contains a zincbinding domain that is structurally related to the GDP/GTP exchange factors of Rab-like GTPases (2). However, regulation of RIG-I activity is underexplored, and the mechanism for its activity remains unknown.Casein kinase II (CK2) is a highly conserved serine-threonine kinase that uses both ATP and GTP as phosphate donors. It is constitutively activated and ubiquitously expressed. It usually presents as a tetrameric holoenzyme complex of two catalytic subunits (alpha and/or alphaЈ) and two regulatory beta subunits, phosphorylates more than 300 substrates, and controls a wide range of processes, including the regulation of the cell cycle, apoptosis, transformation, and circadian rhythm (20). Recent evidences suggest a potentially important role for CK2 in the control of the inflammatory response (11, 13). However, whether CK2 is involved in viral infections has not been studied.Reversible phosphorylation is an important regulatory mechanism for many biological processes in eukaryotic organisms, especially for signal pathways. The phosphorylation state of a protein is controlled dynamically by both protein kinases and phosphatases. Protein phosphorylation can lead to activation or inactivation of the substrate, depending on its nature. We here report that RIG-I activity is regulated by phosphorylation and dephosphorylation during viral infection. MATERIALS AND METHODSPlasmids. Flag-or hemagglutinin (HA)-tagged RIG-I, HA-CARD (amino acids [aa] 1 to 234), Flag-⌬CARD (aa 234 to 956), and HA-MDA5 were kindly provided by Hongbing Shu (Wuhan University, China), Flag-CK2 was a gift from

ganglion (DRG) sensory neurons, including >90% of C-nociceptors (pain-sensing neurons) and C-low-threshold mechanoreceptors, as well as a lower percentage of Ad-nociceptors and Ab afferents. At age 10 weeks (n ¼ 5) and at age 26 weeks (n ¼ 5), mice were perfused transcardially with paraformaldehyde, and the right knees were collected, post fixed and decalcified. Twenty-mm thick frozen sections were collected at mid-joint level. Consecutive sections were stained with hematoxylin & eosin. Age-matched heterozygous C57BL/6 Pirt-GCaMP3 mice were used to confirm innervation patterns. These mice express the green fluorescent calcium indicator, GCaMP3, in~90% of all sensory DRG neurons (including the Na v 1.8 population), and not in other peripheral or central tissues, through the Pirt promoter. Results: Examination of the knees of 10-week old Na V 1.8-TdTomato mice revealed areas of dense innervation by Na V 1.8-expressing sensory fibers, most notably the bone marrow, the lateral synovium, and the connective tissue layer (epiligament) surrounding the cruciate ligaments, including the areas of attachment. Other structures, such as the medial synovium and the collagenous substance of the cruciate ligaments, were less densely innervated. Na V 1.8 nociceptors were also present in the outer third of the lateral meniscus. The articular cartilage, the inner two thirds of the lateral meniscus, and the medial meniscus did not show innervation. Figure 1 shows an example of these features in one mouse-but these findings were remarkably reproducible in n ¼ 5 mice. Assessment of Na V 1.8 signal in knees of 26-week old mice revealed marked changes in innervation density (not shown). Compared to 10-week old knees, 26-week old knees showed a dramatic decline in Na V 1.8-expressing nociceptors in the lateral synovium, as well as in the epiligament and attachment areas of the cruciate ligaments. Similar age-related changes in the innervation were also detected in the knees of 26-week old Pirt-GCaMP3 mice compared to 10-week old knees, providing independent evidence that the chosen markers are specific for nerve fibers. Conclusions: This study reproducibly shows, for the first time, that the nociceptive innervation of specific murine knee tissues dramatically declines with age. Remarkably, this occurs quite early on in the life of the mouse, where we find dense innervation at 10 weeks and a marked decline by 26 weeks. Ongoing studies are aimed at monitoring innervation with more advanced age. The biological significance of these findings needs to be explored, as well as the relationship with pathogenesis of osteoarthritis.

Osteoarthritis (OA) is the most common joint disease, characterized by progressive destruction of the articular cartilage. The surface of joint cartilage is the first defensive and affected site of OA, but our knowledge of genesis and homeostasis of this superficial zone is scarce. EGFR signaling is important for tissue homeostasis. Immunostaining revealed that its activity is mostly dominant in the superficial layer of healthy cartilage but greatly diminished when OA initiates. To evaluate the role of EGFR signaling in the articular cartilage, we studied a cartilage-specific Egfr-deficient (CKO) mouse model (Col2-Cre EgfrWa5/flox). These mice developed early cartilage degeneration at 6 mo of age. By 2 mo of age, although their gross cartilage morphology appears normal, CKO mice had a drastically reduced number of superficial chondrocytes and decreased lubricant secretion at the surface. Using superficial chondrocyte and cartilage explant cultures, we demonstrated that EGFR signaling is critical for maintaining the number and properties of superficial chondrocytes, promoting chondrogenic proteoglycan 4 (Prg4) expression, and stimulating the lubrication function of the cartilage surface. In addition, EGFR deficiency greatly disorganized collagen fibrils in articular cartilage and strikingly reduced cartilage surface modulus. After surgical induction of OA at 3 mo of age, CKO mice quickly developed the most severe OA phenotype, including a complete loss of cartilage, extremely high surface modulus, subchondral bone plate thickening, and elevated joint pain. Taken together, our studies establish EGFR signaling as an important regulator of the superficial layer during articular cartilage development and OA initiation.EGFR | articular cartilage | chondrocyte | lubrication | osteoarthritis

Background Exosomes are emerging as important mediators of the cross-talk between tumor cells and the microenvironment. The communication between tumor-derived exosomes and macrophages has a critical role in facilitating tumor progression. However, the mechanisms by which exosomes modulate tumor development in lung cancer are not fully understood. Methods Short hairpin RNA mediated knockdown or exogenous expression of TRIM59 combined with in vitro and in vivo assays were performed to prove the functional significance of TRIM59. Western blotting, real-time PCR, co-immunoprecipitation, immunofluorescence (IF) staining assays, proximity ligation assay (PLA), ubiquitination assays, lactate secretion and lipid droplets content measurement, and rescue experiments were used to evaluate the mechanism. Lewis lung carcinoma (LLC) cells were injected via subcutaneously or tail vein into C57BL/6 wild-type (WT) and transgenic mice to assess the role of TRIM59 in vivo. Results We demonstrated that tripartite motif-containing 59 (TRIM59) was expressed in lung cancer cells-derived exosomes, and can be transferred to macrophages through the exosomes. Activated macrophages by TRIM59 promote lung cancer progression in vitro and in vivo. Mechanistic investigations revealed that TRIM59 physically interacts with abhydrolase domain containing 5 (ABHD5) and directly induced the ubiquitination of ABHD5 and led to its proteasome-dependent degradation. ABHD5, an lipolytic co-activator, deficiency induced metabolic reprogramming and enabled NLRP3 inflammasome activation in macrophages. Further studies showed that the exacerbation of NLRP3 inflammasome activation by ABHD5 deficiency, provides a positive feedback loop to promote cancer progression by preferentially secrete the proinflammatory cytokine IL-1β. Conclusions Collectively, these data indicate that tumor-derived exosomal TRIM59 converts macrophages to tumor-promoting functions of macrophages via regulating ABHD5 proteasomal degradation, to activate NLRP3 inflammasome signaling pathway to promote lung cancer progression by IL-1β secretion. Our findings also indicate that tumor-derived exosomal TRIM59 has an important role in intercellular communication for fostering an inflammatory microenvironment and promoting lung metastasis.

Since focal stress on the SBP underlying sites of cartilage damage increases during late stages of OA, these findings establish mechanical loading-induced attenuation of sclerostin expression and elevation of bone formation along the SBP surface as the major mechanisms characterizing subchondral bone phenotypes associated with severe late-stage OA in mice.

No abstract

Epstein–Barr virus (EBV), a ubiquitous human herpesvirus, can latently infect the human population. EBV is associated with several types of malignancies originating from lymphoid and epithelial cell types. EBV latent antigen 3C (EBNA3C) is essential for EBV-induced immortalization of B-cells. The Moloney murine leukemia provirus integration site (PIM-1), which encodes an oncogenic serine/threonine kinase, is linked to several cellular functions involving cell survival, proliferation, differentiation, and apoptosis. Notably, enhanced expression of Pim-1 kinase is associated with numerous hematological and non-hematological malignancies. A higher expression level of Pim-1 kinase is associated with EBV infection, suggesting a crucial role for Pim-1 in EBV-induced tumorigenesis. We now demonstrate a molecular mechanism which reveals a direct role for EBNA3C in enhancing Pim-1 expression in EBV-infected primary B-cells. We also showed that EBNA3C is physically associated with Pim-1 through its amino-terminal domain, and also forms a molecular complex in B-cells. EBNA3C can stabilize Pim-1 through abrogation of the proteasome/Ubiquitin pathway. Our results demonstrate that EBNA3C enhances Pim-1 mediated phosphorylation of p21 at the Thr145 residue. EBNA3C also facilitated the nuclear localization of Pim-1, and promoted EBV transformed cell proliferation by altering Pim-1 mediated regulation of the activity of the cell-cycle inhibitor p21/WAF1. Our study demonstrated that EBNA3C significantly induces Pim-1 mediated proteosomal degradation of p21. A significant reduction in cell proliferation of EBV-transformed LCLs was observed upon stable knockdown of Pim-1. This study describes a critical role for the oncoprotein Pim-1 in EBV-mediated oncogenesis, as well as provides novel insights into oncogenic kinase-targeted therapeutic intervention of EBV-associated cancers.