TGF-ß Initiates ß-Catenin-Mediated CTGF Secretory Pathway in Old Bovine Nucleus Pulposus Cells: A Potential Mechanism for Intervertebral Disc Degeneration.

We have recently demonstrated that overexpression of Smurf2 under the control of type II collagen alpha 1 (Col2a1) promoter induces an intervertebral disc degeneration phenotype in Col2a1-Smurf2 transgenic mice. The chondrocyte-like cells that express type II collagen and Smurf2 in the transgenic mouse discs are prone to degenerate. However, how the chondrocyte-like cells contribute to disc degeneration is not known. Here, we utilized primary old bovine nucleus pulposus (NP) cells as substitutes for the chondrocyte-like cells in Col2a1-Smurf2 transgenic mouse discs to identify mechanism. We found that 35% of the cells were senescent; TGF-ß treatment of the cells induced a rapid moderate accumulation of ß-catenin, which interacted with connective tissue growth factor (CTGF/CCN2) in the cytoplasm and recruited it to the membrane for secretion. The TGF-ß-initiated ß-catenin-mediated CTGF secretory cascade did not occur in primary young bovine NP cells; however, when Smurf2 was overexpressed in young bovine NP cells, the cells became senescent and allowed this cascade to occur. These results suggest that Smurf2-induced disc degeneration in Col2a1-Smurf2 transgenic mice occurs through activation of CTGF secretory pathway in senescent disc cells.

The human cytomegalovirus UL26 protein antagonizes NF-κB activation.

UNLABELLED: Viral infection frequently triggers activation of host innate immune pathways that attempt to limit viral spread. The NF-κB pathway is a critical component that governs this response. We have found that the human cytomegalovirus (HCMV) U(L)26 protein antagonizes NF-κB activation. Upon infection, an HCMV strain lacking the U(L)26 gene (ΔU(L)26) induced the nuclear translocation of the NF-κB RelB subunit and activated expression and secretion of interleukin-6 (IL-6), an NF-κB target gene. The ΔU(L)26 mutant was also more sensitive to challenge with tumor necrosis factor alpha (TNF-α), a canonical NF-κB inducer. Further, expression of U(L)26 in the absence of other viral proteins blocked NF-κB activation induced by either TNF-α treatment or infection with Sendai virus (SeV). Our results indicate that U(L)26 expression is sufficient to block TNF-α-induced NF-κB nuclear translocation and IκB degradation. Last, U(L)26 blocks TNF-α-induced IκB-kinase (IKK) phosphorylation, a key step in NF-κB activation. Combined, our results indicate that U(L)26 is part of a viral program to antagonize innate immunity through modulation of NF-κB signaling. IMPORTANCE: The NF-κB signaling pathway regulates innate immunity, an integral host process that limits viral pathogenesis. Viruses have evolved mechanisms to modulate NF-κB signaling to ensure their replication. HCMV is a major cause of birth defects and disease in immunosuppressed populations. HCMV is known to actively target the NF-κB pathway, which is important for HCMV infection. Our results indicate that the HCMV U(L)26 gene is a key modulator of NF-κB pathway activity. We find the U(L)26 gene is both necessary and sufficient to block NF-κB activation upon challenge with antiviral cytokines. Further, U(L)26 attenuates the phosphorylation and activation of a key NF-κB activating kinase complex, IKK. Our study provides new insight into how HCMV targets the NF-κB pathway. Given its importance to viral infection, the mechanisms through which viruses target the NF-κB pathway highlight areas of vulnerability that could be therapeutically targeted to attenuate viral replication.

Distinct domains within the human cytomegalovirus U(L)26 protein are important for wildtype viral replication and virion stability.

The human cytomegalovirus (HCMV) U(L)26 gene encodes a virion protein that is important for high titer viral replication. To identify specific domains within the U(L)26 protein that contribute to viral infection, we created a panel of site-directed U(L)26 mutant viruses and assessed their impact on phenotypes attributed to U(L)26. We find that the C-terminal 38 amino acids of the U(L)26 protein are absolutely necessary for U(L)26 function. A stop-insertion mutant that produced a truncated U(L)26 protein lacking this region behaved identically to U(L)26-null viruses. This included reduced accumulation of IE1 protein at early time points, smaller plaque size, reduced virion stability, and growth with similarly attenuated kinetics. This C-terminal truncation decreased the amount of U(L)26 packaged into the virion resulting in reduced delivery of U(L)26 to newly infected cells. Further, this C-terminal truncated U(L)26 exhibited substantially reduced nuclear localization compared to wildtype U(L)26. Translation of U(L)26 mRNA is initiated from two separate in frame methionines that give rise to a long and a short isoform of U(L)26. We find that the N-terminal 34 amino acids, which are unique to the long isoform of U(L)26, are also important for the function of the U(L)26 protein. A viral mutant that produces only the short isoform of U(L)26 and lacks these N-terminal 34 amino acids exhibits delayed IE1 accumulation, and demonstrates intermediate defects in viral plaque size, virion stability and viral growth kinetics. Ablation of the short U(L)26 isoform in the presence of the long U(L)26 isoform did not impact any of the in vitro phenotypes tested. These experiments highlight important domains within the U(L)26 protein that contribute to HCMV infection.