The human cytomegalovirus (HCMV) UL26 gene encodes a virion protein that is important for high titer viral replication. To identify specific domains within the UL26 protein that contribute to viral infection, we created a panel of site-directed UL26 mutant viruses and assessed their impact on phenotypes attributed to UL26. We find that the C-terminal 38 amino acids of the UL26 protein are absolutely necessary for UL26 function. A stop-insertion mutant that produced a truncated UL26 protein lacking this region behaved identically to UL26-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 UL26 packaged into the virion resulting in reduced delivery of UL26 to newly infected cells. Further, this C-terminal truncated UL26 exhibited substantially reduced nuclear localization compared to wildtype UL26. Translation of UL26 mRNA is initiated from two separate in frame methionines that give rise to a long and a short isoform of UL26. We find that the N-terminal 34 amino acids, which are unique to the long isoform of UL26, are also important for the function of the UL26 protein. A viral mutant that produces only the short isoform of UL26 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 UL26 isoform in the presence of the long UL26 isoform did not impact any of the in vitro phenotypes tested. These experiments highlight important domains within the UL26 protein that contribute to HCMV infection.
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‐β treatment of the cells induced a rapid moderate accumulation of β‐catenin, which interacted with connective tissue growth factor (CTGF/CCN2) in the cytoplasm and recruited it to the membrane for secretion. The TGF‐β‐initiated β‐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. © 2018 The Authors JBMR Plus published by Wiley Periodicals, Inc. on behalf of American Society for Bone and Mineral Research.
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