Ufmylation of UFBP1 Is Dispensable for Endoplasmic Reticulum Stress Response, Embryonic Development, and Cardiac and Intestinal Homeostasis.

Protein modification by ubiquitin fold modifier 1 (UFM1), termed ufmylation, regulates various physiological and pathological processes. Among emerging UFM1 targets, UFM1 binding protein 1 (UFBP1) is the first identified ufmylation substrate. Recent clinical and animal studies have demonstrated the pivotal roles of UFBP1 in development, hematopoiesis, intestinal homeostasis, chondrogenesis, and neuronal development, which has been linked to its function in maintaining endoplasmic reticulum (ER) homeostasis. However, the importance of UFBP1 ufmylation in these cellular and physiological processes has yet to be determined. It has been proposed that ufmylation of lysine 268 (267 in humans) in UFBP1 plays a critical role in mediating the effects of the ufmylation pathway. In this study, we for the first time probe the pathophysiological significance of UFBP1 ufmylation in vivo by creating and characterizing a mouse UFBP1 knockin (KI) model in which the lysine 268 of UFBP1, the amino acid accepting UFM1, was mutated to arginine. Our results showed that the K268R mutation reduced the total ufmylated proteins without altering the expression levels of individual ufmylation enzymes in mouse embryonic fibroblasts. The K268R mutation did not alter ER stress-stimuli-induced ER stress signaling or cell death in mouse embryonic fibroblasts. The homozygous KI mice were viable and morphologically indistinguishable from their littermate wild-type controls up to one year of age. Serial echocardiography revealed no cardiac functional impairment of the homozygous KI mice. Furthermore, the homozygous KI mice exhibited the same susceptibility to dextran sulfate sodium (DSS) -induced colitis as wild-type mice. Taken together, these results suggest that UFBP1 K268 is dispensable for ER stress response, embryonic development, cardiac homeostasis under physiological conditions, and intestinal homeostasis under pathological conditions. Our studies call for future investigations to understand the biological function of UFBP1 ufmylation and offer a new mouse model to determine the roles of UFBP1 ufmylation in different tissues under stress conditions.

Cytoplasmic Relocalization and Colocalization with Viroplasms of Host Cell Proteins, and Their Role in Rotavirus Infection.

Rotavirus replicates in the cytoplasm of infected cells in unique virus-induced cytoplasmic inclusion bodies called viroplasms (VMs), which are nucleated by two essential viral nonstructural proteins, NSP2 and NSP5. However, the precise composition of the VM, the intracellular localization of host proteins during virus infection, and their association with VMs or role in rotavirus growth remained largely unexplored. Mass spectrometry analyses revealed the presence of several host heterogeneous nuclear ribonucleoproteins (hnRNPs), AU-rich element-binding proteins (ARE-BPs), and cytoplasmic proteins from uninfected MA104 cell extracts in the pulldown (PD) complexes of the purified viroplasmic proteins NSP2 and NSP5. Immunoblot analyses of PD complexes from RNase-treated and untreated cell extracts, analyses of coimmunoprecipitation complexes using RNase-treated infected cell lysates, and direct binding assays using purified recombinant proteins further demonstrated that the interactions of the majority of the hnRNPs and ARE-BPs with viroplasmic proteins are RNA independent. Time course immunoblot analysis of the nuclear and cytoplasmic fractions from rotavirus-infected and mock-infected cells and immunofluorescence confocal microscopy analyses of virus-infected cells revealed a surprising sequestration of the majority of the relocalized host proteins in viroplasms. Analyses of ectopic overexpression and small interfering RNA (siRNA)-mediated downregulation of expression revealed that host proteins either promote or inhibit viral protein expression and progeny virus production in virus-infected cells. This study demonstrates that rotavirus induces the cytoplasmic relocalization and sequestration of a large number of nuclear and cytoplasmic proteins in viroplasms, subverting essential cellular processes in both compartments to promote rapid virus growth, and reveals that the composition of rotavirus viroplasms is much more complex than is currently understood.IMPORTANCE Rotavirus replicates exclusively in the cytoplasm. Knowledge on the relocalization of nuclear proteins to the cytoplasm or the role(s) of host proteins in rotavirus infection is very limited. In this study, it is demonstrated that rotavirus infection induces the cytoplasmic relocalization of a large number of nuclear RNA-binding proteins (hnRNPs and AU-rich element-binding proteins). Except for a few, most nuclear hnRNPs and ARE-BPs, nuclear transport proteins, and some cytoplasmic proteins directly interact with the viroplasmic proteins NSP2 and NSP5 in an RNA-independent manner and become sequestered in the viroplasms of infected cells. The host proteins differentially affected viral gene expression and virus growth. This study demonstrates that rotavirus induces the relocalization and sequestration of a large number of host proteins in viroplasms, affecting host processes in both compartments and generating conditions conducive for virus growth in the cytoplasm of infected cells.