No abstract

The products of numerous open reading frames (ORFs) present in the genome of human cytomegalovirus (CMV) have not been characterized. Here, we describe the identification of a new CMV protein localizing to the nuclear envelope and in cytoplasmic vesicles at late times postinfection. Based on this distinctive localization pattern, we called this new protein nuclear rim-associated cytomegaloviral protein, or RASCAL. Two RASCAL isoforms exist, a short version of 97 amino acids encoded by the majority of CMV strains and a longer version of 176 amino acids encoded by the Towne, Toledo, HAN20, and HAN38 strains. Both isoforms colocalize with lamin B in deep intranuclear invaginations of the inner nuclear membrane (INM) and in novel cytoplasmic vesicular structures possibly derived from the nuclear envelope. INM infoldings have been previously described as sites of nucleocapsid egress, which is mediated by the localized disruption of the nuclear lamina, promoted by the activities of viral and cellular kinases recruited by the lamina-associated proteins UL50 and UL53. RASCAL accumulation at the nuclear membrane required the presence of UL50 but not of UL53. RASCAL and UL50 also appeared to specifically interact, suggesting that RASCAL is a new component of the nuclear egress complex (NEC) and possibly involved in mediating nucleocapsid egress from the nucleus. Finally, the presence of RASCAL within cytoplasmic vesicles raises the intriguing possibility that this protein might participate in additional steps of virion maturation occurring after capsid release from the nucleus.

To investigate the immunoregulatory effects of interferon (IFN)-β on CD4+ T cells, we examined the response of CD4+ T cells from IFN-β(+/+) and IFN-β(-/-) mice to CD3/CD28 activation and to differentiation to Th17 lineage, analyzing the expression of signaling effectors, cell surface receptors, production of IL-17, and gene expression profiles. We provide evidence of increased phosphorylation of the membrane proximal kinase associated with TCR activation, ZAP-70, in IFN-β(-/-) T cells compared with IFN-β(+/+) T cells. Anti-CD3/anti-CD28 antibody stimulation of whole splenocytes or CD4+ T cells from IFN-β(-/-) mice results in secretion of IL-17A, in contrast to identical stimulation of cells from IFN-β(+/+) mice, which fails to increase IL-17A production. After CD3/CD28 activation, IFN-β(-/-) CD4+ T cells express higher levels of IRF-4, required for Th17 differentiation, and increased expression of CCR6, IL-23R, IL-6R, and CXCR4, compared with activated IFN-β(+/+) T cells. Notably, cell surface expression of IL-6R and IL-23R is significantly higher in the IFN-β(-/-) CD4+ T cells, with an increased number of double-positive CCR6+IL-23R+ and IL-6R+IL-23R+ CD4+ T cells. On polarization to Th17 lineage, CD4+ T cells from IFN-β(-/-) mice exhibit a more Th17-primed transcriptome compared with CD4+ T cells from IFN-β(+/+) mice. Indeed, when CD4+ T cells from IFN-β(+/+) mice are polarized to Th17 lineage in the presence of IFN-β, many Th17-associated genes are down-regulated. Employing a MOG-peptide-induced experimental autoimmune encephalomyelitis model of multiple sclerosis, we identify a greater proportion of Th17 cells in the lymph nodes of IFN-β(-/-) mice compared with IFN-β(+/+) mice, and increased numbers of CD4+ T cells in the central nervous system of IFN-β(-/-) mice, regardless of the stage of disease. Taken together, our data indicate an immunoregulatory role for IFN-β in the suppression of Th17 cells.