Herpesviral G Protein-Coupled Receptors Activate NFAT to Induce Tumor Formation via Inhibiting the SERCA Calcium ATPase

G protein-coupled receptors (GPCRs) constitute the largest family of proteins that transmit signal to regulate an array of fundamental biological processes. Viruses deploy diverse tactics to hijack and harness intracellular signaling events induced by GPCR. Herpesviruses encode multiple GPCR homologues that are implicated in viral pathogenesis. Cellular GPCRs are primarily regulated by their cognate ligands, while herpesviral GPCRs constitutively activate downstream signaling cascades, including the nuclear factor of activated T cells (NFAT) pathway. However, the roles of NFAT activation and mechanism thereof in viral GPCR tumorigenesis remain unknown. Here we report that GPCRs of human Kaposi’s sarcoma-associated herpesvirus (kGPCR) and cytomegalovirus (US28) shortcut NFAT activation by inhibiting the sarcoplasmic reticulum calcium ATPase (SERCA), which is necessary for viral GPCR tumorigenesis. Biochemical approaches, entailing pharmacological inhibitors and protein purification, demonstrate that viral GPCRs target SERCA2 to increase cytosolic calcium concentration. As such, NFAT activation induced by vGPCRs was exceedingly sensitive to cyclosporine A that targets calcineurin, but resistant to inhibition upstream of ER calcium release. Gene expression profiling identified a signature of NFAT activation in endothelial cells expressing viral GPCRs. The expression of NFAT-dependent genes was up-regulated in tumors derived from tva-kGPCR mouse and human KS. Employing recombinant kGPCR-deficient KSHV, we showed that kGPCR was critical for NFAT-dependent gene expression in KSHV lytic replication. Finally, cyclosporine A treatment diminished NFAT-dependent gene expression and tumor formation induced by viral GPCRs. These findings reveal essential roles of NFAT activation in viral GPCR tumorigenesis and a mechanism of “constitutive” NFAT activation by viral GPCRs.     

The linker region joining the catalytic and the regulatory domains of CnA is essential for binding to NFAT

Calcineurin (CN) is an important regulator of developmental processes and in adults controls the immune response through its regulation of nuclear factor of activated T cells (NFAT). The physical interaction between CN and NFATs is an essential step in the activation of NFAT-dependent genes by calcium signals. Using deletional and substitutional analyses, we have identified a 13-amino acid region within CN that is essential for the interaction with NFAT and with two other CN-binding proteins, AKAP79 and Cabin-1. The interaction of CN with these proteins is selectively disrupted by substitution of specific amino acid residues within this region, indicating that NFAT and other CN-interacting proteins bind differentially to CN. This selectivity suggests that the region identified in CN could be a potential molecular target for immunosuppressive and other therapeutic interventions in diseases involving the CN/NFAT pathway.     

Multifunctional roles for the N-terminal basic motif of Alfalfa mosaic virus coat protein: nucleolar/cytoplasmic shuttling, modulation of RNA-binding activity, and virion formation

In addition to virion formation, the coat protein (CP) of Alfalfa mosaic virus (AMV) is involved in the regulation of replication and translation of viral RNAs, and in cell-to-cell and systemic movement of the virus. An intriguing feature of the AMV CP is its nuclear and nucleolar accumulation. Here, we identify an N-terminal lysine-rich nucleolar localization signal (NoLS) in the AMV CP required to both enter the nucleus and accumulate in the nucleolus of infected cells, and a C-terminal leucine-rich domain which might function as a nuclear export signal. Moreover, we demonstrate that AMV CP interacts with importin-α, a component of the classical nuclear import pathway. A mutant AMV RNA 3 unable to target the nucleolus exhibited reduced plus-strand RNA synthesis and cell-to-cell spread. Moreover, virion formation and systemic movement were completely abolished in plants infected with this mutant. In vitro analysis demonstrated that specific lysine residues within the NoLS are also involved in modulating CP-RNA binding and CP dimerization, suggesting that the NoLS represents a multifunctional domain within the AMV CP. The observation that nuclear and nucleolar import signals mask RNA-binding properties of AMV CP, essential for viral replication and translation, supports a model in which viral expression is carefully modulated by a cytoplasmic/nuclear balance of CP accumulation.     

Phosphorylation by MAPK regulates simian immunodeficiency virus Vpx protein nuclear import and virus infectivity

Transport of the viral genome into the nucleus required phosphorylation of components in the preintegration complex by virion-associated host cellular kinases. In this study, we showed that ERK-2/MAPK is associated with simian immunodeficiency virus (SIV) virions and regulated the nuclear transport of Vpx and virus replication in non-proliferating target cells by phosphorylating Vpx. Suppression of the virion-associated ERK-2 activity by MAPK pathway inhibitors impaired both Vpx nuclear import and viral infectivity without affecting virus particle maturation and release. In addition, mutation analysis indicated that the inactivation of Vpx phosphorylation precluded nuclear import and reduced virus replication in macrophage cultures, even when functional integrase and Gag matrix proteins implicated in viral preintegration complex nuclear import are present. In this study, we also showed that co-localization of Vpx with Gag precursor in the cytoplasm is a prerequisite for Vpx incorporation into virus particles. Substitution of hydrophobic Leu-74 and Ile-75 with serines in the helical domain abrogated Vpx nuclear import, and its incorporation into virus particles, despite its localization in the cytoplasm, suggested that the structural integrity of helical domains is critical for Vpx functions. Taken together, these studies demonstrated that the host cell MAPK signal transduction pathway regulated an early step in SIV infection.     

Transportin-SR2 imports HIV into the nucleus

BACKGROUND: The human immunodeficiency virus type 1 (HIV-1) and other lentiviruses have the capacity to infect nondividing cells like macrophages. This requires import of the preintegration complex (PIC) through the nuclear pore. Although many cellular and viral determinants have been proposed, the mechanism leading to nuclear import is not yet understood. RESULTS: Using yeast two-hybrid and pull-down, we identified and validated transportin-SR2 (TRN-SR2) as a bona fide binding partner of HIV-1 integrase. We confirmed the biological relevance of this interaction by RNAi. Depletion of TRN-SR2 interfered with the replication of HIV-1 and HIV-2 but not MoMLV in HeLaP4 cells. Knockdown of TRN-SR2 in primary macrophages likewise interfered with HIV-1 replication. Using Q-PCR, we pinpoint this block in replication to the early steps of the viral lifecycle. A reduction in 2-LTR formation suggests a block in PIC nuclear import upon siRNA-mediated knockdown. Different lines of evidence clearly proved that the late steps of viral replication are not affected. In an in vivo nuclear-import assay using labeled HIV-1 particles, the defect in nuclear import after depletion of TRN-SR2 was directly visualized. In comparison with control cell lines, the great majority of siRNA-treated cells did not contain any PIC in the nucleus. CONCLUSION: Our data clearly demonstrate that TRN-SR2 is the nuclear-import factor of HIV.     

The formation of cysteine-linked dimers of BST-2/tetherin is important for inhibition of HIV-1 virus release but not for sensitivity to Vpu

Background

The integrase (IN) of human immunodeficiency virus type 1 (HIV-1) has been implicated in different steps during viral replication, including nuclear import of the viral pre-integration complex. The exact mechanisms underlying the nuclear import of IN and especially the question of whether it bears a functional nuclear localization signal (NLS) remain controversial.

Results

Here, we studied the nuclear import pathway of IN by using multiple in vivo and in vitro systems. Nuclear import was not observed in an importin α temperature-sensitive yeast mutant, indicating an importin α-mediated process. Direct interaction between the full-length IN and importin α was demonstrated in vivo using bimolecular fluorescence complementation assay (BiFC). Nuclear import studies in yeast cells, with permeabilized mammalian cells, or microinjected cultured mammalian cells strongly suggest that the IN bears a NLS domain located between residues 161 and 173. A peptide bearing this sequence -NLS-IN peptide- inhibited nuclear accumulation of IN in transfected cell-cycle arrested cells. Integration of viral cDNA as well as HIV-1 replication in viral cell-cycle arrested infected cells were blocked by the NLS-IN peptide.

Conclusion

Our present findings support the view that nuclear import of IN occurs via the importin α pathway and is promoted by a specific NLS domain. This import could be blocked by NLS-IN peptide, resulting in inhibition of viral infection, confirming the view that nuclear import of the viral pre-integration complex is mediated by viral IN.