Daniel R. Nussenzveig

Influenza virus targets the mRNA export machinery and the nuclear pore complex

The NS1 protein of influenza A virus is a major virulence factor that is essential for pathogenesis. NS1 functions to impair innate and adaptive immunity by inhibiting host signal transduction and gene expression, but its mechanisms of action remain to be fully elucidated. We show here that NS1 forms an inhibitory complex with NXF1/TAP, p15/NXT, Rae1/mrnp41, and E1B-AP5, which are key constituents of the mRNA export machinery that interact with both mRNAs and nucleoporins to direct mRNAs through the nuclear pore complex. Increased levels of NXF1, p15, or Rae1 revert the mRNA export blockage induced by NS1. Furthermore, influenza virus down-regulates Nup98, a nucleoporin that is a docking site for mRNA export factors. Reduced expression of these mRNA export factors renders cells highly permissive to influenza virus replication, demonstrating that proper levels of key constituents of the mRNA export machinery protect against influenza virus replication. Because Nup98 and Rae1 are induced by interferons, down-regulation of this pathway is likely a viral strategy to promote viral replication. These findings demonstrate previously undescribed influenza-mediated viral–host interactions and provide insights into potential molecular therapies that may interfere with influenza infection.

Viral interactions with the nuclear transport machinery : Discovering and disrupting pathways

Viruses have been invaluable tools for discovering key pathways of nucleocytoplasmic transport. Conversely, disruption of specific nuclear transport pathways, are crucial for the productive life cycle of some viruses. The major cellular mRNA export pathway, which uses TAP (NXF1)/p15(NXT) as receptor, was discovered as a result of TAP interaction with CTE‐containing RNAs from Mason‐Pfizer Monkey Virus. In addition, CRM1 or exportin 1, which is a transport receptor that mediates nuclear export of proteins, snRNAs, rRNAs and a small subset of mRNAs, was discovered as an interacting partner of the Rev protein of HIV1. Viruses may disrupt the nuclear transport machinery to prevent host antiviral response. VSV Matrix (M) protein inhibits mRNA export by forming a complex with the mRNA export factor Rae1 whereas poliovirus inhibits nuclear import of proteins by probably degrading Nup62 and Nup153. Hence, this review focuses on viruses as tools and as disruptors of nucleocytoplasmic trafficking. IUBMB Life, 57: 65‐72, 2005

NFAR-1 and -2 modulate translation and are required for efficient host defense

We report here that the alternatively spliced nuclear factors associated with double-stranded RNA, NFAR-1 (90 kDa) and -2 (110 kDa), are involved in retaining cellular transcripts in intranuclear foci and can regulate the export of mRNA to the cytoplasm. Furthermore, the NFAR proteins were found to remain associated with exported ribonucleoprotein complexes. Loss of NFAR function, which was embryonic-lethal, caused an increase in protein synthesis rates, an effect augmented by the presence of the mRNA export factors TAP, p15, or Rae1. Significantly, NFAR depletion in normal murine fibroblasts rendered these cells dramatically susceptible to vesicular stomatitis virus replication. Collectively, our data demonstrate that the NFARs exert influence on mRNA trafficking and the modulation of translation rates and may constitute an innate immune translational surveillance mechanism important in host defense countermeasures against virus infection.

Vesicular stomatitis virus inhibits mitotic progression and triggers cell death

Vesicular stomatitis virus (VSV) infects and kills a wide range of cell types; however, the mechanisms involved in VSV‐mediated cell death are not fully understood. Here we show that VSV infection interferes with mitotic progression, resulting in cell death. This effect requires the interaction of VSV matrix (M) protein with the Rae1–Nup98 complex in mitosis, which is associated with a subset of ribonucleoproteins (RNPs). VSV displaced Rae1 from spindle poles, caused spindle abnormalities and triggered substantial cell death during metaphase. These effects were attenuated in cells infected with VSV expressing a mutant M protein that does not bind efficiently to the Rae1–Nup98–RNP complex. In cells that progressed to late mitosis, M protein prevented proper nuclear formation and chromatin decondensation. VSV is an oncolytic (anti‐tumour) agent as it preferentially replicates and kills tumour cells. As tumour cells have a high mitotic index, VSV‐mediated mitotic cell death probably contributes to its oncolytic activity.

Desensitization of the response to thyrotropin-releasing hormone in Xenopus oocytes is an amplified process that precedes calcium mobilization

Consecutive challenges with thyrotropin-releasing hormone (TRH) of oocytes expressing the TRH receptor (TRH-R) resulted in a pronounced desensitization, manifested as a decrease in chloride current amplitude and an increase in response latency. Exposure to low concentrations of TRH resulted in a marked decrease in the amplitude of the subsequent response to a higher concentration of the agonist, even though the second challenge was given before the onset of the response to the first challenge (within 3–15 s). Cellular calcium concentration ([Ca]i) did not increase within this interval, suggesting that calcium was not involved in the desensitization process. The latency of the second response, however, was either unchanged or shortened, implying additive effects of processes initiated by the first challenge. A longer interval (30 s) between the two challenges brought about a more pronounced decrease in amplitude and a prolongation of response latency. The calcium mobilization initiated by a second challenge with a high concentration of the agonist exhibited a longer latency, a lower rate of [Ca]i increase and a lower amplitude. Stimulation of coexpressed cholinergic-muscarinic ml receptors with a low concentration of acetylcholine resulted in a pronounced desensitization of the TRH response (heterologous desensitization). Activation of protein kinase C by β-phorbol 12-myristate,13-acetate resulted in a dose-dependent inhibition of the response to TRH, suggesting that protein kinase C was involved in desensitization. Chelerythrine, a specific inhibitor of protein kinase C, abolished a large part of the desensitization. A mutant of the TRH-R that lacks protein kinase C concensus phosphorylation sites in the C-terminal region, exhibited desensitization. Hence, desensitization is not targeted at this part of the receptor molecule. Our results suggest that a very low receptor occupancy activates an amplification step that results in heterologous desensitization. This process is mediated, at least partly, by the activation of protein kinase C, acting on a target proximal to calcium mobilization.

Sec13 Regulates Expression of Specific Immune Factors Involved in Inflammation In Vivo

The Sec13 protein functions in various intracellular compartments including the nuclear pore complex, COPII-coated vesicles, and inside the nucleus as a transcription regulator. Here we developed a mouse model that expresses low levels of Sec13 (Sec13H/−) to assess its functions in vivo, as Sec13 knockout is lethal. These Sec13 mutant mice did not present gross defects in anatomy and physiology. However, the reduced levels of Sec13 in vivo yielded specific immunological defects. In particular, these Sec13 mutant mice showed low levels of MHC I and II expressed by macrophages, low levels of INF-γ and IL-6 expressed by stimulated T cells, and low frequencies of splenic IFN-γ+CD8+ T cells. In contrast, the levels of soluble and membrane-bound TGF-β as well as serum immunoglobulin production are high in these mice. Furthermore, frequencies of CD19+CD5-CD95+ and CD19+CD5-IL-4+ B cells were diminished in Sec13H/− mice. Upon stimulation or immunization, some of the defects observed in the naïve mutant mice were compensated. However, TGF-β expression remained high suggesting that Sec13 is a negative modulator of TGF-β expression and of its immunosuppressive functions on certain immune cells. In sum, Sec13 regulates specific expression of immune factors with key functions in inflammation.

Human calcitonin receptor is directly targeted to and retained in the basolateral surface of MDCK cells

The human calcitonin receptor (hCTR) is expressed in polarized cells of the kidney, bone, and nervous system. In the kidney, hCTRs are found in cells of the distal nephron to which blood-borne calcitonin has access only at the basolateral surface. We expressed hCTR subtypes 1 and 2 in Madin-Darby canine kidney (MDCK) cells to establish a cell model useful for delineating the molecular mechanisms underlying hCTR polarity. Selective cell surface incubation demonstrated functional polarity of hCTRs by equilibrium binding or cross-linking of radioiodinated salmon calcitonin (125I-sCT) and cAMP accumulation stimulated by sCT. We estimated that at the steady state there are 40-fold more hCTRs on the basolateral than on the apical side. Domain-selective cell surface biotinylation followed by immunoblotting of streptavidin-agarose-fractionated biotinylated glycoproteins independently confirmed the polarized distribution of FLAG epitope-tagged hCTR-2 in the basolateral domain. Confocal microscopy of immunostained receptors revealed that hCTRs are concentrated on a lateral subdomain of the basolateral membrane. Cell surface arrival assay of newly formed receptors demonstrated that direct delivery to the basolateral domain is the mechanism by which hCTRs become polarized. Measurement of receptor turnover on the basolateral surface showed that retention contributes to hCTR distribution at the steady state.The human calcitonin receptor (hCTR) is expressed in polarized cells of the kidney, bone, and nervous system. In the kidney, hCTRs are found in cells of the distal nephron to which blood-borne calcitonin has access only at the basolateral surface. We expressed hCTR subtypes 1 and 2 in Madin-Darby canine kidney (MDCK) cells to establish a cell model useful for delineating the molecular mechanisms underlying hCTR polarity. Selective cell surface incubation demonstrated functional polarity of hCTRs by equilibrium binding or cross-linking of radioiodinated salmon calcitonin (125I-sCT) and cAMP accumulation stimulated by sCT. We estimated that at the steady state there are 40-fold more hCTRs on the basolateral than on the apical side. Domain-selective cell surface biotinylation followed by immunoblotting of streptavidin-agarose-fractionated biotinylated glycoproteins independently confirmed the polarized distribution of FLAG epitope-tagged hCTR-2 in the basolateral domain. Confocal microscopy of immunostained receptors revealed that hCTRs are concentrated on a lateral subdomain of the basolateral membrane. Cell surface arrival assay of newly formed receptors demonstrated that direct delivery to the basolateral domain is the mechanism by which hCTRs become polarized. Measurement of receptor turnover on the basolateral surface showed that retention contributes to hCTR distribution at the steady state.

Functional Properties and Cell Biology of Renal and Vascular Receptors of Atrial Natriuretic Factor

Atrial natriuretic factor (ANF) is secreted mainly by the heart atria in response to volume expansion and increases in atrial pressure or stretch. The hormone has important effects in several tissues, resulting in modulation of plasma volume, extracellular fluid volume, and blood pressure (for review, see [1,2]). There are two general classes of ANF receptors in peripheral tissues. Biological receptors proper (B-ANF receptors) mediate the ANF-induced increase in cGMP and many, if not all, of the physiological effects of the hormone [3]. These receptors were shown to contain guanylate cyclase in the cytoplasmic domain of their molecules [4]. This is a first example of a biological receptor which directly catalyzes the formation of a small second messenger upon hormone binding [4]. The other class of ANF receptors is widely distributed, at orders of magnitude larger in density than B-ANF receptors, in several tissues and cells, including kidney cortex, endothelial and vascular smooth muscle cells, and fibroblasts. The functional and biochemical nature of this receptor (named C-ANF receptor) has been fully characterized [5,6]. C-ANF receptors do not mediate any of the known effects of the hormone, but have an important role in the removal of ANF from the circulation [5]. This is a first example of a separate class of clearance receptors for a circulating polypeptide hormone. The extracellular domains of B-ANF receptors and C-ANF receptors have considerable homology [4], explaining the finding that both receptors have a similar very high affinity for native ANF1–28 [3,7].