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  • Sendai virus particle production: basic requirements and role of the SYWST motif present in HN cytoplasmic tail. 20633915

    Sendai virus (SeV) HN protein is dispensable for virus particle production. HN incorporation into virions strictly depends on a cytoplasmic domain SYWST motif. HNAFYKD, with SYWST replaced with the analogous sequence of measles virus (MeV) H (AFYKD), is not incorporated in virus particles produced by LLCMK2 cells, although it is normally expressed at the plasma membrane. Unlike HNSYWST, HNAFYKD is not internalized to late endosomes, raising the possibility that HN internalization is required for uptake into virus particles. Various mosaic MeV-H containing increasing amounts of the SeV-HN all failed to be taken up in SeV virions. However, when co-expressed with HNAFYKD these MeV-H chimera induced HNAFYKD uptake into virions showing that internalization is not a prerequisite for HN uptake into particles. We propose that HN incorporation in virus particles requires first neutralization by HN of a putative inhibitor of infectious particle formation.
    Document Type:
    Reference
    Product Catalog Number:
    MAB8905
    Product Catalog Name:
    Anti-Measles Blend Antibody, hemagglutinin, clones CV1, CV4

  • Targeting of the Sendai virus C protein to the plasma membrane via a peptide-only membrane anchor. 17229713

    Several cellular proteins are synthesized in the cytosol on free ribosomes and then associate with membranes due to the presence of short peptide sequences. These membrane-targeting sequences contain sites to which lipid chains are attached, which help direct the protein to a particular membrane domain and anchor it firmly in the bilayer. The intracellular concentration of these proteins in particular cellular compartments, where their interacting partners are also concentrated, is essential to their function. This paper reports that the apparently unmodified N-terminal sequence of the Sendai virus C protein (MPSFLKKILKLRGRR . . .; letters in italics represent hydrophobic residues; underlined letters represent basic residues, which has a strong propensity to form an amphipathic alpha-helix in a hydrophobic environment) also function as a membrane targeting signal and membrane anchor. Moreover, the intracellular localization of the C protein at the plasma membrane is essential for inducing the interferon-independent phosphorylation of Stat1 as part of the viral program to prevent the cellular antiviral response.
    Document Type:
    Reference
    Product Catalog Number:
    07-224
    Product Catalog Name:
    Anti-phospho-STAT2 (Tyr689) Antibody

  • Characterization of the amino acid residues of sendai virus C protein that are critically involved in its interferon antagonism and RNA synthesis down-regulation. 15220418

    Sendai virus (SeV) encodes two accessory proteins, V and C, in the alternative reading frames in the P gene that are accessed transcriptionally (V) or translationally (C). The C protein is expressed as a nested set of four C-coterminal proteins, C', C, Y1, and Y2, that use different initiation codons. Using HeLa cell lines constitutively expressing the various C proteins, we previously found that the smallest (the 175-residue Y2) of the four C proteins was fully capable of counteracting the antiviral action of interferons (IFNs) and inhibiting viral RNA synthesis and that the C-terminal half of 106 residues was sufficient for both of these inhibitory functions (A. Kato et al., J. Virol. 75:3802-3810, 2001, and A. Kato et al., J. Virol. 76:7114-7124, 2002). Here, we further generated HeLa cell lines expressing the mutated C (Cm) proteins with charged amino acids substituted for alanine residues at either positions 77 and 80; 114 and 115; 139 and 142; 151, 153, and 154; 156; or 173, 175, and 176. We found that only the mutations at positions 151, 153, and 154 abolished IFN antagonism. All the Cm proteins lost the ability to bind with STAT1 under our assay conditions, regardless of their ability to inhibit IFN signaling. On the other hand, the Cm proteins that altered the tyrosine phosphorylation and dephosphorylation of STAT1 and STAT2 always retained IFN antagonism. Thus, the abnormality of phosphorylation or dephosphorylation appeared to be a cause of the IFN antagonism by SeV C. Regarding viral RNA synthesis inhibition, all mutants but the mutant with replacements at positions 114 and 115 greatly reduced the inhibitory activity, indicating that anti-RNA synthesis by the C protein is governed by amino acids scattered across its C-terminal half. Thus, amino acid sequence requirements differ greatly between IFN antagonism and RNA synthesis inhibition. In addition, we confirmed that another SeV accessory protein, V, does not antagonize IFN.
    Document Type:
    Reference
    Product Catalog Number:
    07-224
    Product Catalog Name:
    Anti-phospho-STAT2 (Tyr689) Antibody

  • AIP1/Alix is a binding partner of Sendai virus C protein and facilitates virus budding. 15994787

    The C protein, an accessory protein of Sendai virus (SeV), has anti-interferon capacity and suppresses viral RNA synthesis. In addition, it is thought that the C protein is involved in virus budding because of the low efficiency of release of progeny virions from C-knockout virus-infected cells and because of the requirement of the C protein for efficient release of virus-like particles. Here, we identified AIP1/Alix, a host protein involved in apoptosis and endosomal membrane trafficking, as an interacting partner of the C protein using a yeast two-hybrid system. The amino terminus of AIP1/Alix and the carboxyl terminus of the C protein are important for the interaction in mammalian cells. Mutant C proteins unable to bind AIP1/Alix failed to accelerate the release of virus-like particles from cells. Furthermore, overexpression of AIP1/Alix enhanced SeV budding from infected cells in a C-protein-dependent manner, while the release of nucleocapsid-free empty virions was also enhanced. Finally, AIP1/Alix depletion by small interfering RNA resulted in suppression of SeV budding. The results of this study suggest that AIP1/Alix plays a role in efficient SeV budding and that the SeV C protein facilitates virus budding through interaction with AIP1/Alix.
    Document Type:
    Reference
    Product Catalog Number:
    MAB1501
    Product Catalog Name:
    Anti-Actin Antibody, clone C4

  • Inducible epithelial resistance against acute Sendai virus infection prevents chronic asthma-like lung disease in mice. 31968123

    Respiratory viral infections play central roles in the initiation, exacerbation and progression of asthma in humans. An acute paramyxoviral infection in mice can cause a chronic lung disease that resembles human asthma. We sought to determine whether reduction of Sendai virus lung burden in mice by stimulating innate immunity with aerosolized Toll-like receptor (TLR) agonists could attenuate the severity of chronic asthma-like lung disease.
    Document Type:
    Reference
    Product Catalog Number:
    Multiple
    Product Catalog Name:
    Multiple

  • The STAT2 activation process is a crucial target of Sendai virus C protein for the blockade of alpha interferon signaling. 12610111

    Sendai virus (SeV) C protein functions as an interferon (IFN) antagonist and renders cells unresponsive to both alpha/beta IFN (IFN-alpha/beta) and IFN-gamma. We have recently found the physical association of the C protein with signal transducer and activator of transcription 1 (STAT1) in infected cells. However, involvement of the C-STAT1 interaction in the blockade of IFN signaling has remained unclear. We generated here a series of C mutant proteins that retained or lost the STAT1-binding capacity and examined their effects on IFN-alpha signaling. All of the C mutant proteins with no STAT1-binding capacity lost the ability to inhibit the IFN-alpha response. In contrast, the C mutant proteins retaining the STAT1-binding capacity suppressed IFN-alpha-stimulated tyrosine phosphorylation of both STAT2 and STAT1 to various degrees. Remarkably, their anti-IFN-alpha capacities correlated well with the inhibitory effect on phosphorylation of STAT2 rather than STAT1. In infected cells, the levels of tyrosine-phosphorylated (pY) STAT2 were below the detection level irrespective of duration of IFN-alpha stimulation, whereas the levels of pY-STAT1 strikingly increased after long-term IFN-alpha stimulation. These results suggest that the STAT2 activation process is a crucial target for the blockade of IFN-alpha signaling. An in vitro binding assay with extracts from (STAT1-deficient) U3A and (STAT1-expressing) U3A-ST1 cells suggested the requirement of STAT1 for the C-STAT2 interaction. Furthermore, expression of STAT1 enhanced the inhibitory effect of the C protein on STAT2 activation in U3A cells. The C protein thus appears to participate in the inhibitory process for STAT2 activation through the STAT1 interaction.
    Document Type:
    Reference
    Product Catalog Number:
    07-224
    Product Catalog Name:
    Anti-phospho-STAT2 (Tyr689) Antibody

  • Minimal features of efficient incorporation of the hemagglutinin-neuraminidase protein into sendai virus particles. 24155372

    Two transmembrane glycoproteins form spikes on the surface of Sendai virus, a member of the Respirovirus genus of the Paramyxovirinae subfamily of the Paramyxoviridae family: the hemagglutinin-neuraminidase (HN) and the fusion (F) proteins. HN, in contrast to F, is dispensable for viral particle production, as normal amounts of particles can be produced with highly reduced levels of HN. This HN reduction can result from mutation of an SYWST motif in its cytoplasmic tail to AFYKD. HNAFYKD accumulates at the infected cell surface but does not get incorporated into particles. In this work, we derived experimental tools to rescue HNAFYKD incorporation. We found that coexpression of a truncated HN harboring the wild-type cytoplasmic tail, the transmembrane domain, and at most 80 amino acids of the ectodomain was sufficient to complement defective HNAFYKD incorporation into particles. This relied on formation of disulfide-bound heterodimers carried out by the two cysteines present in the HN 80-amino-acid (aa) ectodomain. Finally, the replacement of the measles virus H cytoplasmic and transmembrane domains with the corresponding HN domains promoted measles virus H incorporation in Sendai virus particles.
    Document Type:
    Reference
    Product Catalog Number:
    MAB8905
    Product Catalog Name:
    Anti-Measles Blend Antibody, hemagglutinin, clones CV1, CV4

  • Role of matrix and fusion proteins in budding of Sendai virus. 11689619

    Paramyxoviruses are assembled at the surface of infected cells, where virions are formed by the process of budding. We investigated the roles of three Sendai virus (SV) membrane proteins in the production of virus-like particles. Expression of matrix (M) proteins from cDNA induced the budding and release of virus-like particles that contained M, as was previously observed with human parainfluenza virus type 1 (hPIV1). Expression of SV fusion (F) glycoprotein from cDNA caused the release of virus-like particles bearing surface F, although their release was less efficient than that of particles bearing M protein. Cells that expressed only hemagglutinin-neuraminidase (HN) released no HN-containing vesicles. Coexpression of M and F proteins enhanced the release of F protein by a factor greater than 4. The virus-like particles containing F and M were found in different density gradient fractions of the media of cells that coexpressed M and F, a finding that suggests that the two proteins formed separate vesicles and did not interact directly. Vesicles released by M or F proteins also contained cellular actin; therefore, actin may be involved in the budding process induced by viral M or F proteins. Deletion of C-terminal residues of M protein, which has a sequence similar to that of an actin-binding domain, significantly reduced release of the particles into medium. Site-directed mutagenesis of the cytoplasmic tail of F revealed two regions that affect the efficiency of budding: one domain comprising five consecutive amino acids conserved in SV and hPIV1 and one domain that is similar to the actin-binding domain required for budding induced by M protein. Our results indicate that both M and F proteins are able to drive the budding of SV and propose the possible role of actin in the budding process.
    Document Type:
    Reference
    Product Catalog Number:
    MAB1501
    Product Catalog Name:
    Anti-Actin Antibody, clone C4

  • Identification of amino acid positions associated with neuraminidase activity of the hemagglutinin-neuraminidase glycoprotein of Sendai virus. 1846501

    Identification of amino acid positions associated with neuraminidase activity on the hemagglutinin-neuraminidase (HN) glycoprotein of paramyxoviruses has been difficult because neuraminidase-inhibiting antibodies are not neutralizing and thus, escape mutants have not been isolated. Instead, many investigators have correlated an altered neuraminidase (NA) activity of natural virus variants, such as plaque-size variants, with sequence changes in the HN protein. To identify regions on the HN glycoprotein of Sendai virus (SV) that are associated with NA activity, we investigated NA activity of three plaque-size variants which potentially differed from the standard SV (SV/std). NA activity was measured by the ability of virus to elute from chicken erythrocytes as a result of cleaving sialic acid receptors, and by the ability of virus to cleave sialic acid from the small trisaccharide neuraminlactose and the larger substrate fetuin in an in vitro assay. Virions purified from each of the isolated plaques had a HN content and hemagglutinating activity similar to that of SV/std, yet each variant eluted much more rapidly from chicken erythrocytes than SV/std. In vitro NA activity of the plaque-size variants was 1.6 to 3.8 times greater than that of SV/std, providing supporting evidence for the elution data. Although all plaque-size variants showed elevated NA activity, there was no correlation of activity with plaque size. Sequence analysis showed that one of the variants had an amino acid change from glutamic acid to valine at position 165 and from lysine to glutamic acid at position 461, while a second variant had only the change at position 461. A third variant had a nearby change at position 468, from threonine to lysine. Taken together, these data support the conclusion that the amino acid residues at positions 461-468 and 165 are involved in neuraminidase activity of SV.
    Document Type:
    Reference
    Product Catalog Number:
    LP1
    Product Catalog Name:
    VLDL, human

  • Generation of induced pluripotent stem cells from a small amount of human peripheral blood using a combination of activated T cells and Sendai virus. 22422317

    Induced pluripotent stem cells (iPSCs) have become important cell sources for genetic disease models, and they have the potential to be cell sources for future clinical therapies. However, invasive tissue sampling reduces the number of candidates who consent to donate cells for iPSC generation. In addition, integrated transgenes can potentially insert at inappropriate points in the genome, and in turn have a direct oncogenic effect. Technical modifications using a combination of activated T cells and a temperature-sensitive mutant of Sendai virus (SeV) can avoid invasive tissue sampling and residual transgene issues in generating iPSCs. Such advances may increase the number of consenting patients for cell donations. Here we present a detailed protocol for the generation of iPSCs from a small amount of human peripheral blood using a combination of activated T cells and mutant SeV encoding human OCT3/4, SOX2, KLF4 and c-MYC; T cell-derived iPSCs can be generated within 1 month of blood sampling.
    Document Type:
    Reference
    Product Catalog Number:
    MAB4360
    Product Catalog Name:
    Anti-TRA-1-60 Antibody, clone TRA-1-60