Identification and characterization of a monoclonal antibody to an antigen expressed on activated macrophages.

Koestler, Thomas P.; Rieman, David J.; Muirhead, Kathy; Greig, Russell; Poste, George

doi:10.1073/pnas.81.14.4505

Cited by 25 publications

(16 citation statements)

References 35 publications

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“…Mouse anti-M1 monoclonal antibody was generated as described previously (18). Rabbit anti-NP polyclonal antibody was produced by immunizing a mouse with purified His-NP, as described previously (24).…”

Section: Cells and Reagentsmentioning

confidence: 99%

A Nuclear Export Signal in the Matrix Protein of Influenza A Virus Is Required for Efficient Virus Replication

Cao

Liu

et al. 2012

J Virol

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The influenza A virus matrix 1 protein (M1) shuttles between the cytoplasm and the nucleus during the viral life cycle and plays an important role in the replication, assembly, and budding of viruses. Here, a leucine-rich nuclear export signal (NES) was identified specifically for the nuclear export of the M1 protein. The predicted NES, designated the Flu-A-M1 NES, is highly conserved among all sequences from the influenza A virus subtype, but no similar NES motifs are found in the M1 sequences of influenza B or C viruses. The biological function of the Flu-A-M1 NES was demonstrated by its ability to translocate an enhanced green fluorescent protein (EGFP)-NES fusion protein from the nucleus to the cytoplasm in transfected cells, compared to the even nuclear and cytoplasmic distribution of EGFP. The translocation of EGFP-NES from the nucleus to the cytoplasm was not inhibited by leptomycin B. NES mutations in M1 caused a nuclear retention of the protein and an increased nuclear accumulation of NEP during transfection. Indeed, as shown by rescued recombinant viruses, the mutation of the NES impaired the nuclear export of M1 and significantly reduced the virus titer compared to titers of wild-type viruses. The NES-defective M1 protein was retained in the nucleus during infection, accompanied by a lowered efficiency of the nuclear export of viral RNPs (vRNPs). In conclusion, M1 nuclear export was specifically dependent on the Flu-A-M1 NES and critical for influenza A virus replication. Influenza A virus is a negative-strand RNA virus composed of eight segmented strands of an RNA genome (21). The viral structural components include the viral envelope, the transmembrane proteins (hemagglutinin [HA], neuraminidase [NA], and M2), M1, NS2, and the viral ribonucleoprotein (vRNP), which contains viral RNA (vRNA), nucleoprotein (NP), and viral polymerase proteins (PB1, PB2, and PA) (44).M1 is the most abundant protein in the virus particle, sustaining the virion structure by forming a shell connecting the viral envelope and the nucleocapsid (40). M1 is synthesized in the late stages of infection, shuttles between the nucleus and the cytoplasm (45), and plays multiple roles in various steps of the influenza virus life cycle.Newly synthesized M1 is transported from the cytoplasm into the nucleus via a nuclear localization signal (NLS) located in its N-terminal domain (residues 101 to 105) (38, 48). In the nucleus, M1 is involved in the blocking of the transcription of viral mRNA by binding to vRNPs (4, 49). Aside from terminating viral transcription, M1 also plays an important role in the nuclear export of vRNPs. Indeed, the nuclear presence of M1 is required for vRNPs to be transported to the cytoplasm (8, 27), where the vRNPs are subsequently transported to the budding site. First, M1 binds to histones in the nucleus (51) and may participate in releasing vRNPs from the nuclear matrix. Second, M1 likely bridges the NEP and vRNPs, forming a complex that is in turn exported from the nucleus by the nuclear export signal (NES) ...

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Section: Cells and Reagentsmentioning

confidence: 99%

A Nuclear Export Signal in the Matrix Protein of Influenza A Virus Is Required for Efficient Virus Replication

Cao

Liu

et al. 2012

J Virol

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“…Several monoclonal antibodies specific for murine macrophage-surface antigens have been reported (12,19,(20)(21)(22). Some of these were directed against activated macrophages (12,19,21,22).…”

Section: Discussionmentioning

confidence: 99%

“…Some of these were directed against activated macrophages (12,19,21,22). In this study we established two hybridomas: B12 monoclonal antibody reacted with all macrophages, while C14 antibody reacted well with resident and thioglycollate-, glycogen-, and peptone-elicited macrophages, but little with BCG-and glucan-elicited macrophages, which mediate LDMC with loach egg lectin and release a cytotoxin (Table 2).…”

Section: Discussionmentioning

confidence: 99%

Antigens Associated with Various Cytotoxic Activities of Murine Peripheral Macrophages

Okutomi

Satoh

Oshima

et al. 1986

Microbiology and Immunology

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BCG-or glucan-elicited murine peripheral macrophages released a cytotoxin in the presence of loach egg lectin, whereas proteose peptone-, glycogen-, or thioglycollate-elicited or resident macrophages did not. The macrophages that released cytotoxin coincided with those that showed lectin-dependent macrophagemediated cytolysis (LDMC) in response to loach egg lectin.The cytotoxin released by BCG-elicited macrophages in response to loach egg lectin had a molecular weight of 55 K daltons.The macrophages that released cytotoxin and other cytotoxic macrophages such as those that showed LDMC-and antibody-dependent macrophage-mediated cytolysis (ADMC) were examined by using several antibodies to surface antigens of macrophages.The results showed that murine peripheral macrophages could be divided into three types. Resident macrophages (Type I) which had common macrophage antigens (Mac-1 and B12) showed only LDMC in response to wheat germ agglutinin. Some elicited macrophages (Type II) were asialo GM1-positive and showed both ADMC and LDMC in response to wheat germ agglutinin. Activated macrophages (Type III) showed LDMC in response to loach egg lectin and cytotoxin-release, but had no antigen detectable with monoclonal anti-macrophage antibody (C14). These three types of macrophages were clearly distinguished diagrammatically by their roof-shaped, rocket-shaped and irregular-shaped profiles of activities and antigens.These data suggest that several selected surface antigens of macrophages are associated with distinct cytotoxic stages of peripheral macrophages.Macrophages are important effector cells in recognition and destruction of tumor cells (2, 9). We have been studying mediator-dependent macrophagemediated cytolysis in a murine syngeneic system (24, 25). We found that there are two types of mediator-dependent cytolysis : antibody-dependent macrophage-mediated cytolysis (ADMC) with strict specificity (28, 29) and lectin-dependent macrophage-mediated cytolysis (LDMC) with broad specificity (14). Moreover, we showed that the mechanisms of these two types of cytotoxicity are different not only in their recognition steps, but also in their cytolytic processes (7).Recently we found that an animal lectin purified from loach eggs can induce cytolysis in cooperation with BCG-elicited macrophages (27) and that a soluble cytotoxin participates in target cell killing (17). It is unknown at which stage of 1037

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“…Anti-NP rabbit polyclonal antibody and anti-M1 mouse monoclonal antibody were obtained by immunizing animals with hexahistidinetagged NP and M1 as previously described (23,24). Anti-c-myc (9E10) and anti-FLAG (M2) mouse monoclonal antibodies were purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA) and Sigma-Aldrich (St. Louis, MO), respectively.…”

Section: Methodsmentioning

confidence: 99%

Characteristics of Nucleocytoplasmic Transport of H1N1 Influenza A Virus Nuclear Export Protein

Gao

Wang

Cao

et al. 2014

J Virol

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The influenza A virus nuclear export protein (NEP) plays crucial roles in the nuclear export of the viral ribonucleoprotein complex through the chromosome region maintenance 1 (CRM1)-mediated cellular protein transport system. However, the detailed mechanism of NEP nucleocytoplasmic trafficking remains incompletely understood. Here, we investigated the subcellular localization of NEP from two strains of H1N1 influenza A virus and found that 2009 swine-origin H1N1 influenza A virus A/California/04/2009 (CA04) NEP displayed a distinct cellular distribution pattern, forming unique nuclear aggregates, compared to A/WSN/33 (H1N1) (WSN) NEP. Characterization of the nucleocytoplasmic transport pathways of these two NEPs showed that they both enter the nucleus by passive diffusion but are exported through the nuclear export receptor CRM1-mediated pathway with different efficiencies. The two identified nuclear export signals (NESs) on the two NEPs functioned similarly despite differences in their amino acid sequences. Using a two-hybrid assay, we confirmed that the CA04 NEP interacts less efficiently with CRM1 and that a threonine residue at position 48 is responsible for the nuclear aggregation. The present study revealed the dissimilarity in subcellular NEP transport processes between the 2009 pandemic (H1N1) influenza A virus CA04 and the laboratory-adapted H1N1 virus WSN and uncovered the mechanism responsible for this difference. IMPORTANCEBecause the efficiency of the nucleocytoplasmic transport of viral components is often correlated with the viral RNA polymerase activity, propagation, and host range of influenza viruses, the present study investigated the subcellular localization of NEP from two strains of H1N1 influenza virus. We found that the NEPs of both A/California/04/2009 (H1N1) (CA04) and A/WSN/33 (H1N1) (WSN) enter the nucleus by passive diffusion but are exported with different efficiencies, which were caused by weaker binding activity between the CA04 NEP and CRM1. The results of the present study revealed characteristics of the nuclear import and export pathways of NEP and the mechanism responsible for the differences in the cellular distribution of NEP between two H1N1 strains.

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Identification and characterization of a monoclonal antibody to an antigen expressed on activated macrophages.

Cited by 25 publications

References 35 publications

A Nuclear Export Signal in the Matrix Protein of Influenza A Virus Is Required for Efficient Virus Replication

A Nuclear Export Signal in the Matrix Protein of Influenza A Virus Is Required for Efficient Virus Replication

Antigens Associated with Various Cytotoxic Activities of Murine Peripheral Macrophages

Characteristics of Nucleocytoplasmic Transport of H1N1 Influenza A Virus Nuclear Export Protein

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