Establishment of an artificial replication system for influenza virus and studies on the mechanism of the antiviral activities of the murine Mx1 protein on influenza virus.
Item
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Title
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Establishment of an artificial replication system for influenza virus and studies on the mechanism of the antiviral activities of the murine Mx1 protein on influenza virus.
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Identifier
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AAI9224822
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identifier
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9224822
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Creator
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Huang, Taosheng.
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Contributor
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Adviser: Mark Krystal
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Date
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1992
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Language
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English
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Publisher
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City University of New York.
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Subject
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Biology, Microbiology | Biology, Molecular | Engineering, Biomedical
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Abstract
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Until recently, influenza virus, as a negative sense RNA virus, had not been adaptable to many of the powerful molecular techniques available today. However, during the past few years, Dr. Palese and his colleagues in collaboration with Dr. Krystal have developed a system to study influenza virus replication and packaging. In their experiments, a DNA vector was used to produce an influenza virus-like RNA (IVACAT-1) in vitro. This RNA product has the 3{dollar}\sp\prime{dollar} and 5{dollar}\sp\prime{dollar} noncoding region of the NS gene flanking the antisense sequences of a coding region of the chloramphenicol acetyltransferase (CAT) gene. When mixed with purified polymerase prepared from influenza virus and transfected into cells, the RNA can be replicated and packaged by superinfecting influenza virus. This system now makes analysis of regulatory sequences in RNA feasible. However, the need for superinfecting influenza virus make it difficult to examine the functions of the individual viral proteins involved. To address this, a recombinant vaccinia virus-driven replication system for influenza virus was developed, in which the synthetic influenza virus like RNA (IVACAT-1) was replicated and expressed by influenza virus helper proteins supplied through infection with recombinant vaccinia viruses. We found that the minimum subset of influenza virus proteins needed for specific replication and expression of the ribonucleoprotein was the three polymerase proteins (PB2, PB1, and PA) and the nucleoprotein (NP) and that addition of NS1 and NS2 did not have significant effects on replication and transcription in this system.;In the second part of this thesis, the antiviral mechanism of the murine Mx1 protein was studied. The murine Mx1 protein is an interferon-inducible protein which confers to cells the selective resistance to influenza virus infection. Using the recombinant vaccinia virus-driven replication system, we found that IVACAT-1 RNP was expressed in Mx1 expressing cells infected with recombinant vaccinia viruses expressing influenza polymerase proteins (PB2, PB1, and PA) and NP.;In the third part of this thesis, we constructed a vector to express M1 protein of influenza virus. Expression of M1 protein was obtained at very high levels. Purification of M1 protein from bacteria was achieved through its solubility in acidic chloroform:methanol solution. Antibody raised against bacteria expressed M1 protein specifically recognized the matrix protein from influenza virus infected cellular extract. With this antibody, the intracellular localization was examined. We found that the M protein migrated to the nucleus during the course of virus infection. Bacterial-expressed M1 protein is able to inhibit viral polymerase activity in in vitro transcription reactions. Availability of antibody and a large amount of bacterial-expressed M1 protein should allow further examination of the function of the M1 protein and the dissection of functional domains.
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Type
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dissertation
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Source
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PQT Legacy CUNY.xlsx
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degree
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Ph.D.
