Functional analysis of 2C protein in hepatitis A virus and poliovirus genome replication,

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Title Functional analysis of 2C protein in hepatitis A virus and poliovirus genome replication,
Publication Type dissertation
School or College School of Medicine
Department Pathology
Author Cho, Michael Wan
Date 1994-03
Description The growth properties of hepatitis A virus (HAV) in cultured cells and the biochemical properties of two viral proteins involved in genome replication, 2C and 3Dpol, were examined. The replication of HAV, unlike that of poliovirus and other picornaviruses, was nonlytic and characterized by a protracted growth cycle which terminated in a persistent infection. Replication was sensitive to millimolar concentrations of guanidine. Rapid completion of the replication cycle of HAV subsequent to reversal of guanidine inhibition demonstrated that a major factor in the apparant slow growth of the virus was asynchrony in replication. Biochemical analysis of recombinant 2C expressed in Escherichia coli was hampered by the high toxicity of the protein, low expression level, and marked insolubility. These problems were overcome by expressing the protein in either recombinant baculovirus or vaccinia virus systems, from which adequate amounts of soluble protein could be extracted by maintaining nonionic detergents in the preparation. The detergent, however, posed a subsequent problem in purification of the protein. Preliminary data showed that poliovirus 2C binds and hydrolyzes nucleoside triphosphates (NTP). No RNA helicase activity was detected. Unexpectedly, poliovirus 3Dpol exhibited an RNA duplex unwinding activity which was dependent on chain elongation, but not hydrolysis of the gamma-phosphate of NTP. Thus, the activity exhibited by 3D$/sp[/rm pol]$ is a strand displacement reaction, rather than an RNA helicase activity. Examination of human cells expressing poliovirus 2C or 2BC by immunofluorescence and electron microscopy demonstrated that these proteins, in the absence of other viral proteins, associated with specific cytoplasmic membranes. Additionally, they induced reorganization of membranes including formation of vesicles which morphologically resembled those found in poliovirus-infected cells. 2C, but not 2BC, induced the formation of tubular membrane structures with a myelin-like arrangement in the lumen of rough endoplasmic reticulum. This property of 2C was dependent on having intact NTP binding motifs in the protein. We speculate that these membrane reorganizations play a role in viral RNA replication.
Type Text
Publisher University of Utah
Subject Persistent Infection; Biochemical Analysis: Morphologically
Subject MESH Hepatovirus; Polioviruses; Hepatitis A; Hepatitis A virus
Dissertation Institution University of Utah
Dissertation Name PhD
Language eng
Relation is Version of Digital reproduction of "Functional analysis of 2C protein in hepatitis A virus and poliovirus genome replication". Spencer S. Eccles Health Sciences Library. Print version of "Functional analysis of 2C protein in hepatitis A virus and poliovirus genome replication". available at J. Willard Marriott Library Special Collection. QR6.5 1994 .C48.
Rights Management © Michael Wan Cho.
Format application/pdf
Format Medium application/pdf
Format Extent 5,305,076 bytes
Identifier undthes,4430
Source Original: University of Utah Spencer S. Eccles Health Sciences Library (no longer available).
Funding/Fellowship Genetics predoctoral training grant award from National Institutes of Health.
Master File Extent 5,305,134 bytes
ARK ark:/87278/s6251m31
Setname ir_etd
ID 191961
Reference URL https://collections.lib.utah.edu/ark:/87278/s6251m31
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