Expression of a recombinant DNA gene coding for the vesicular stomatitis virus nucleocapsid protein

Sprague, Judy; Condra, Jon H.; Arnheiter, H; Lazzarini, Robert A.

doi:10.1128/jvi.45.2.773-781.1983

Cited by 164 publications

(56 citation statements)

References 24 publications

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“…Bacterially expressed NP protein of influenza virus was shown to bind well to both viral and nonviral RNA substrates when assayed in vitro [4]. The N protein of VSV bound tightly to cellular RNAs when synthesized in the absence of other viral proteins in an in vivo expression system [28], and it bound to endogenous reticulocyte RNAs or added RNA species when expressed by in vitro translation [16]. The measles virus NP protein, expressed in vivo by a vaccinia virus recombinant, has recently been shown to assemble into structures very similar to authentic measles nucleocapsids, as determined by both electron microscopy and equilibrium sedimentation on CsC1 gradients [27].…”

Section: Discussionmentioning

confidence: 99%

Localization of an RNA-binding domain in the nucleocapsid protein of the coronavirus mouse hepatitis virus

Masters

1992

Archives of Virology

109

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The interaction between the nucleocapsid (N) protein of mouse hepatitis virus (MHV) and RNA was studied in an effort to define portions of the N molecule that participate in binding to RNA. N mRNAs transcribed from SP6 and T7 vectors were translated in a rabbit reticulocyte lysate. Analysis of synthesized N protein in a nondenaturing gel system showed that it bound in vitro to an endogenous RNA in the reticulocyte lysate but not to its own mRNA. A set of deletion mutants was constructed in order to localize the RNA-binding activity of the N protein. It was found that removal of as much as 135 amino-terminal or 57 carboxy-terminal amino acids from the molecule had little or no effect on RNA binding. Moreover, deletion mutants lacking both termini still retained RNA-binding ability. By contrast, internal deletions or truncations extending beyond these two limits effectively abolished RNA binding by N protein. Thus, the RNA-binding region of N has been mapped to the second (central) of the three structural domains of the molecule.

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Section: Discussionmentioning

confidence: 99%

Localization of an RNA-binding domain in the nucleocapsid protein of the coronavirus mouse hepatitis virus

Masters

1992

Archives of Virology

109

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“…Encapsidation competent N synthesized in a cell-free system was not sufficient for extensive structure-function analysis (Patton et al 1983). Native viral protein obtained after dissociating from RNA (Blumberg et al 1984) or recombinant protein obtained by over-expressing within eukaryotic cellular milieu (Sprague et al 1983) forms aggregates. Bacterially over-expressed protein, recovered by denaturation/ refolding, required high salt to prevent precipitation, therefore, compromising the scope for wide biochemical applications ).…”

Section: Chandipura Virus N Protein and Encapsidationmentioning

confidence: 99%

Reviewing Chandipura: A Vesiculovirus in Human Epidemics

et al. 2007

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Chandipura virus, a member of the rhabdoviridae family and vesiculovirus genera, has recently emerged as human pathogen that is associated with a number of outbreaks in different parts of India. Although, the virus closely resembles with the prototype vesiculovirus, Vesicular Stomatitis Virus, it could be readily distinguished by its ability to infect humans. Studies on Chandipura virus while shed light into distinct stages of viral infection; it may also allow us to identify potential drug targets for antiviral therapy. In this review, we have summarized our current understanding of Chandipura virus life cycle at the molecular detail with particular interest in viral RNA metabolisms, namely transcription, replication and packaging of viral RNA into nucleocapsid structure. Contemporary research on otherwise extensively studied family member Vesicular Stomatitis Virus has also been addressed to present a more comprehensive picture of vesiculovirus life cycle. Finally, we reveal examples of protein economy in Chandipura virus life-cycle whereby each viral protein has evolved complexity to perform multiple tasks.

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“…The plasmid designated JC 1 19 is an SV40-based expression vector obtained from Dr. J. Rose (Yale University) and has been described previously Bergmann, 1982, 1983;Sprague et al, 1983). The plasmid designated pMBP1, containing cDNA for rat 14K MBP, was obtained from Dr. L. Hood (Caltech) and has been described previously (Roach et al, 1983).…”

Section: Plasmidsmentioning

confidence: 99%

“…The cDNA for rat 14K MBP was excised from the plasmid pMBP-1 (Roach et al, 1983) with EcoRI. The ends of the cDNA were made blunt with DNA polymerase, BglII linkers were added, and the linked cDNA was ligated with BarnHI-digested JC119, which contains the SV40 origin of replication, late promoter, splice signals, and polyadenylation site (Sprague et al, 1983). The orientation and arrangement of MBP cDNA sequences in the recombinant plasmids, shown in Fig.…”

Section: Construction Of Mbp Expression Vectormentioning

confidence: 99%

Expression and Localization of Myelin Basic Protein in Oligodendrocytes and Transfected Fibroblasts

Barbarese

Barry

Chou

et al. 1988

Journal of Neurochemistry

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Myelin basic protein (MBP) is a major structural component of myelin. It is expressed exclusively in myelinating glia (oligodendrocytes in the CNS and Schwann cells in the PNS) and is localized to the cytoplasmic surface of the plasma membrane and myelin membrane produced by these cells. The work described here concerns the mechanism of plasma membrane localization of MBP in myelinating glial cells and whether it involves differentiated functions specific to these cells or general functions of plasma membrane assembly common to all cells. To this end, the subcellular localization of endogenous MBP in mouse oligodendrocytes was compared with that of transiently expressed MBP in monkey fibroblasts (Cos-1 cells) transfected with an MBP expression vector containing cDNA for rat 14K MBP. The steady-state levels of MBP-specific RNA and of MBP polypeptide expressed in the transfected fibroblasts were comparable to the levels expressed in oligodendrocytes in primary culture. MBP localization was analyzed in whole cells by immunofluorescence and in specific intracellular compartments by subcellular fractionation. The results show that MBP expressed in wild-type oligodendrocytes is localized to the plasma membrane. In contrast, MBP expressed in transfected fibroblasts appears dispersed in the cytoplasm and is distributed uniformly among the various subcellular fractions.(ABSTRACT TRUNCATED AT 250 WORDS)

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Expression of a recombinant DNA gene coding for the vesicular stomatitis virus nucleocapsid protein

Cited by 164 publications

References 24 publications

Localization of an RNA-binding domain in the nucleocapsid protein of the coronavirus mouse hepatitis virus

Localization of an RNA-binding domain in the nucleocapsid protein of the coronavirus mouse hepatitis virus

Reviewing Chandipura: A Vesiculovirus in Human Epidemics

Expression and Localization of Myelin Basic Protein in Oligodendrocytes and Transfected Fibroblasts

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