TY - JOUR
T1 - Characterization of Nora Virus Structural Proteins via Western Blot Analysis
AU - Ericson, Brad L.
AU - Carlson, Darby J.
AU - Carlson, Kimberly A.
N1 - Funding Information:
The authors thank Drs. Hultmark and Ekströmfor their gift of the original Nora virus stocks. In addition, they thank Angela Wang, Andrew Prososki, Morgan Abert, Kirsten Lipps, Tad Fuchs, and Alexis Page for technical help and Tom Bargar of the UNMC Electron Microscopy Center for his work. The research was supported by grants to K. A. Carlson from the National Center for Research Resources (5P20RR016469) and the National Institute for General Medical Science (8P20GM103427), a component of the National Institutes of Health.
Publisher Copyright:
© 2016 Brad L. Ericson et al.
PY - 2016
Y1 - 2016
N2 - Nora virus is a single stranded RNA picorna-like virus with four open reading frames (ORFs). The coding potentials of the ORFs are not fully characterized, but ORF3 and ORF4 are believed to encode the capsid proteins (VP3, VP4a, VP4b, and VP4c) comprising the virion. To determine the polypeptide composition of Nora virus virions, polypeptides from purified virus were compared to polypeptides detected in Nora virus infected Drosophila melanogaster. Nora virus was purified from infected flies and used to challenge mice for the production of antisera. ORF3, ORF4a, ORF4b, and ORF4c were individually cloned and expressed in E. coli; resultant recombinant proteins purified and were used to make monospecific antisera. Antisera were evaluated via Western blot against whole virus particles and Nora virus infected fly lysates. Viral purification yielded two particle types with densities of ∼1.31 g/mL (empty particles) and ∼1.33 g/mL (complete virions). Comparison of purified virus polypeptide composition to Nora virus infected D. melanogaster lysate showed the number of proteins in infected cell lysates is less than purified virus. Our results suggest the virion is composed of 6 polypeptides, VP3, VP4a, two forms of VP4b, and two forms of VP4c. This polypeptide composition is similar to other small RNA insect viruses.
AB - Nora virus is a single stranded RNA picorna-like virus with four open reading frames (ORFs). The coding potentials of the ORFs are not fully characterized, but ORF3 and ORF4 are believed to encode the capsid proteins (VP3, VP4a, VP4b, and VP4c) comprising the virion. To determine the polypeptide composition of Nora virus virions, polypeptides from purified virus were compared to polypeptides detected in Nora virus infected Drosophila melanogaster. Nora virus was purified from infected flies and used to challenge mice for the production of antisera. ORF3, ORF4a, ORF4b, and ORF4c were individually cloned and expressed in E. coli; resultant recombinant proteins purified and were used to make monospecific antisera. Antisera were evaluated via Western blot against whole virus particles and Nora virus infected fly lysates. Viral purification yielded two particle types with densities of ∼1.31 g/mL (empty particles) and ∼1.33 g/mL (complete virions). Comparison of purified virus polypeptide composition to Nora virus infected D. melanogaster lysate showed the number of proteins in infected cell lysates is less than purified virus. Our results suggest the virion is composed of 6 polypeptides, VP3, VP4a, two forms of VP4b, and two forms of VP4c. This polypeptide composition is similar to other small RNA insect viruses.
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U2 - 10.1155/2016/9067848
DO - 10.1155/2016/9067848
M3 - Article
C2 - 27298753
AN - SCOPUS:85019135628
SN - 2090-908X
VL - 2016
JO - Scientifica
JF - Scientifica
M1 - 9067848
ER -