Characterization of Nora Virus Structural Proteins via Western Blot Analysis

Abstract

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.

Exploring Aβ Proteotoxicity and Therapeutic Candidates Using Drosophila melanogaster

“…Protein levels ( Figure 1 D): When studying proteotoxicity, it is of interest to investigate the levels of toxic proteins in the flies. This is commonly carried out by a Western blot, which is a useful method to analyze the presence of various proteins in a crude tissue sample by relying on specific antibodies for detection [ 14 , 25 ]. Additionally, an estimate of the protein concentration is generated by comparing the intensity of the migrated bands.…”

Analysis of immune-related genes during Nora virus infection of <em>Drosophila melanogaster</em> using next generation sequencing

“…ORF2 encodes a replicative cassette, which consists of a helicase, protease and RNA-dependent RNA polymerase [1] . ORF3 encodes VP3 and is essential for the stability of the viral capsid during environmental stress, such as heat or protease exposure [5] , [6] . ORF4 encodes VP4, which is processed into three major proteins: VP4A, VP4B and VP4C [7] .…”

In vitro assembly and evaluation of Nora virus VLPs

“…The milk/TBST mixture was removed and fresh 5% non-fat dry milk/TBST with primary antibody (anti-Nora virus, 1:500 dilution) was added for 3 hours and incubated at room temperature. Preparation of the primary antibody was described in Ericson et al, 2016. Detection was achieved with the secondary antibody, goat antimouse IgG Fc alkaline phosphatase conjugate (1:5,000 dilution, Pierce) and developed with NBT/BCIP (Pierce).…”

“…ORF 4 encodes approximately 931 amino acids and consists of a polyprotein, which is cleaved proteolytically to make the major structural proteins (Ekström et al, 2011). These viral proteins are VP4A (37 kDa), VP4B (32 kDa), and VP4C (49 kDa) (Ericson et al, 2016). Even though VP3, VP4A, VP4B, and VP4C are components of the virus, there is little known as to how the capsid assembles and the importance played by these proteins in viral structure and/or stability.…”