Studies on the Rabies Virus RNA Polymerase: 3. Two‐Dimensional Electrophoretic Analysis of the Multiplicity of Non‐Catalytic Subunit (P Protein)

Abstract

The genome of rabies virus encodes two kinds of phosphoproteins; one is the nucleoprotein (N) which encapsidates viral genomic RNA, and the other is a nominal phosphoprotein (P; formerly called M1), that is thought to work multifunctionally for viral RNA synthesis. Newly synthesized N proteins are mostly associated with P proteins, which is thought to be a prerequisite step for the N protein to be used for encapsidating viral genomic and anti-genomic RNAs specifically. Phosphorylation of the N protein occurs during or after the encapsidation (20). The P protein also works for viral RNA synthesis with the viral large (L) protein (catalytic subunit of the viral RNA polymerase), but the role(s) of the P protein and the mechanism of its phosphorylation remained to be elucidated precisely (18,33).The P protein of another rhabdovirus family (vesicular stomatitis virus; VSV) has been studied extensively (see a review by Banerjee and Barik; 1). The P protein was shown to serve as a molecular chaperone for the viral 32 P]phosphate implied that p40 was a hyperphosphorylated form. We further examined here these proteins by two-dimensional (2-D) gel electrophoresis and immunoblotting, showing that a major component, p37, was composed of multiply modified subcomponents of different pIs (termed p37-1, p37-2, p37-3, etc., based on their acidity) in the virion and infected cells, but the unmodified precursor (termed p37-0) was little in amount. The viral nucleocapsid (NC)-bound P proteins were composed of multiple forms of p37 (the major one was p37-1) and also a minor component, p40-1. P proteins which were bound to newly synthesized free N proteins were mostly composed of p37-1, indicating that hyperphosphorylation of P proteins occurred after their being used for the encapsidation. Treatment of the infected cells with okadaic acid induced accumulation of the more acidic forms of P proteins, suggesting that heterogeneity in the full-sized P proteins is a reflection of their dynamic aspects of multiple cycles of phosphorylations and dephosphorylations in the cell. Two-D gel analyses demonstrated also that p40 was not so acidic as we expected, and implied that our previous data of apparent hyperphosphorylation of p40 was due to very frequently recycled utilization of the protein, and preformed non-labeled P proteins were also 32 P-phosphorylated in a radiolabeling period and were converted to the p40.Key words: rabies virus P protein, multiple components of P protein, non-catalytic subunit of rabies virus RNA polymerase, okadaic acid, phosphorylation and dephosphorylation, isoelectric focusing, 2-D gel electrophoresis

Further Studies on the Hyperphosphorylated Form (p40) of the Rabies Virus Nominal Phosphoprotein (P)

“…Two-D gel electrophoresis was performed as described previously (6). In brief, cells were dissolved in isoelectric focusing (IEF) buffer (9.5 M urea, 2% Triton X-100, 5% 2-mercaptoethanol, 2% Pharmalyte, pIϭ4.5-5.4), and applied to the gels prepared in capillary tubes for the first dimensional separation and, after a brief run (10 min) at 500 V, IEF was performed for 3.5 hr at 750 V. Then, the first dimensional IEF gel was taken out from each capillary tube and put onto the top of SDS-PAGE slab gel for the second dimensional separation.…”

“…The amount of p40 in the cell was very little, but became detectable when the infected cells were incubated in the presence of phosphatase inhibitor (okadaic acid; OKA) (6,17). Our previous two-dimensional gel electrophoretic studies revealed that p40 was not as phosphorylated as expected, but rather it was implied that p40 was very quickly dephosphorylated to p37 and recycled rapidly and repeatedly (6).…”

“…ly found as the NC-associated form in the cell as well as in the virion, while the soluble free p40 was little in amount (6,17). The amount of p40 in the cell was very little, but became detectable when the infected cells were incubated in the presence of phosphatase inhibitor (okadaic acid; OKA) (6,17).…”

“…Furthermore, although we could detect the p40 in the cell even in the presence of staurosporine (our unpublished data), we cannot tell whether such p40 was the same one that was produced in the absence of the agent, which might be elucidated by two-dimensional (2-D) gel electrophoresis. In addition, it remained yet unclear whether the precursor of p40 is the unphosphorylated form or the phosphorylated form of p37 (6,17).…”

“…The rabies virus P protein is composed of subcomponents that show different electrophoretic mobilities in SDS-PAGE; one such component is a 40-kDa minor component (p40) and others are 37-kDa major components (p37) of different pIs (5,6,17,20 Abstract: We investigated possible mechanisms involved in production of a hyperphosphorylated form (p40) of rabies virus P protein, to which two dimensional (2-D) gel electrophoresis was applied. The P gene products produced in Escherichia coli cells could be detected as a single spot of unphosphorylated 37-kDa form (termed as p37-0) in a 2-D gel.…”