Studies on the Rabies Virus RNA Polymerase: 2. Possible Relationships between the Two Forms of the Non‐Catalytic Subunit (P Protein)

Abstract

We investigated the relationship between the two forms of rabies virus P protein, a non‐catalytic subunit of rabies virus RNA polymerase. The two displayed different electrophoretic mobilities as 37‐ and 40‐kDa polypeptides, hence termed as p37 and p40, respectively. Double labeling experiments with [3H]leucine and [32P]orthophosphate demonstrated that p40 was much more phosphorylated than p37. Treatment of the virion proteins with alkaline phosphatase eliminated only p40, and not 37‐kDa polypeptide. The p37 was a major product of the P gene, and was accumulated in the infected cell and incorporated into the virion. On the other hand, p40 was apparently detected only in the virion, and little detected in the cells. Treatment of infected cells with okadaic acid, however, resulted in significant accumulation of p40 in the cell, suggesting that p40 was continuously produced in the cell but dephosphorylated quickly. We detected both 37‐ and 40‐kDa products in P cDNA‐transfected animal cells, while only a 37‐kDa product was produced in Escherichia coli. Incubation of 37‐kDa products from E. coli with the lysates of animal cells in vitro resulted in the production of a 40‐kDa product, which was also shown to be suppressed by the heparin. From these results, it is suggested that p40 is produced by the hyperphosphorylation of a 37‐kDa polypeptide, which depends on certain heparin‐sensitive cellular enzyme(s) and occurs even in the absence of the other viral gene products, and that p40 is reverted quickly to p37 in the infected cells, probably being dependent on some virus‐induced factor(s).

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

“…In our previous study (31), p40 was suggested to be a hyperphosphorylated form of P protein based on the observation of its relatively much higher specific radioactivity than that of p37 in the double radiolabeling experiments with [ 32 P]orthophosphate and [ 3 H]leucine. The number of phosphate groups in each p37 molecule was calculated to be one or two (average was 1.4), while that of p40 was about ten.…”

“…For expression of the rabies virus P protein in E. coli, we used a fulllength P cDNA clone which was transferred into the NdeI site of an inducible expression vector pET3a (Novagen) as described previously (31). Expression was done in E. coli BL21(DE3) pLysS (Novagen) by adding isopropyl-β-D-thiogalactopyranoside (IPTG) at a final concentration of 1 mM according to Studier et al (30).…”

“…We recently investigated possible relationships between the two forms of full-sized rabies virus P protein (p37 and p40) (31). Although only a trace amount of p40 was detected in the cell, treatment of the infected cultures with okadaic acid (OKA) resulted in accumulation of p40, suggesting that p40 was continuously produced all the time, but dephosphorylated quickly in the cell.…”

“…Although only a trace amount of p40 was detected in the cell, treatment of the infected cultures with okadaic acid (OKA) resulted in accumulation of p40, suggesting that p40 was continuously produced all the time, but dephosphorylated quickly in the cell. In vitro phosphorylation assays strongly suggested that p37 serves as a precursor for p40 production, which was shown to occur even in the absence of the other viral gene products and was suggested to be catalyzed by cellular heparin-sensitive enzyme(s) (31). More recently, Gupta et al (14) suggested that a cellular heparin-sensitive 71-kDa kinase (termed the rabies virus protein kinase; RVPK) is involved in p40 production, while a staurosporine-sensitive kinase (PKC gamma isomer) is involved in the phosphorylation of p37 without affecting the electrophoretic mobility.…”

“…As to the heterogeneity of the rabies virus P protein, we reported previously that, in addition to a major form (37 kDa) of the P protein, the virion contained another minor, but full-sized, component which was recognized by anti-P antibody and termed p40 according to its relative electrophoretic mobility in SDS-PAGE (16,31). The p40 was very little in amount in the cell, and was more phosphorylated than a major 37-kDa component (p37) of the P protein (6,20,32).…”