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

Abstract

The rabies virus nominal phosphoprotein (P) is a virus genome-encoded molecular chaperone, which helps the viral nucleoprotein (N) to specifically and correctly encapsidate the viral genome-sized RNA (21). Before encapsidating the viral RNA, the N protein is complexed with the P protein in a certain manner (13,14). During the encapsidation process, the P protein changes its N-binding site from the N-terminal side to the C-terminal, and also changes its conformation (7,18). The P protein binding to the nucleocapsid (NC) seemed to be enhanced and/or stabilized by phosphorylation of the N protein (19). The P protein is also thought to collaborate with the viral catalytic large (L) protein in viral RNA synthesis (11). For this, the NCassociated P protein might mediate the L protein binding to the NC as suggested for the assembly of RNA synthetic apparatus of some other negative-stranded RNA viruses (3, 9, 15).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. The 37-kDa proteins in the virus-infected cells are composed of some phosphorylated forms, including a major p37-1 and more phosphorylated minor forms (e.g., p37-2, p37-3, etc.), but little p37-0 is detected (Eriguchi et al., 2002). When the E. coli-produced P protein analogues were incubated with BHK-21 cell lysates, heparin-sensitive phosphorylation occurred as described previously (Takamatsu et al., 1998), giving an additional 40-kDa spot. However, such a p40-like derivative displayed a little more basic pI value than that of the authentic p40 produced in the infected cells; hence, the former was termed p40-0 (pIϭ4.78), while the latter, p40-1 (pIϭ4.73). In contrast, p40 produced in the P cDNAtransfected animal cell was detected at the p40-1 position. In addition, staurosporine did not affect the p40-1 production in virus-infected nor the P cDNA-transfected animal cells, while the agent reduced production of hyperphosphorylated forms of p37, resulting in accumulation of p37-1, but not of p37-0. These results suggest that, although p37-0 may become a substrate for the heparin-sensitive protein kinase (PK) in vitro, only p37-1 is a substrate for p40 production catalyzed by heparin-sensitive PK in animal cells, and staurosporine-sensitive PK is involved in the production of more phosphorylated forms of p37, but not in p37-1 production from p37-0.

Structural Difference Recognized by a Monoclonal Antibody #404‐11 between the Rabies Virus Nucleocapsid (NC) Produced in Virus Infected Cells and the NC‐Like Structures Produced in the Nucleoprotein (N) cDNA‐Transfected Cells

“…After brief centrifugation [10 min at 5,000 rpm (Լ5,000ϫg)] in a microfuge, the lysates were applied to ultra-centrifugation at 46,000 rpm (200,000ϫg) for 2 hr through a 20% sucrose cushion. The NC pellet was subjected to treatment with alkaline phosphatase as described previously (22). In brief, the pellet was lysed in an alkaline buffer (pH 9.0) containing 1% NP-40, and divided into two equal parts.…”

Development and evaluation of an anti-rabies virus phosphoprotein-specific monoclonal antibody for detection of rabies neutralizing antibodies using RFFIT

“…P protein is a multi-functional protein interacting with N (N-P) and is a key component and regulatory protein of the P-L complex for viral genome replication [ 17 ]. P protein is detected in both the virus and virus-infected host cells, in two forms: a major hypo-phosphorylated 37-kDa form and a minor hyper-phosphorylated 40-kDa form [ 18 ]. mAbs targeting P protein have been developed in some studies [ 19 , 20 ].…”