Expression of MIP-1alpha (CCL3) by a recombinant rabies virus enhances its immunogenicity by inducing innate immunity and recruiting dendritic cells and B cells.

Previously, we showed that overexpression of MIP-1alpha in mouse brain further decreased rabies virus (RABV) pathogenicity (L. Zhao, H. Toriumi, Y. Kuang, H. Chen, and Z. F. Fu, J. Virol., 83:11808-11818, 2009). In the present study, the immunogenicity of recombinant RABV expressing MIP-1alpha (rHEP-MIP1alpha) was determined. It was found that intramuscular immunization of BALB/c mice with rHEP-MIP1alpha resulted in a higher level of expression of MIP-1alpha at the site of inoculation, increased recruitment of dendritic cells (DCs) and mature B cells into the draining lymph nodes and the peripheral blood, and higher virus-neutralizing antibody titers than immunization with the parent rHEP and recombinant RABVs expressing RANTES (CCL5) or IP-10 (CXCL10). Our data thus demonstrate that expression of MIP-1alpha not only reduces viral pathogenicity but also enhances immunogenicity by recruiting DCs and B cells to the site of immunization, the lymph nodes, and the blood.

The roles of chemokines in rabies virus infection: overexpression may not always be beneficial.

It was found previously that induction of innate immunity, particularly chemokines, is an important mechanism of rabies virus (RABV) attenuation. To evaluate the effect of overexpression of chemokines on RABV infection, chemokines macrophage inflammatory protein 1alpha (MIP-1alpha), RANTES, and IP-10 were individually cloned into the genome of attenuated RABV strain HEP-Flury. These recombinant RABVs were characterized in vitro for growth properties and expression of chemokines. It was found that all the recombinant viruses grew as well as the parent virus, and each of the viruses expressed the intended chemokine in a dose-dependent manner. When these viruses were evaluated for pathogenicity in the mouse model, it was found that overexpression of MIP-1alpha further decreased RABV pathogenicity by inducing a transient innate immune response. In contrast, overexpression of RANTES or IP-10 increased RABV pathogenicity by causing neurological diseases, which is due to persistent and high-level expression of chemokines, excessive infiltration and accumulation of inflammatory cells in the central nervous system, and severe enhancement of blood-brain barrier permeability. These studies indicate that overexpression of chemokines, although important in controlling virus infection, may not always be beneficial to the host.

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.

We investigated structural changes in the rabies virus (HEP-Flury strain) nucleocapsid (NC) during the virus replication, for which we used two anti-nucleoprotein (N) monoclonal antibodies (mAbs), #404-11 (specific for a conformation-dependently exposed linear epitope) and #1-7-11 (specific for a conformational epitope which is exposed after the nucleocapsid formation). Both mAbs recognized the N protein of the viral NC, but not of the RNA-free N-P complex. The 1-7-11 and 404-11 epitopes could be mapped to the N-terminal and the C-terminal regions of N protein, respectively. Immunoprecipitation studies demonstrated that treatment of the NC either with the alkaline phosphatase or sodium deoxycholate (DOC) resulted in dissociation of most P proteins from the NC and in the reduced reactivity to mAb #404-11, but not to mAb #1-7-11. NC-like structures produced in the N cDNA-transfected cells displayed strong reactivity to mAb #1-7-11; however, reactivity to mAb #404-11 was very weak. And, coexpression with viral phosphoprotein (P) resulted in little increase in reactivity to mAb #404-11 of the NC-like structures, while the reactivity was significantly increased by cotransfection with P and the viral minigenome whose 3'- and 5'-end structures were derived from the viral genome. From these results, we assume that, although the 404-11 epitope is a linear one, the epitope-containing region is exposed only when N proteins encapsidate properly the viral RNA in collaboration with the P protein. Further, exposure of the 404-11 epitope region might be function-related, and be regulated by association and dissociation of the P protein.

Further studies on the hyperphosphorylated form (p40) of the rabies virus nominal phosphoprotein (P).

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 (pI=4.78), while the latter, p40-1 (pI=4.73). 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.

Association of rabies virus nominal phosphoprotein (P) with viral nucleocapsid (NC) is enhanced by phosphorylation of the viral nucleoprotein (N).

We investigated possible role(s) of N protein phosphorylation in the rabies virus replication process. A large amount of P proteins are associated with the viral nucleocapsid (NC) in the infected cell, the amount which was greatly decreased by phosphatase-treatment of the isolated NC, indicating that the phosphate group of N and/or P proteins is essential for their stable association with the NC. Immunoprecipitation studies were performed on the coexpressed normal N or phosphorylation deficient N(S389A) and P proteins, demonstrating that the P protein associated with phosphorylation-deficient NC-like structures was much less in amount than that associated with the wild type NC. Similar results were also obtained with a mutant P protein, PDeltaN19, which lacked the N-terminal 19 amino acids and was capable of binding to the NC-like structures but incapable of forming the RNA-free N-P complexes. Immunoprecipitation studies with mAb #402-13 further suggested that the NC-specific linear 402-13 epitope was exposed even on the P proteins which were associated with the phosphorylation-deficient NC-like structures, but such association was very weak as demonstrated by greatly decreased amounts of coprecipitated NC-like structures. From these results, we assume that the phosphorylation of N protein enhances the association between the 402-13 epitope-positive P protein and the NC probably by stabilizing such P-NC binding.

Characterization of a slow-migrating component of the rabies virus matrix protein strongly associated with the viral glycoprotein.

We investigated multiple forms of rabies virus matrix (M) protein. Under non-reducing electrophoretic conditions, we detected, in addition to major bands of monomer forms (23- and 24-kDa) of M protein, an M antigen-positive slow-migrating minor band (about 54 kDa) in both the virion and infected cells. Relative contents of the 54-kDa and monomer components in the virion were about 20-30% and 70-80% of the whole M protein, respectively, while the content of the 54-kDa component was smaller (about 10-20% of the total M protein) in the cell than in the virion. The 54-kDa components could be extracted from the infected cells with sodium deoxycholate, but they were quite resistant to extraction with 1% nonionic detergents by which most monomer components were solubilized. The 54-kDa component was precipitated more efficiently than the monomer by a monoclonal antibody (mAb; #3-9-16), which recognized a linear epitope located at the N-terminal of the M protein. The mAb #3-9-16 coprecipitated the viral glycoprotein (G), which was demonstrated to be due to strong association between the G and 54-kDa component of the M protein. Monomers and the 54-kDa polypeptide migrated to the same isoelectric point (pI) in twodimensional (2-D) gel electrophoresis, implicating that the 54-kDa component was composed of component(s) of the same pI as that of the M protein monomers. From these results, we conclude that the M antigen-positive 54-kDa polypeptide is a homodimer of M protein, taking an N-terminal-exposed conformation, and is strongly associated with the viral glycoprotein. Possible association with a membrane microdomain of the cell will be discussed.

Monoclonal antibody #3-9-16 recognizes one of the two isoforms of rabies virus matrix protein that exposes its N-terminus on the virion surface.

We investigated behaviors of the rabies virus matrix (M) protein using a monoclonal antibody (mAb), #3-9-16, that recognized a linear epitope located at the N-terminus of the protein. Based on the reactivity with this mAb, M proteins could be divided into at least two isoforms; an ordinary major form (Malpha) whose 3-9-16 epitope is hidden, and an N-terminal-exposed epitope-positive form (Mbeta). The Mbeta protein accounted for about 25-30% of the total M proteins in the virion, while its content in the cell ranged from 10 to 15% of total M protein. Fluorescent antibody (FA) staining showed that the Mbeta antigen distributed in the Golgi area where the colocalized viral glycoprotein antigen was also detected. Mbeta antigen was shown to be exposed on the surface of infected cells by both immunoprecipitation and FA staining with the mAb, whereby the cells might have become sensitive to the mAb-dependent complement-mediated cytolysis. Similarly, the Mbeta antigen was shown to be exposed on the virion surface, and the infectivity of the virus was destroyed by the mAb in the presence of a complement. Together with these results, we think that the M protein molecule takes either of two conformations, one (Mbeta) of which exposes the 3-9-16 epitope located in the N-terminal region of the M protein, that are also exposed on the surface of the virion and infected cells, whereby it might play a certain important role(s) in the virus replication process differently from the other form (Malpha), probably through its intimate association with the Golgi area and/or the cell membrane.

Structural relationship between nucleocapsid-binding activity of the rabies virus phosphoprotein (P) and exposure of epitope 402-13 located at the C terminus.

The structural changes of the nominal phosphoprotein (P) of rabies virus using a monoclonal antibody, mAb #402-13, was investigated. This mAb recognized a linear epitope that was mapped roughly to a C-terminal region of the P protein, ranging from aa 256 to 297. The P gene products were detected by the mAb in immunoblot assays, the products of which were produced either in BHK-21 cells or in Escherichia coli cells. The mAb, however, detected very low levels of P gene products in immunoprecipitation assays. The mAb recognized the nucleocapsid (NC)-associated P proteins but recognized free P protein and free N-P complex produced in the infected cells much less efficiently. When the P proteins were released from the NC, however, they were no longer recognized by the mAb. Similar results were obtained from BHK-21 cells co-transfected with P and N cDNAs. Furthermore, studies with C-terminally truncated P protein mutants revealed that the NC-binding ability of the P protein was dependent on the presence of the C-terminal epitope region. From these results, it is thought that the 402-13 epitope region is concealed when the P protein is present in a free form or free N-P complex but is exposed when it is associated with the NC. The C-terminal epitope region seemed to be essential for the P protein to be associated with the NC but not for the formation of free N-P complexes with newly synthesized N protein.

Studies on the rabies virus RNA polymerase: 3. Two-dimensional electrophoretic analysis of the multiplicity of non-catalytic subunit (P protein).

We described previously (Takamatsu et al., 1998. Microbiol. Immunol. 42: 761-771) the rabies virus P protein as being composed of several components of different sizes, among which the full-sized major components were termed as p40 and p37 according to their electrophoretic mobilities, and radiolabeling studies with [32P]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 32P-phosphorylated in a radiolabeling period and were converted to the p40.