GLYCOPROTEIN-M AND GLYCOPROTEIN-N OF PSEUDORABIES VIRUS FORM A DISULFIDE-LINKED COMPLEX

Genes homologous to the herpes simplex virus UL49.5 open reading frame are conserved throughout the Herpesviridae. In the alphaherpesvirus p seudorabies virus (PrV), the UL49.5 product is an O-glycosylated struc tural protein of the viral envelope, glycoprotein N (gN) (A. Jons, H. Granzow, R. Kuchling, and T. C. Mettenleiter, J. Virol. 70:1237-1241, 1996). For functional characterization of gN, a gN-negative PrV mutant , PrV-gN beta, and the corresponding rescuant, PrV-gN beta R, were con structed, gN-negative PrV was able to productively replicate on noncom plementing cells, and one-step growth in cell culture was only slightl y reduced compared to that of wild-type PrV. However, penetration was significantly delayed. In indirect immunofluorescence assays with rabb it serum directed against baculovirus-expressed gN, specific staining of wild-type PrV-infected cells occurred only after permeabilization o f cells, whereas live cells failed to react with the antiserum. This i ndicates the lack of surface accessibility of gN in the plasma membran e of a PrV-infected cell. Western blot analyses and radioimmunoprecipi tation experiments under reducing and nonreducing conditions led to th e discovery of a heteromeric complex composed of gM and gN. The comple x was stable in the absence of 2-mercaptoethanol but dissociated after the addition of the reducing agent, indicating that the partners are linked by disulfide bonds. Finally, gN was absent from gM-negative PrV virions, whereas gM was readily detected in virions in the absence of gN. Thus, gM appears to be required for virion localization of gN.