A study on pseudorabies virus VP22 and flexible tegument assembly

The alphaherpesviruses are neurotropic in nature, able to reside in the peripheral nervous system of the host. The extracellular form of the virus is the virion and is required for transmission of infection from one host to the next. Pseudorabies virus (PRV), like other alphaherpesviruses, is also capable of spreading between cells within a host. Components of the virion, for example, are required for the movement of PRV infection across synaptically connected neurons. The tegument is a poorly understood structure within the virion. The Us2 and VP22 tegument proteins of Pseudorabies virus are dispensable for virus growth, neurovirulence and neuroinvasion. The steady state localization of PRV VP22 in infected cells was demonstrated to be in the nucleus. While only its non-phosphorylated form was detected in extracellular virions, VP22 was found to be a major constituent of the tegument. Analysis of fluorescent VP22 fusion proteins in virions revealed its incorporation into the tegument structure to be flexible, in the presence or absence of a fluorescent capsid structure. Cellular actin, along with Us2 and at least two other tegument proteins, was found to compensate for the loss of VP22 in virions. Further studies revealed that actin was present in the tegument layer.; Differentiated PC12 cells, similar to primary sympathetic neurons, were infected with a PRV strain expressing a fluorescent VP22 fusion protein. VP22 puncta, exhibiting a range of fluorescence intensities, were shown to enter the axon-like projections of these cells and traffic away from the cell body late in infection. The infection of primary sympathetic neuronal cultures was used to demonstrate that fluorescent VP22 puncta entered axons, in the presence or absence of a fluorescent capsid structure. Treatment of infected neurons with the fungal metabolite brefeldin A (BFA) prevented axonal entry of fluorescent tegument and capsid structures, regardless of whether they were together or separate. The secretory pathway of infected neurons was disrupted by BFA treatment, evident by the mislocalization of proteins that reside in the Golgi structure. Thus, disruption of the secretory pathway was shown to coincide with a block in axonal entry of fluorescent tegument and capsid structures.