Proteomics Analysis Identifies IRSp53 and Fascin as Critical for PRV Egress and Direct Cell-Cell Transmission.

Pseudorabies virus (PRV) has been widely used as a live trans-synaptic tracer for mapping neuronal circuits. Systematically identifying mature PRV virion proteomes and defining co-purified host proteins are necessary to fully understand the detailed mechanism underlying PRV transmission processes. Here, a PRV virion purification strategy based on sorting with flow cytometry is developed and the mature extracellular and intracellular PRV virion proteomes using LC coupled with MS/MS are characterized. In addition to viral proteins, a large number of host proteins are also identified, including proteins related to actin cytoskeletal dynamics and membrane protrusion. How many of these host proteins are true virion components are unknown and the majority of these may not be. Through functional analysis, it is found that IRSp53 and fascin are critical for the egress process and play a role in direct cell-cell transmission. Moreover, it is shown that CDC42 and Rac1 are also involved in the production of mature extracellular virions. The results suggest that the formation of the filopodia-like cytoskeleton and the rearrangement of the membrane, which are both associated with IRSp53 and fascin, may be important for the transmission of viruses used in neuronal tracing.

Quantitative proteomics study of host response to virulent and attenuated pseudorabies virus infection in mouse brain.

Bartha, the pseudorabies virus (PRV) vaccine strain, is widely used in studies of neuronal circuit-tracing, due to its attenuated virulence and retrograde spreading. However, we know little regarding the molecular mechanisms of PRV infection and spreading between structurally connected neurons. In this study, we systematically analyzed the host brain proteomes after acute infection with PRV, attempting to identified the proteins involved in the processes. Mice were injected with PRV-Bartha and PRV-Becker (PRV-Bartha's wild-type parent strain) in the olfactory system, the proteomes of the brain and synaptosome were analyzed and compared at various infection intervals using mass spectrometry-based proteomics techniques. In all, we identified >100 PRV-infection regulated proteins at the whole-tissue level and the synaptosome level. While at whole-tissue level, bioinformatics analyses mapped most of the regulations to the inflammation pathways, at the synaptosome level, most of those to synaptic transmission, cargo transport and cytoskeleton organization. We established regulated protein networks demonstrating distinct cellular regulation pattern between the global and the synaptosome levels. Moreover, we identified a series of potentially PRV-strain-specific regulated proteins with diverse biological functions. This study may provide new clues for molecular mechanisms for PRV infection and spread.

ORF7 of Varicella-Zoster Virus Is Required for Viral Cytoplasmic Envelopment in Differentiated Neuronal Cells.

Although a varicella-zoster virus (VZV) vaccine has been used for many years, the neuropathy caused by VZV infection is still a major health concern. Open reading frame 7 (ORF7) of VZV has been recognized as a neurotropic gene in vivo, but its neurovirulent role remains unclear. In the present study, we investigated the effect of ORF7 deletion on VZV replication cycle at virus entry, genome replication, gene expression, capsid assembly and cytoplasmic envelopment, and transcellular transmission in differentiated neural progenitor cells (dNPCs) and neuroblastoma SH-SY5Y (dSY5Y) cells. Our results demonstrate that the ORF7 protein is a component of the tegument layer of VZV virions. Deleting ORF7 did not affect viral entry, viral genome replication, or the expression of typical viral genes but clearly impacted cytoplasmic envelopment of VZV capsids, resulting in a dramatic increase of envelope-defective particles and a decrease in intact virions. The defect was more severe in differentiated neuronal cells of dNPCs and dSY5Y. ORF7 deletion also impaired transmission of ORF7-deficient virus among the neuronal cells. These results indicate that ORF7 is required for cytoplasmic envelopment of VZV capsids, virus transmission among neuronal cells, and probably the neuropathy induced by VZV infection.IMPORTANCE The neurological damage caused by varicella-zoster virus (VZV) reactivation is commonly manifested as clinical problems. Thus, identifying viral neurovirulent genes and characterizing their functions are important for relieving VZV related neurological complications. ORF7 has been previously identified as a potential neurotropic gene, but its involvement in VZV replication is unclear. In this study, we found that ORF7 is required for VZV cytoplasmic envelopment in differentiated neuronal cells, and the envelopment deficiency caused by ORF7 deletion results in poor dissemination of VZV among neuronal cells. These findings imply that ORF7 plays a role in neuropathy, highlighting a potential strategy to develop a neurovirulence-attenuated vaccine against chickenpox and herpes zoster and providing a new target for intervention of neuropathy induced by VZV.

A subchronic intravenous toxicity study of magnesium fructose-1,6-diphosphate in beagle dogs.

Magnesium fructose-1,6-diphosphate is a novel agent of antimyocardial ischaemia. In the present study, the subchronic toxicity of magnesium fructose-1,6-diphosphate was investigated after 13-week repeated intravenous administration in beagle dogs. The animals received doses of 0, 75, 150 and 300 mg/kg/day (three males and three females for each dose). During the study period, clinical signs, mortality, body weights, food consumption, electrocardiogram, urinalysis, haematology, clinical biochemistry, macroscopic findings, organ weights and histopathology were examined. The administration of magnesium fructose-1,6-diphosphate resulted in increased incidence of clinical signs, including salivation and emesis. These effects were transient and were noted in almost all dogs given 300 mg/kg/day and occasionally noted in the 150 mg/kg/day dose-treated animals. Serum magnesium in the 150 mg/kg/day and 300 mg/kg/day dose-treated animals was significantly increased after 6- and 13-week administration, but recovered at the end of a 2-week recovery period. At 6 weeks, a statistically significant decrease in serum electrolytes, including sodium and potassium, was observed in the treatment groups. There were no other treatment-related findings. Under the conditions of the present study, magnesium fructose-1,6-diphosphate did not show any evidence of target organ toxicity. The no-observed-adverse-effect level for 13-week intravenous administration of magnesium fructose-1,6-diphosphate to beagle dogs was considered 75 mg/kg/day based on observations of clinical signs and serum electrolytes.