| Herpes Simplex Virus Type 1 is a causative agent for a number of diseases in humans. The assembly process initiates in the nuclei of infected cells, where viral DNA replication and capsid assembly takes place and viral genomes are packaged into capsids which are then thought to bud into the inner nuclear membrane, generating enveloped particles in the perinuclear space. These particles then exit the nucleus and traffic out of the cell.;Using a reversible temperature sensitive mutant ts Prot.A, we have been able to synchronize viral assembly. We have also developed rapid biochemical assays to measure DNA packaging and envelopment of HSV-1. These assays were based on the hypothesis that the packaged genomes would be resistant to DnaseI treatment, compared to unpackaged DNA. Using the synchronized assembly system and the assays we developed, we have been able to determine the kinetics of DNA packaging and envelopment of HSV-1 in infected cells. We also observed that in the absence of ATP, no infectious viral particles are produced and that DNA packaging and capsid assembly are ATP dependent processes.;Once the packaged capsids exit the nucleus, the route that it takes to exit the cell is a contentious issue. One view, the single envelopment model, is that the perinuclear virions traffic out of the cell via the classical secretory pathway. An alternative view is that the initial envelope the virions acquired from the inner nuclear membrane fuses with the outer nuclear membrane to release naked capsids into the cytoplasm which then bud into an organelle such as the Golgi, Trans Golgi network (TGN) or endosomes, and acquire their final envelope.;To distinguish between the two models, we used the fungal metabolite Brefeldin A (BFA). BFA is an agent that blocks endoplasmic reticulum (ER) to Golgi trafficking. If the first model is true, then there should be an increase in naked cytoplasmic capsids.;Surprisingly, however, we observed none of the predicted results. Under our conditions, packaged capsids become trapped within the nucleoplasm and fail to bud into the perinuclear space and traffic into the cytoplasm. We conclude that the immediate effect of BFA upon the assembly pathway of HSV is to block capsid envelopment at the inner nuclear membrane. |
