Establishment of a quiescent herpes simplex virus type 1 infection in neurally-differentiated PC12 cells

Rat pheochromocytoma (PC12) cells differentiated with nerve growth factor (Nd-PC12) were used to investigate the establishment of a non-productive herpes simplex virus type 1 (HSV-1) infection that is reversible. The results of this work are as follows: (i) Nd-PC12 cultures could be maintained as long term (>7 weeks) non-dividing cultures only when plated on collagen-coated dishes in the absence of serum; (ii) Infection of Nd-PC12 with HSV-1 strains KOS and 17 in the transient presence of acycloguanosine (ACV) resulted in all cultures free of detectable levels of infectious virus at the time of ACV removal and ACV was not needed to maintain the non-productive quiescent state in the subsequent 8 weeks; (iii) These persistently infected and quiescent (QIF)-PC12 cultures demonstrated both spontaneous and forskolin-inducible virus production, at low (5%) and high frequencies (92 - 100%), respectively during the first 2 weeks post-ACV withdrawal. (iv) In contrast to other in vitro models, HSV-1 failed to reactivate following removal of nerve growth factor. (v) A high percentage of QIF-PC12 cultures (50-100%) produced virus in response to forskolin treatment as long as 7 weeks post-ACV withdrawal. (vi) Expression of HSV-1 productive genes (i.e. alpha 0, alpha 4, alpha 27, U-L,30 and U-L,18) dropped precipitously in the presence of ACV and remained undetectable or continued to decline following its removal, whereas the levels of LAT and the host gene G3PDH remained relatively constant throughout the 31 day study period as measured by RT-PCR. These results indicate that QIF-PC12 cells offer a novel, neuronal cell culture system that may enhance our ability to study HSV-1 reactivation from a cryptic, latent-like, non-productive state in the absence of replication inhibitors.