Replication of herpes simplex virus type 1 within trigeminal ganglia is required for high frequency but not high viral genome copy number latency

The replication properties of a thymidine kinase-negative (TK-) mutant of h erpes simplex virus type 1 (Hm-1) were exploited to examine the relative co ntributions of replication at the body surface and within trigeminal gangli a (TG) on the establishment of latent infections. The replication of a TK- mutant, 17/tBTK(-), was reduced by similar to 12-fold on the mouse cornea c ompared to the rescued isolate 17/tBRTK(+), and no replication of 17/tBTK(- ) in the TG of these mice was detected. About 1.8% of the TG neurons of mic e infected with 17/tBTK(-) harbored the latent viral genome compared to 23% of those infected with 17/tBRTK(+). In addition, the latent sites establis hed by the TK- mutant contained fewer copies of the HSV-1 genome (average, 2.3/neuron versus 28/neuron), On the snout, sustained robust replication of 17tBTK(-) in the absence of significant replication within the TG resulted in a modest increase in the number of latent sites, Importantly, these lat ently infected neurons displayed a wild-type latent-genome copy number prof ile, with some neurons containing hundreds of copies of the TK- mutant geno me, As expected, the replication of the TK- mutant appeared to be blocked p rior to DNA replication in most ganglionic neurons in that (i) virus replic ation was severely restricted in ganglia, (ii) the number of neurons expres sing HSV proteins was reduced 30-fold compared to the rescued isolate, (iii ) cell-to-cell spread of virus was not detected within ganglia, and (iv) th e proportion of infected neurons expressing late proteins was reduced by 89 % compared to the rescued strain. These results demonstrate that the viral TK gene is required for the efficient establishment of latency. This requir ement appears to be primarily for efficient replication within the ganglion , which leads to a sixfold increase in the number of latent sites establish ed. Further, latent sites with high genome copy number can be established i n the absence of significant virus genome replication in neurons. This sugg ests that neurons can be infected by many HSV virions and still enter the l atent state.