ICP0, ICP4, or VP16 expressed from adenovirus vectors induces reactivationof latent herpes simplex virus type 1 in primary cultures of latently infected trigeminal ganglion cells

In a previous study, we demonstrated that infected-cell polypeptide 0 (ICP0 ) is necessary for the efficient reactivation of herpes simplex virus type 1 (HSV-1) in primary cultures of latently infected trigeminal ganglion (TC) cells (W. P. Halford and P. A. Schaffer, J. Virol. 75:3240-3249, 2001), Th e present study was undertaken to determine whether ICP0 is sufficient to t rigger HSV-1 reactivation in latently infected TG cells. To test this hypot hesis, replication-defective adenovirus vectors that express wild-type and mutant forms of ICP0 under the control of a tetracycline response element ( TRE) promoter were constructed. Similar adenovirus vectors encoding wild-ty pe ICP4, wild-type and mutant forms of the HSV-1 origin-binding protein (OB P), and wild-type and mutant forms of VP16 were also constructed. The TRE p romoter was induced by coinfection of Vero cells with the test vector and a n adenovirus vector that expresses the reverse tetracycline-regulated trans activator in the presence of doxycycline. Northern blot analysis demonstrat ed that transcription of the OEP gene in the adenovirus expression vector i ncreased as a function of doxycycline concentration over a range of 0.1 to 10 muM, Likewise, Western blot analysis demonstrated that addition of 3 muM doxycycline to adenovirus vector-infected Vero cells resulted in a 100-fol d increase in OBP expression. Wild-type forms of ICP0, ICP 1, OBP, and VP16 expressed from adenovirus vectors were functional based on their ability t o complement plaque formation in Vero cells by replication-defective HSV-1 strains with mutations in these genes. Adenovirus vectors that express wild -type forms of ICP0, ICP4 or VP16 induced reactivation of HSV-1 in 86% +/- 5%, 86% +/- 5%, and 97% +/- 5% of TG cell cultures, respectively (means +/- standard deviations). In contrast, vectors that express wild-type OBP or m utant forms of ICP0, OBP, or VP16 induced reactivation in 5% +/- 5%, 8% +/- 0%, 0% +/- 0%, and 13% +/- 6% of TG cell cultures, respectively. In contro l infections, an adenovirus vector expressed green fluorescent protein effi ciently in TG neurons but did not induce HSV-1 reactivation, Therefore, exp ression of ICP0, ICP4, or VP16 is sufficient to induce HSV-1 reactivation i n latently infected TG cell cultures. We conclude that this system provides a powerful tool for determining which cellular acid viral proteins are suf ficient to induce HSV-1 reactivation from neuronal latency.