Disruption of PML-associated nuclear bodies by IE1 correlates with efficient early stages of viral gene expression and DNA replication in human cytomegalovirus infection

In human cytomegalovirus (HCMV) infection, both of the major immediate-earl y proteins IE1(IE68, UL123) and IE2(IE86, UL122) target to PML protein-asso ciated nuclear bodies known as PODs or ND10 at very early times after infec tion. IE1 causes a redistribution of both PML and IE1 from the PODs into a nuclear diffuse form, whereas IE2 initially localizes adjacent to PODs but later associates with viral DNA replication compartments. The peripheries o f PODs are also believed to be sites for initiation of both viral IE transc ription and DNA replication. However, because IE1 is nonessential at high m ultiplicity of infection (m.o.i.) in HF cells, the exact role of these proc esses in viral infection has been enigmatic. Therefore, we investigated the effects of overexpression of PML in the presence or absence of IE1 on the intranuclear distribution of IE2 and formation of viral DNA replication com partments, as well as on the levels of delayed-early and late viral transcr iption and protein accumulation. Infection with wild-type HCMV(Towne) and t he IE1-deleted derivative HCMV(CR208), which fails to disrupt PODs, was com pared in a pair of related astrocytoma/glioblastoma cell lines, the U373-Ne o control and a variant U373-PML that constitutively overexpresses PML(560) in much larger than normal PODs. IFA studies on the localization patterns for IE1, IE2, and PML showed that, although the numbers of IE2-positive cel ls were not significantly reduced in either the wild-type virus-infected U3 73-PML cell line or in Delta IE1-infected control cells, POD disruption by IE1 in wild-type virus infection was delayed by up to 6 h in U373-PML cells compared to control cells. Furthermore, there was considerable enhancement of IE2 colocalization with PODs in Delta IE1-infected U373-PML cells. Form ation of viral DNA replication compartments in the U373-PML cell line was a lso greatly delayed, measured at fivefold lower after wild-type virus infec tion and 12-fold lower after infection with Delta IE1 than in the control c ell line at 48 h at an m.o.i. of 1.0. The levels of representative early an d late viral proteins detected by Western blotting were suppressed by fivef old and 22-fold at 24 and 72 h, respectively, in the U373-PML cell line, ev en with high m.o.i. wild-type HCMV infection. Decreased viral protein level s also occurred when control cells were infected with the Delta IE1 virus a nd these two effects were additive in the U373-PML cell line. Similarly, wh en U373-PML cells were infected with recombinant HCMV expressing an extrage nic luciferase reporter gene under the control of viral early (Pol) or late (pp28) promoters, their transcriptional activation was reduced up to fivef old at both high and low m.o.i, compared to that of the control cells. Over all, these results suggest that POD disruption by IE1 and subsequent redist ribution of both PML and IE1 at very early times after infection may play a n important role in the efficient utilization of cellular transcription and replication machinery by HCMV and contribute to rapid progression of the H CMV lytic cycle. (C) 2000 Academic Press.