Translocation and Colocalization of ICP4 and ICP0 in Cells Infected with Herpes Simplex Virus 1 Mutants Lacking Glycoprotein E, Glycoprotein I, or the Virion Host Shutoff Product of the U_L41 Gene

Abstract

In wild-type herpes simplex virus 1-infected cells, the major regulatory protein ICP4 resides in the nucleus whereas ICP0 becomes dynamically associated with proteasomes and late in infection is translocated and dispersed in the cytoplasm. Inhibition of proteasomal function results in retention or transport of ICP0 to the nucleus. We report that in cells infected with mutants lacking glycoprotein E (gE), glycoprotein I (gI), or the product of the U L 41 gene, both ICP4 and ICP0 are translocated to the cytoplasm and coaggregate in small dense structures that, in the presence of proteasomal inhibitor MG132, also contain proteasomal components. In an earlier study concerning the redistribution of the IP3 receptor I in infected cells, we noted that the infected cell protein no. 4 (ICP4) is translocated into the cytoplasm in cells infected with mutant viruses lacking glycoprotein E or U L 41, the gene encoding the virion host shutoff protein (vhs) (18). This was an unexpected finding. ICP4 is one of several major regulatory proteins encoded by herpes simplex virus 1 (HSV-1). The protein acts both as a repressor and as a transactivator. Studies of temperature-sensitive mutants have shown that ICP4 is required throughout the replicative cycle of the virus (5, 6). Unlike ICP0, which late in infection with wild-type virus is translocated to the cytoplasm, ICP4 is thought to be a nuclear protein and in fact resides exclusively in the nucleus in wild-type virus-infected cells. This report centers on the role of gE, gI, and U L 41 proteins in the localization of ICP4 and ICP0 proteins. Relevant to this report are the following.(i) In wild-type virus-infected cells, there is not significant colocalization of ICP0 and ICP4. ICP0 initially colocalizes with the ND10 structure (12, 30, 31), fills the nucleus, and eventually, between 9 and 12 h after infection, is present in a diffuse form entirely in the cytoplasm (16,19,20,28). Export of ICP0 to the cytoplasm is blocked by exposure of cells 2 h after infection to proteasomal inhibitor MG132 (13,28). A striking feature of the translocation process is that exposure of infected cells to MG132 after ICP0 has been exported to the cytoplasm results in the relocation of ICP0 to the nucleus (28).ICP0 is also exported to the cytoplasm in cells infected with mutants lacking ICP4 (⌬ICP4) (16,28). Unlike the situation with wild-type virus-infected cells, ICP0 is translocated at earlier times after infection and forms small dense structures dispersed throughout the cytoplasm or arranged around the nucleus. In the presence of MG132, added late after infection, ICP0 is retained in the cytoplasm in similar small dense structures that also contain proteasomal components (28). Relevant to this report is the observation that a D199A substitution in ICP0 prevents the translocation of ICP0 to the cytoplasm (52,53). This observation suggests that the translocation of ICP0 is an active process involving an interactive partner.Last, ICP0 and ICP4 have been reported to colocalize in the cytoplasm in ce...

Nuclear retention of ICP0 in cells exposed to HDAC inhibitor or transfected with DNA before infection with herpes simplex virus 1

“…To obtain the 10-Kb repeats of the Lac operator, the pLacO-SV2-neo plasmid was digested with the SalI/XhoI. In all experiments the cells were exposed to 10 PFU of HSV-1(F) per cell in medium 199 at 24 h after mock transfection or transfection with DNA (14).…”

“…The ICP4, ICP0, and ICP8 mouse monoclonal antibodies (Goodwin Institute for Cancer Research, Plantation, FL) were used at a 1:1,000 dilution. The ICP0 exon 2 rabbit polyclonal antibody was used at a 1:2,000 dilution (14). The CoREST rabbit polyclonal antibody (Upstate) was used at a 1:1,000 dilution.…”

“…The cells were fixed in 4% paraformaldehyde, at times indicated in the Results, permeabilized, blocked with 0.1% Triton X-100 in PBS in the presence of 10% horse serum and 1% BSA (PBS-TBH solution), and reacted with primary antibodies, diluted in PBS-TBH, as described earlier (14). The ICP4, ICP0, and ICP8 mouse monoclonal antibodies (Goodwin Institute for Cancer Research, Plantation, FL) were used at a 1:1,000 dilution.…”

“…The sources, properties, and propagation of HEp-2, rabbit skin cells, HEK293, U2OS and the telomerase transformed human embryonic lung (HEL) cells, and the properties of the limited passage HSV-1(F) strain were described elsewhere (14).…”

ICP0 enables and monitors the function of D cyclins in herpes simplex virus 1 infected cells

“…The sources, properties, and propagation of HEp-2, U2OS, and Vero cells were reported elsewhere (13,24). The properties of the parent Fig.…”