Nuclear localization of tegument-delivered pp71 in human cytomegalovirus-infected cells is facilitated by one or more factors present in terminally differentiated fibroblasts

Abstract

Herpesviral virions contain a tegument layer that consists primarily of viral proteins. The delivery of fully functional proteins to infected cells upon virion envelope fusion to the plasma membrane allows herpesviruses to modulate cellular activities prior to viral gene expression. Certain tegument proteins can also regulate viral processes. For example, the pp71 tegument protein encoded by the UL82 gene of human cytomegalovirus (HCMV) stimulates viral immediate early (IE) gene expression and thus acts to initiate the productive lytic infectious cycle. In terminally differentiated fibroblasts infected with HCMV, tegument-delivered pp71 traffics to the nucleus and degrades the cellular transcriptional corepressor Daxx to initiate viral IE gene expression and lytic replication. However, when HCMV infects incompletely differentiated cells, tegument-delivered pp71 remains in the cytoplasm, allowing the nucleus-localized Daxx protein to silence viral IE gene expression and promote the establishment of a latent infection in certain cell types. We sought to determine whether undifferentiated cells block the trafficking of tegument-delivered pp71 to the nucleus or whether differentiated cells facilitate the nuclear transport of tegument-delivered pp71. Heterogenous cell fusion experiments demonstrated that tegument-delivered pp71 found in the cytoplasm of undifferentiated NT2 cells could be driven into the nucleus by one or more factors provided by fully differentiated fibroblasts. Our data raise the intriguing possibility that latency is the default program launched by HCMV upon viral entry into cells and that lytic infection is initiated only in certain (differentiated) cells that can facilitate the delivery of incoming pp71 to the nucleus.

FIG. 1.

Tegument-delivered pp71 is nuclear in differentiated but not undifferentiated cells. NHDFs (A, B, and C) and NT2 cells (D, E, F, and G) were plated on coverslips, infected at an MOI of 1 for 6 h, and then analyzed by indirect immunofluorescence for pp71, IE1, or pp65 and Oct4, as indicated. The boxed area of the merged panel of row D is shown as an enlarged image in row E for easier visualization of pp71 localization. In coculture experiments, NHDFs were transfected with either GFP and p14 (H) or GFP and an empty vector (I). Six hours later, they were overseeded with NT2 cells. These heterogenous cell cultures were fixed 15 h later and then analyzed by indirect immunofluorescence for Oct4. Nuclei were counterstained with Hoechst dye. NHDF nuclei in the second and fourth panels of row I are marked with asterisks.

FIG. 2.

Experimental design for fusion experiments. (A) NHDFs were transfected with either GFP and p14, GFP and an empty vector, or p14 alone, plated on coverslips, and then cultured for 6 h. Separately, NT2 cells were infected at an MOI of 5 for 4 h, collected with trypsin, and subsequently coplated with the transfected NHDFs. Cells were incubated for 15 h to allow syncytium formation and then fixed and analyzed by indirect immunofluorescence. (B) NT2 cells were transfected with either GFP and p14 or GFP and an empty vector, plated on coverslips, and then cultured for 22 h. Separately, NT2 cells were infected at an MOI of 5 for 4 h, collected with trypsin, and subsequently coplated with the transfected NT2 cells. Cells were incubated for 18 h to allow syncytium formation and then fixed and analyzed by indirect immunofluorescence.

FIG. 3.

pp71 localizes to the nuclei of NHDF-NT2 syncytia but not NT2-NT2 syncytia. NHDFs transfected with GFP and p14 (A and B) or GFP and an empty vector (C and D) were overseeded with infected NT2 cells (MOI = 5) and then fixed and analyzed for pp71 (A and C) or Oct4 (B and D) by indirect immunofluorescence. NT2 cells transfected with p14 and GFP (E and F) were overseeded with infected NT2 cells (MOI = 5) and then fixed and analyzed for pp71 (E) or Oct4 (F) expression by indirect immunofluorescence. Nuclei were counterstained with Hoechst dye. Asterisks in panel D indicate GFP-positive, Oct4-negative NHDF nuclei. Arrows in panel E and H point to punctate pp71. To allow for easier visualization of pp71, enlargements of the merged images from panels C (#) and D (##) are shown as panels G and H, respectively.

FIG. 4.

Quantification of pp71 localization and IE gene expression in syncytia. Syncytia formed by HCMV-infected NT2 cells and either NHDFs (left) or NT2 cells (right) were examined for pp71 localization and IE gene expression. The average percentages of syncytia with tegument-delivered pp71 in the cytoplasm (bars labeled C) or nucleus (bars labeled N) or that express IE1 (bars labeled IE) are shown for 3 independent experiments, in which a total of at least 250 syncytia were analyzed. Error bars represent 1 standard deviation.

FIG. 5.

pp71 can activate IE gene expression when transported to the nuclei of NHDF-NT2 syncytia. NHDFs transfected with either GFP and an empty vector (A) or GFP and p14 (B) were overseeded with infected NT2 cells (MOI = 5) and examined for IE1 expression by indirect immunofluorescence. (C) NT2 cells were transfected with GFP and p14, overseeded with infected NT2 cells (MOI = 5), and examined for IE1 expression by indirect immunofluorescence. (D) NHDFs transfected with GFP and p14 were overseeded with NT2 cells infected with UV-inactivated virus (MOI = 5) and then examined for pp71 by indirect immunofluorescence. Nuclei were counterstained with Hoechst dye.

FIG. 6.

Tegument-delivered pp71 localizes to the nuclei of Oct4-positive syncytia and colocalizes with Daxx. NHDFs transfected with p14 were overseeded with NT2 cells infected with wild-type (A and B) or UV-inactivated (C and D) virus (MOI = 5) and then fixed and analyzed for pp71 and Oct4 (A), pp71 and IE1 (B), or pp71 and Daxx (C and D) by indirect immunofluorescence. Nuclei were counterstained with Hoechst dye. (D) Enlargement of the boxed area in panel C.

FIG. 7.

Daxx is degraded in NHDF-NT2 syncytia. NHDFs transfected with p14 were overseeded with mock-infected (lane M) or HCMV-infected (lane V) NT2 cells. Fifteen hours after being overseeded, cells were trypsinized and lysed and Western blotting was performed. Syncytial lysates were blotted for Daxx and tubulin (Tub).

FIG. 8.

Tegument-delivered pp71 remains cytoplasmic in NT2 cells treated with leptomycin B (LMB). NT2 cells were either untreated (A and C) or pretreated (B, D, E, and F) for 1 h with LMB and then mock infected (A, B, and E) or infected with HCMV (C, D, and F) at an MOI of 2. pp71 and either NF-κB (A, B, C, and D) or Oct4 (E and F) were visualized by indirect immunofluorescence. Nuclei were counterstained with Hoechst dye.

FIG. 9.

Model for nuclear localization of tegument-delivered pp71. Tegument-delivered pp71 could be actively blocked from entering the nuclei of undifferentiated cells (A), efficiently exported from the nuclei of undifferentiated cells (B), or driven into the nuclei of differentiated cells (C) by a positively acting, dominant factor (X). Data presented here support the model depicted in panel C.