The tegument protein UL71 of human cytomegalovirus is involved in late envelopment and affects multivesicular bodies

Abstract

Morphogenesis of human cytomegalovirus (HCMV) is still only partially understood. We have characterized the role of HCMV tegument protein pUL71 in viral replication and morphogenesis. By using a rabbit antibody raised against the C terminus of pUL71, we could detect the protein in infected cells, as well as in virions showing a molecular mass of approximately 48 kDa. The expression of pUL71, detected as early as 48 h postinfection, was not blocked by the antiviral drug foscarnet, indicating an early expression. The role of pUL71 during virus replication was investigated by construction and analysis of a UL71 stop mutant (TBstop71). The mutant could be reconstituted on noncomplementing cells proving that pUL71 is nonessential for virus replication in human fibroblasts. However, the inhibition of pUL71 expression resulted in a severe growth defect, as reflected by an up to 16-fold reduced extracellular virus yield after a high-multiplicity infection and a small-plaque phenotype. Ultrastructural analysis of cells infected with TBstop71 virus revealed an increased number of nonenveloped nucleocapsids in the cytoplasm, many of them at different stages of envelopment, indicating that final envelopment of nucleocapsids in the cytoplasm was affected. In addition, enlarged multivesicular bodies (MVBs) were found in close proximity to the viral assembly compartment, suggesting that pUL71 affects MVBs during virus infection. The observation of numerous TBstop71 virus particles attached to MVB membranes and budding processes into MVBs indicated that these membranes can be used for final envelopment of HCMV.

Fig. 1.

Characterization of HCMV pUL71 by Western blot analysis in infected and transfected cells, as well as in purified virus particles. (A) The presence of pUL71 was assayed by using a polyclonal pUL71 antibody directed against the C terminus of pUL71 in lysates of HFFs infected with HCMV wild-type virus at 5 days postinfection (wild type), mock-infected HFFs (HFF), and Cos7 cells transiently transfected with pEF-UL71 24 h posttransfection (UL71aa1-361) and control cells (Cos7). Values for the prestained molecular weight marker, Precision Plus (Bio-Rad), are displayed to the right. (B) Western blot analysis of lysates of gradient purified wild-type virus (virions), HFFs infected with wild-type virus 5 days postinfection (wild type), and mock-infected cells (HFF). Viral proteins pUL71, MCP, pp65, and cellular actin were detected with respective antibodies. (C) Expression kinetics of viral proteins in HFFs infected with wild-type virus. Viral proteins pUL71, MCP, and pp65 were detected at the indicated times postinfection. Detection of cellular actin was used to ensure comparable loading. (D) Expression of viral proteins pUL71, MCP, and pp65 in absence or presence of foscarnet (FOS, 400 μmol) in HFFs infected with wild-type virus at 72 and 96 h postinfection. Actin was used as loading control. Note that in the presence of foscarnet, the expression of MCP and pp65, but not of pUL71, is impaired.

Fig. 2.

Characterization of the start codon of UL71. (A) Sequence of the promoter region of UL71 from nucleotides 136991 to 137186 of the TB40-BAC4 sequence (40). Predicted promoters within this sequence are underlined and putative transcriptional start is indicated by a dot. Possible start codons of pUL71 are boxed, and numbers above correspond to the amino acids relative to the annotated start codon indicated by the number 1. The arrow indicates the start and direction of pUL71 translation as annotated (accession number EF999921.1). (B) Western blot analysis of HFFs infected with HCMV wild-type virus at 5 days postinfection (wild type), mock-infected HFFs (HFF), Cos7 cells (Cos7), and Cos7 cells transiently transfected with the pUL71 expression plasmids in panel A as indicated.

Fig. 3.

Generation of UL71 stop mutant TBstop71. (A) Position of the introduced stop codon and the frameshift mutation at the 5′ prime of UL71. In TBrev71-BAC, the stop and frameshift mutation was restored to the original UL71 sequence with a silent mutation (indicated by the gray box). (B) Restriction fragment length polymorphism analysis of BAC-DNAs of TB40-BAC4 (wild type), TBstop71-BAC, and TBrev71-BAC with restriction enzymes EcoRI and HindIII. The three BAC-DNAs were used to reconstitute the respective viruses. Lanes M, molecular marker (1-kb DNA ladder; Invitrogen).

Fig. 4.

Western blot analysis of HFFs infected with wild-type, TBstop71, and TBrev71 viruses at 5 days postinfection and mock-infected control cells. Cell lysates were separated on a 10% sodium dodecyl sulfate-polyacrylamide gel and analyzed by Western blot analysis. Actin and viral proteins were detected with the respective antibodies.

Fig. 5.

Growth characterization of TBstop71 virus. (A and B) Growth kinetics of wild-type (□), TBstop71 (▴), and TBrev71 (•) viruses. HFFs were infected with an MOI of 3. Culture supernatants (A) and cell-associated virus particles (B) were sampled at the indicated times postinfection, and virus yields were determined by titration on HFFs. Day 0 values represent the inocula. Each data point and error bar represents the mean ± the standard error of at least three independent experiments. (C and D) Multistep growth kinetic of wild-type (□), TBstop71 (▴), and TBrev71 (•) viruses. HFFs were infected with an MOI of 0.01. Culture supernatants (C) and cell-associated virus particles (D) were sampled at the indicated times postinfection, and virus yields were determined by titration on HFFs. Day 0 values represent the inocula. Each data point and error bar represents the mean ± the standard error of at least three independent experiments. (E) Plaque assay of wild-type, TBstop71, and TBrev71 virus-infected HFFs. Cells were infected with 100 PFU. After 24 h, the cells were overlaid with methylcellulose and fixed with methanol at 9 days postinfection. Infected cells were detected by an anti-IE1/2 and UL44 monoclonal antibody. The proteins were visualized by a Cy3-conjugated secondary goat anti-mouse antibody. Nuclei were marked with DAPI. The plaque areas of wild-type, TBstop71, and TBrev71-virus infected cells were analyzed with the Axio-Observer.Z1 fluorescence microscope using a ×10 objective lens. For the statistical analysis, the relative plaque areas of wild-type, TBstop71, and TBrev71 virus-infected cells were measured by the program ImageJ. The mean percentage of the areas and standard error were determined by counting at least 50 plaques in each experiment. (The experiments were repeated at least two times; ***, P < 0.0001.)

Fig. 6.

Ultrastructural analysis of wild-type (A, C, and E) and TBstop71 (B, D, and F) virus-infected MRC-5 cells at 5 days postinfection. (A) A typical assembly compartment of an HCMV wild-type infected cell in close proximity to the nucleus (N). MVBs (arrowheads) with diameters of not more than 1 μm can be observed in the cytoplasm (C). (B) In a TBstop71 virus-infected cell, many enlarged MVBs (arrowhead) with diameters of several μm can be seen located around the assembly compartment. (C) No virus particles are attached at the MVB membrane, although in some cases viral particles or dense bodies were found inside of MVBs. (D) Many viral particles are attached to MVB membranes. Some of them are in a budding process into or are already inside the lumen of the MVB. (E) Few viral particles in a budding process into small crescent-shaped vesicles (arrow). Note that the majority of virus particles are fully enveloped within small vesicles. (F) Larger vesicles (arrow) are used as multiple budding sites by TBstop71 virus particles.

Fig. 7.

Three-dimensional reconstruction of membranes and viral capsids from tomography of TBstop71 virus-infected HFFs at 5 days postinfection. (A) Single slice from the three-dimensional tomogram with a large vesicle in which multiple budding processes of TBstop71 virus particles are observed. (B) Membranes used as budding sites as well as viral capsids were reconstructed and superimposed on the original micrograph. Membranes are depicted in light blue; viral capsids are indicated in dark blue. (C) Reconstruction of the budding processes only. Bars, 200 nm. Note that areas with high mass density, such as membranes, occur bright in the dark-field STEM tomograms. See Movie S1 at http://www.uniklinik-ulm.de/struther/institute/virologie/home/forschung/publikationen/suppl.html.

Fig. 8.

Subcellular localization of gB, pp28, and pp150 in wild-type and TBstop71 virus-infected HFFs (MOI = 0.1 to 0.5). Cells were fixed with 4% PFA in PBS at 7 days postinfection. Proteins were detected by the respective primary antibodies visualized by goat anti-mouse antibodies conjugated to Alexa Fluor 555. Nuclei were marked with DAPI.

Fig. 9.

Subcellular localization of MVB marker CD63 and viral pp28 in wild-type and TBstop71 virus-infected HFFs (MOI = 0.1 to 0.5). Cells were fixed with 4% PFA in PBS at 7 days postinfection. CD63 and pp28 were detected by the respective MAbs and visualized by isotype-specific secondary antibodies conjugated to Alexa Fluor 488 (for pp28) and 555 (for CD63). Cell nuclei were visualized by using DAPI.