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. 2003 Mar;77(5):3191-203.
doi: 10.1128/jvi.77.5.3191-3203.2003.

Envelopment of human cytomegalovirus occurs by budding into Golgi-derived vacuole compartments positive for gB, Rab 3, trans-golgi network 46, and mannosidase II

M Homman-Loudiyi  1 , K Hultenby, W Britt, C Söderberg-Nauclér
Affiliations

Envelopment of human cytomegalovirus occurs by budding into Golgi-derived vacuole compartments positive for gB, Rab 3, trans-golgi network 46, and mannosidase II

M Homman-Loudiyi et al. J Virol. 2003 Mar.

Erratum in

  • J Virol. Arch. 2003 Jul;77(14):8179

Abstract

Although considerable progress has been made towards characterizing virus assembly processes, assignment of the site of tegumentation and envelopment for human cytomegalovirus (HCMV) is still not clear. In this study, we examined the envelopment of HCMV particles in human lung fibroblasts (HF) HL 411 and HL 19, human umbilical vein endothelial cells, human pulmonary arterial endothelial cells, and arterial smooth muscle cells at different time points after infection by electron microscopy (EM), immunohistochemistry, and confocal microscopy analysis. Double-immunofluorescence labeling experiments demonstrated colocalization of the HCMV glycoprotein B (gB) with the Golgi resident enzyme mannosidase II, the Golgi marker TGN (trans-Golgi network) 46, and the secretory vacuole marker Rab 3 in all cell types investigated. Final envelopment of tegumented capsids was observed at 5 days postinfection by EM, when tegumented capsids budded into subcellular compartments located in the cytoplasm, in close proximity to the Golgi apparatus. Immunogold labeling and EM analysis confirmed staining of the budding compartment with HCMV gB, Rab 3, and mannosidase II in HL 411 cells. However, the markers Rab 1, Rab 2, Rab 7, Lamp 1 (late endosomes and lysosomes), and Lamp 2 (lysosomes) neither showed specific staining of the budding compartment in the immunogold labeling experiments nor colocalized with gB in the immunofluorescent colocalization experiments in any cell type studied. Together, these results suggest that the final envelopment of HCMV particles takes place mainly into a Golgi-derived secretory vacuole destined for the plasma membrane, which may release new infectious virus particles by fusion with the plasma membrane.

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Figures

FIG. 1.
FIG. 1.
HCMV buds into cytoplasmic vacuoles. Structural EM was performed on HCMV-infected human lung fibroblasts at 5 dpi. An overview of the HCMV replication cycle at 5 dpi in an HCMV-infected HF is shown. (A) Virus particles undergoing envelopment in cytoplasmic vacuoles are indicated by arrows. PM, plasma membrane; NM, nuclear membrane. (B) Three morphologically different capsids, A, B, and C, are formed in the nucleus. (C and D) Tegumented capsids bud into cytoplasmic vacuoles (V) to acquire their envelope. (E) Mature virus particles (VP) and dense bodies (DB) exit the cell by fusion of the transporting vacuole and the plasma membrane to release its content to the extracellular space.
FIG. 2.
FIG. 2.
Immunofluorescent staining localizing HCMV protein expression in HCMV-infected HF at 5 dpi. The tegument proteins pp28 (B), pp71 (C), and pp150 (D) are found in the cytoplasm and in high concentrations around the nuclear membrane (pp28 in panel B) and in the Golgi region (pp150 in panel D). A possible cytoplasmic site of tegument protein acquisition is showed in the EM micrographs (A and J), where tegumented capsids accumulate around structures of dense stained material. The viral glycoproteins gL (F), gH (G), and gB (H) are expressed in the Golgi apparatus and in punctuate structures in the cytoplasm. Notable is the dot-like staining underneath the plasma membrane and in the cytoplasm close to the Golgi apparatus. Naked A, B, and C capsids that have traversed the nuclear membrane lose the acquired membrane when entering the cytoplasm (I). The different cell types HL 411 (E), HL 19 (K), HUVEC (L), and HPAEC (N) showed the same mechanism of envelopment by budding into cytoplasmic vacuoles. PM, plasma membrane; NM, nuclear membrane.
FIG. 3.
FIG. 3.
Colocalization of gB (red) with the subcellular markers (green) mannosidase II (Man 2), Rab 3, Rab 4, and TGN 46 in HL 411. Colocalization was performed to identify the intracellular site of gB accumulation. Mannosidase II, Rab 3, and TGN 46 showed clear colocalizationwith gB in distinct vacuole compartments in the cytoplasm and in the Golgi region. The cytoplasmic, punctuate colocalization staining pattern of Rab 3, TGN 46, and mannosidase II is enlarged to show the distribution of cytoplasmic vacuoles (yellow dots). Rab 4 colocalized with gB to a lesser extent, but only in compartments next to the nuclear membrane or the Golgi stack. Uninfected cells stained with the same markers are shown in the left columns. The colocalization of Rab 3, mannosidase II, and TGN 46 with gB was confirmed in HPAEC, HUVEC, HL 19, and ASMC (bottom panels).
FIG. 4.
FIG. 4.
Colocalization of gB and the markers Rab 1, Rab 2, Rab 5, and Rab 6 in HCMV-infected HF cells at 5 dpi. Colocalization was performed to identify the intracellular site of gB accumulation. Rab 1, Rab 2, Rab 5, and Rab 6 did not show colocalization with gB. The four markers demonstrated a weak colocalization with gB in the Golgi apparatus but not in any intracellular compartment.
FIG. 5.
FIG. 5.
Immunogold localization of subcellular markers in HCMV-infected HF. Immunogold labeling was performed to identify the vacuole compartment detected by confocal microscopy. Secondary antibodies marked with colloidal gold were used to visualize the staining of gB (A), mannosidase II (B) Rab 3 (C), and Rab 5 (D). The HCMV gB, Rab 3, and mannosidase II antibodies stained the envelope membrane of virus particles (VP), dense bodies (DB), and the vacuole membrane (V).
FIG. 6.
FIG. 6.
Model of the HCMV envelopment and exit pathway. Naked nucleocapsids, formed in the nucleus, traverse the nuclear membrane and become tegumented through the exit pathway by an unknown process. Viral glycoproteins are released from the Golgi apparatus within small transport vacuoles. Fusion of these vacuoles results in the formation of large gB-, mannosidase II-, TGN 46-, and Rab 3-positive cytoplasmic vacuoles. The tegumented HCMV capsids become enveloped when budding into the vacuoles, acquiring the vacuole membrane as their envelope. The vacuoles are then transported to the plasma membrane, and virus particles are subsequently released upon fusion of the vacuoles with the cellular plasma membrane.
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