An endothelial cell-specific requirement for the UL133-UL138 locus of human cytomegalovirus for efficient virus maturation

Abstract

Human cytomegalovirus (HCMV) infects a variety of cell types in humans, resulting in a varied pathogenesis in the immunocompromised host. Endothelial cells (ECs) are considered an important target of HCMV infection that may contribute to viral pathogenesis. Although the viral determinants important for entry into ECs are well defined, the molecular determinants regulating postentry tropism in ECs are not known. We previously identified the UL133-UL138 locus encoded within the clinical strain-specific ULb' region of the HCMV genome as important for the latent infection in CD34(+) hematopoietic progenitor cells (HPCs). Interestingly, this locus, while dispensable for replication in fibroblasts, was required for efficient replication in ECs infected with the TB40E or fusion-inducing factor X (FIX) HCMV strains. ECs infected with a virus lacking the entire locus (UL133-UL138(NULL) virus) complete the immediate-early and early phases of infection but are defective for infectious progeny virus production. ECs infected with UL133-UL138(NULL) virus exhibited striking differences in the organization of intracellular membranes and in the assembly of mature virions relative to ECs infected with wild-type (WT) virus. In UL133-UL138(NULL) virus-infected ECs, Golgi stacks were disrupted, and the viral assembly compartment characteristic of HCMV infection failed to form. Further, progeny virions in UL133-UL138(NULL) virus-infected ECs inefficiently acquired the virion tegument and secondary envelope. These defects were specific to infection in ECs and not observed in fibroblasts infected with UL133-UL138(NULL) virus, suggesting an EC-specific requirement for the UL133-UL138 locus for late stages of replication. To our knowledge, the UL133-UL138 locus represents the first cell-type-dependent, postentry tropism determinant required for viral maturation.

Fig 1

UL133-UL138 is required for viral replication in ECs. HMVEC were infected with WT or UL133-UL138_NULL virus of either the TB40E (A) or FIX (B) strain at an MOI of 0.05, and virus yields were measured over a time course by TCID₅₀ on fibroblasts. WT is represented by the filled squares (A) or dark bars (B). UL133-UL138_NULL virus is represented by the open circles (A) or light bars (B). The values plotted are averages from three independent experiments, and the standard deviations are indicated by the error bars for each time point. In some cases, the error bars are too small to be seen. In panel B, the asterisks indicate values at the limit of detection.

Fig 2

UL133-UL138 is not required for virus entry or genome transit to the nucleus in ECs. HMVEC were infected with TB40E-WT or -UL133-UL138_NULL virus at an MOI of 0.02. Infected (GFP⁺) cells were analyzed at 48 hpi by flow cytometry. (A) Representative dot plots with the percentage of infected cells in each infection indicated. (B) The average mean fluorescence intensities for four independent experiments for each infection are shown, and the standard deviations are indicated by the error bars. FSC, forward scatter.

Fig 3

Accumulation of IE, early, and late proteins in infected HMVEC. HMVEC were infected with TB40E-WT or -UL133-UL138_NULL virus at an MOI of 2. (A) Protein lysates harvested over a time course were analyzed by immunoblotting using antibodies specific to the IE1 and IE2 proteins, the UL44 early protein, and the pp28 late protein. β-Actin serves as a loading control. Quantification of average IE1 (B) and pp28 (C) protein levels (normalized to actin) in at least four independent experiments is shown. The standard deviation is indicated. Student's t test indicates that the differences between WT and UL133-UL138_NULL virus at each of the time points for IE1 (P ≥ 0.2) and pp28 (P ≥ 0.1) are not significant.

Fig 4

UL133-UL138 is required for maintaining cytoplasmic membrane organization and for virion maturation. HMVEC were infected with WT or UL133-UL138_NULL virus at an MOI of 4. Cells were fixed, embedded, and sectioned for transmission electron microscopy at 5 dpi. Representative micrographs are shown to illustrate the accumulation of nucleocapsids (A and B), the organization of cytoplasmic membranes (C and D), the formation of MVBs (E and F), and the maturation of cytoplasmic virions (G and H). N and C indicate nucleus and cytoplasm, respectively; DB indicates dense bodies. Open arrowheads, nucleocapsids (A and B); filled arrowheads, Golgi stacks (C) or vesicles (D); filled arrows, MVBs (E and F); open arrows, cytoplasmic virus particles (G and H). Scale bars, 2 μm (A to D) and 0.5 μm (E to H).

Fig 4

UL133-UL138 is required for maintaining cytoplasmic membrane organization and for virion maturation. HMVEC were infected with WT or UL133-UL138_NULL virus at an MOI of 4. Cells were fixed, embedded, and sectioned for transmission electron microscopy at 5 dpi. Representative micrographs are shown to illustrate the accumulation of nucleocapsids (A and B), the organization of cytoplasmic membranes (C and D), the formation of MVBs (E and F), and the maturation of cytoplasmic virions (G and H). N and C indicate nucleus and cytoplasm, respectively; DB indicates dense bodies. Open arrowheads, nucleocapsids (A and B); filled arrowheads, Golgi stacks (C) or vesicles (D); filled arrows, MVBs (E and F); open arrows, cytoplasmic virus particles (G and H). Scale bars, 2 μm (A to D) and 0.5 μm (E to H).

Fig 5

UL133-UL138 is dispensable for mature virion formation in infected fibroblasts. MRC-5 fibroblasts were infected with WT (top panel) or UL133-UL138_NULL (bottom panel) virus at an MOI of 2. At 5 dpi, cells were fixed, embedded, and sectioned for transmission electron microscopy. Representative micrographs are shown to illustrate the presence of Golgi stacks and mature virions in each infection. N and C indicate nucleus and cytoplasm, respectively; DB indicates dense bodies. Filled arrowheads, Golgi stacks; open arrows, virions. Scale bar, 0.5 μm.

Fig 6

UL133-UL138_NULL virus-infected ECs fail to form assembly compartments. HMVEC were infected with WT or UL133-UL138_NULL virus at an MOI of 2 or mock infected. At 144 h postinfection, cells were processed for indirect immunofluorescence using antibodies specific to the GM130 Golgi marker or pp28 viral protein. The nuclei are indicated by DAPI staining. Localization was visualized by confocal microscopy using a Zeiss 510 Meta Confocal microscope. Panel B contains low-magnification fields of cells to demonstrate the range of AC morphology in each infection. Magnification, ×60 (A) and ×40 (B).

Fig 7

Assembly compartment formation in fibroblasts is similar for WT and UL133-UL138_NULL virus infection. MRC-5 fibroblasts were infected with WT or UL133-UL138_NULL virus at an MOI of 2 or mock infected. At 96 hpi, cells were processed for indirect immunofluorescence using antibodies specific to giantin (A), GS27 (B), EEA1 (C), CD63 (D), pp28 (E), or gB (F). Infection of the cells imaged was determined by GFP fluorescence (data not shown). The nuclei are indicated by DAPI staining. Localization was visualized by confocal microscopy using a Zeiss 510 Meta Confocal microscope. Magnification, ×60.

Fig 8

Analysis of cellular membrane organization and viral assembly compartment markers in WT and UL133-UL138_NULL virus infection in ECs. HMVEC were infected with WT or UL133-UL138_NULL virus at an MOI of 2 or mock infected. At 144 hpi, cells were processed for indirect immunofluorescence using antibodies specific to giantin (A), GS27 (B), EEA1 (C), CD63 (D), pp28 (E), or gB (F). Infection of the cells imaged was determined by GFP fluorescence (data not shown). The nuclei are indicated by DAPI staining. Localization was visualized by confocal microscopy using a Zeiss 510 Meta Confocal microscope. Magnification, ×60.