Human cytomegalovirus labeled with green fluorescent protein for live analysis of intracellular particle movements - PubMed (original) (raw)

Human cytomegalovirus labeled with green fluorescent protein for live analysis of intracellular particle movements

Kerstin Laib Sampaio et al. J Virol. 2005 Mar.

Abstract

Human cytomegalovirus (HCMV) replicates in the nuclei of infected cells. Successful replication therefore depends on particle movements between the cell cortex and nucleus during entry and egress. To visualize HCMV particles in living cells, we have generated a recombinant HCMV expressing enhanced green fluorescent protein (EGFP) fused to the C terminus of the capsid-associated tegument protein pUL32 (pp150). The resulting UL32-EGFP-HCMV was analyzed by immunofluorescence, electron microscopy, immunoblotting, confocal microscopy, and time-lapse microscopy to evaluate the growth properties of this virus and the dynamics of particle movements. UL32-EGFP-HCMV replicated similarly to wild-type virus in fibroblast cultures. Green fluorescent virus particles were released from infected cells. The fluorescence stayed associated with particles during viral entry, and fluorescent progeny particles appeared in the nucleus at 44 h after infection. Surprisingly, strict colocalization of pUL32 and the major capsid protein pUL86 within nuclear inclusions indicated that incorporation of pUL32 into nascent HCMV particles occurred simultaneously with or immediately after assembly of the capsid. A slow transport of nuclear particles towards the nuclear margin was demonstrated. Within the cytoplasm, most particles performed irregular short-distance movements, while a smaller fraction of particles performed centripetal and centrifugal long-distance movements. Although numerous particles accumulated in the cytoplasm, release of particles from infected cells was a rare event, consistent with a release rate of about 1 infectious unit per h per cell in HCMV-infected fibroblasts as calculated from single-step growth curves. UL32-EGFP-HCMV will be useful for further investigations into the entry, maturation, and release of this virus.

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Figures

FIG. 1.

(A) Flow chart of the generation of recombinant UL32-EGFP-HCMV-TB40. The boldface letter in the sequence indicates the amino acid exchange (K1054H) at the C terminus of the UL32 open reading frame. (B) Colocalization of pUL32 (red) and EGFP (green) detected by indirect-immunofluorescence assays in viral plaques after infection of fibroblasts with plaque-purified UL32-EGFP-HCMV-TB40.

FIG. 2.

Growth of recombinant UL32-EGFP-HCMV-TB40 in human foreskin fibroblasts. (A) Kinetics of viral antigen expression. Viral immediate-early antigen (pUL122/123), early antigen (pUL44), and late antigen (pUL86) were detected in infected fibroblasts by indirect immunofluorescence. The first time point of appearance is indicated. (B) Single-step growth curves of wild-type HCMV strain TB40 and the recombinant HCMV strain UL32-EGFP-HCMV-TB40. Fibroblast cultures were infected at an MOI of 1, and infectious virus progeny in thesupernatants of infected cultures was determined daily by limiting-dilution analyses. (C) Multistep growth curves of wild-type HCMV strain TB40 and the recombinant HCMV strain UL32-EGFP-HCMV-TB40. Fibroblast cultures were infected at an MOI of 0.08, and infectious virus progeny in the supernatants of infected cultures was determined daily by limiting-dilution analyses. (D) Subcellular colocalization of pUL32 (red) and EGFP (green) detected by indirect-immunofluorescence assays in infected fibroblasts 4 days after infection with UL32-EGFP-HCMV-TB40. For comparison, the inset displays subcellular localization of pUL32 (red) in infected fibroblasts 4 days after infection with wild-type (wt) HCMV-TB40.

FIG. 3.

Microscopic and biochemical analysis of green fluorescent virus UL32-EGFP-HCMV particles. (A) Detection of green fluorescent UL32-EGFP-HCMV-TB40 particles in cultured fibroblasts by merge of fluorescence and phase-contrast micrographs under conditions of viral adsorption. (B) Detection of gradient-purified UL32-EGFP-HCMV-TB40 virions in cultured fibroblasts by merge of fluorescence micrograph and DAPI stain under conditions of viral adsorption. (C) Detection of GFP and pUL32 in infected fibroblasts at 1 h after infection with UL32-GFP-HCMV-TB40 or HCMV-TB40 or a mixture of both virus strains. Almost all UL32-GFP-HCMV-TB40 particles are detected by the GFP antibody. In contrast, no wild-type particles are detected by the GFP antibody, proving the specificity of the staining. In coinfections, the different strains can by discriminated. (D) Ultrastructural localization of the pUL32-EGFP fusion protein after adsorption of gradient-purified UL32-EGFP-HCMV-TB40 virions or wild-type virions to cultured fibroblasts. EGFP was detected by immunogold labeling on ultrathin cryosections. (E) Immunoblotting of protein lysates of UL32-EGFP-HCMV-TB40-particles versus wild-type HCMV-TB40 particles, using a primary antibody against pUL32. (F) Detection of PCR amplification products specific for wild-type virus (634 bp) and recombinant virus (1,378 bp) in DNA preparations from wild-type HCMV-TB40 and recombinant UL32-EGFP-HCMV, using a primer pair spanning the UL31-UL32 transition region.

FIG. 3.

FIG. 4.

Kinetics of appearance and intracellular localization of UL32-EGFP-HCMV-TB40 particles during the replication cycle. (A to D) Appearance of green fluorescent particles in a living infected fibroblast at 44 h (A), 48 h (B), 50 h (C), and 51 h (D) after infection at an MOI of 0.05. (E) Subcellular localization of green fluorescent particles displayed by an overlay of fluorescence microscopy and phase-contrastmicroscopy of a living infected cell at 65 h p.i. (F) Subcellular localization of green fluorescent particles as detected by confocal laser scanning microscopy of an acetone-fixed infected cell at 72 h p.i. Indirect immunofluorescence of lamin B (Cy3, red) and EGFP (Alexa 488, green) versus DNA staining (DAPI, blue) is shown. (G) Intranuclear colocalization of the tegument protein pUL32 and the major capsid protein pUL86 by confocal laser scanning microscopy of a paraformaldehyde-fixed infected cell at 72 h p.i. The native fluorescence of pUL32-EGFP is displayed versus indirect immunofluorescence of pUL86 (Cy3, red) and DNA staining (DAPI, blue). (H) Ultrastructural localization of the pUL32-EGFP fusion protein. Fibroblast cultures at 4 days after infection with UL32-EGFP-HCMV-TB40 or with wild-type virus were fixed and cryosectioned. EGFP was detected by immunogold labeling on ultrathin cryosections. Examples of viral capsids with associated pUL32-EGFP are indicated by arrowheads.

FIG. 4.

FIG. 5.

Live imaging of particle movements in fibroblasts infected by UL32-EGFP-HCMV-TB40 as documented by time-lapse fluorescence microscopy. (A) Centripetal and centrifugal movements in fibroblasts 1 h after infection at an MOI of 10. White arrows, actual position of the particle; gray arrows, starting point of the particle. Selected frames from videos are shown. (B) Particle movements in an infected cell at 4 days after infection at an MOI of 0.1. The light gray arrow points to a particle moving parallel to the cell margin. The black arrow points to the perinuclear aggregation site. The dark gray arrow indicates the starting point of a particle appearing to be leaving the nucleus. The paired white arrows indicate routes of saltatory movements. (C) Statistical analysis of particle movements in fibroblasts 3 days after infection with UL32-EGFP-HCMV-TB40. (D) Time-lapse series suggestive of release of a particle at the cell periphery of a fibroblast at 4 days after infection at an MOI of 0.1. Selected frames from a video are shown.

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Human cytomegalovirus labeled with green fluorescent protein for live analysis of intracellular particle movements - PubMed (original) (raw)