Human cytomegalovirus UL131 open reading frame is required for epithelial cell tropism - PubMed (original) (raw)

Human cytomegalovirus UL131 open reading frame is required for epithelial cell tropism

Dai Wang et al. J Virol. 2005 Aug.

Abstract

Epithelial cells are one of the prominent cell types infected by human cytomegalovirus (HCMV) within its host. However, many cultured epithelial cells, such as ARPE-19 retinal pigmented epithelial cells, are poorly infected by laboratory-adapted strains in cell culture, and little is known about the viral factors that determine HCMV epithelial cell tropism. In this report, we demonstrate that the UL131 open reading frame (ORF), and likely the entire UL131-128 locus, is required for efficient infection of epithelial cells. Repair of the mutated UL131 gene in the AD169 laboratory strain of HCMV restored its ability to infect both epithelial and endothelial cells while compromising its ability to replicate in fibroblasts. ARPE-19 epithelial cells support replication of the repaired AD169 virus as well as clinical isolates of HCMV. Productive infection of cultured epithelial cells, endothelial cells, and fibroblasts with the repaired AD169 virus leads to extensive membrane fusion and syncytium formation, suggesting that the virus may spread through cell-cell fusion.

PubMed Disclaimer

Figures

FIG. 1.

HCMV UL131-128 locus. (A) Diagram of the locus. The positions of transcriptional start sites and poly(A) cleavage sites are indicated. Solid boxes represent the sequence of wild-type ORFs. The location of the point mutation in the AD169 UL131 gene is indicated, and the portion of UL131 that is not expressed is designated by an open box. (B) Amino acid sequence of the N-terminal domain of UL131 in two repaired AD169 derivatives, BAD_r_UL131-Y4 and BAD_r_UL131-C4, and HCMV variant strains. The likely signal peptide cleavage sites are indicated.

FIG. 2.

Characterization of endothelial cell and fibroblast infection of UL131-repaired AD169 derivatives. (A) BAD_wt_, BAD_r_UL131-Y4, and BAD_r_UL131-C4 infection of endothelial cells. HUVECs or human LMVECs were infected at a multiplicity of 1 PFU/cell, and cells were fixed 48 h after infection. Nuclei were stained with DAPI (blue), and infected cells were identified by GFP expression (green) from a marker cassette on the viruses. (B) Syncytium formation induced by BAD_r_UL131 viruses. MRC-5 cells or HUVEC cells were infected at a multiplicity of 0.1 PFU/cell; 5 days after infection, the cells were fixed with 2% paraformaldehyde, the nuclei of the cells were stained with DAPI (blue), and GFP expression (green) demonstrated that fused cells were infected.

FIG. 3.

Growth kinetics of UL131-repaired AD169 derivatives in MRC-5 cells and HUVEC cells. Cells were infected with BAD_wt_ (•), BAD_r_UL131-Y4 (○), or BAD_r_UL131-C4 (▾) at a multiplicity of 1 or 0.01 PFU/cell. Cell-free and cell-associated virus was collected at the indicated times after infection, and infectious virus was quantified by TCID50 assay on MRC-5 cells.

FIG. 4.

Cell tropism of a UL131-rapaired AD169 derivative. (A) Identification of infected HeLa and MCF-7 cells by fluorescence. Cells were infected with BAD_r_UL131-Y4 at a multiplicity of 1 PFU/cell and fixed 48 h after infection. Nuclei were stained with DAPI (blue), and infected cells were identified by IE1 antigen staining (red) and GFP expression (green) from a marker cassette on the viruses. (B) Quantification of UL131-UL128 locus-dependent host cell tropism. Cultures of epithelial cells, endothelial cells, and fibroblasts were infected at a multiplicity of 1 PFU/cell with BAD_wt_ (black bars) or BAD_r_UL131-Y4 (gray bars); 48 h later, cells were fixed with 2% paraformaldehyde, and infected cells were identified by monitoring GFP expression.

FIG.5.

Infection of ARPE-19 and HeLa epithelial cells by UL131-repaired AD169 derivatives. (A) Growth kinetics of BAD_wt_, BAD_r_UL131-Y4, and BAD_r_UL131-C4 in ARPE-19 cells. Cells were infected at a multiplicity of 1 or 0.01 PFU/cell, cell-free (•) and cell-associated (○) viruses were collected at the indicated times, and infectious virus was quantified by TCID50 assay on MRC-5 cells. (B) Induction of cell-cell fusion by BAD_r_UL131 viruses infection in ARPE-19 cells. ARPE-19 cells were infected at a multiplicity of 0.1 PFU/cell, and cultures were observed 3 or 7 days later by phase contrast and fluorescent microscopy. (C) Growth kinetics of BAD_r_UL131-Y4 in HeLa cells. HeLa cells were infected at a multiplicity of 0.01 PFU/cell, cell-free (•) and cell-associated (○) virus was collected at the indicated times, and infectious virus was quantified by TCID50 assay on MRC-5 cells. (D) Cytopathic effect of BAD_r_UL131-Y4 and BAD_r_UL131-C4 in HeLa cells. HeLa cells were infected at a multiplicity of 1 PFU/cell, and cells were observed 3 or 7 days later by fluorescent microscopy.

FIG.6.

Cell tropism of HCMV strains. (A) Diagram of the UL131-UL128 locus in the Towne, Toledo, FIX, and TR strains of HCMV. The positions of transcriptional start sites and poly(A) cleavage sitesare indicated. Solid boxes represent wild-type ORFs. The point mutation in the Towne UL130 gene and the deletion in the Toledo UL128 gene are shown, and the positions of open reading frames whose expression is disrupted are represented by open boxes. (B) Entry of HCMV strains into MRC-5, HUVEC, HeLa, and ARPE-19 cells. Cells were infected at a multiplicity of 1 PFU/cell, and 48 h later, the cultures were fixed with 2% paraformaldehyde, and infected cells were identified by IE1 expression. (C) Production of infectious progeny by HCMV strains in ARPE-19 cells. Cells were infected at a multiplicity of 1 PFU/cell. Six days (BAD_r_UL131-Y4) or 12 days (all other viruses) later, the cells and media were collected together, samples were frozen and thawed three times, and virus titers were determined by TCID50 assay on MRC-5 cells.

References

1. Akter, P., C. Cunningham, B. P. McSharry, A. Dolan, C. Addison, D. J. Dargan, A. F. Hassan-Walker, V. C. Emery, P. D. Griffiths, G. W. Wilkinson, and A. J. Davison. 2003. Two novel spliced genes in human cytomegalovirus. J. Gen. Virol. 84:1117-1122. - PubMed
1. Bacsi, A., J. Aranyosi, Z. Beck, P. Ebbesen, I. Andirko, J. Szabo, L. Lampe, J. Kiss, L. Gergely, and F. D. Toth. 1999. Placental macrophage contact potentiates the complete replicative cycle of human cytomegalovirus in syncytiotrophoblast cells: role of interleukin-8 and transforming growth factor-beta1. J. Interferon Cytokine Res. 19:1153-1160. - PubMed
1. Baines, J. D., P. L. Ward, G. Campadelli-Fiume, and B. Roizman. 1991. The UL20 gene of herpes simplex virus 1 encodes a function necessary for viral egress. J. Virol. 65:6414-6424. - PMC - PubMed
1. Bodaghi, B., M. E. Slobbe-van Drunen, A. Topilko, E. Perret, R. C. Vossen, M. C. van Dam-Mieras, D. Zipeto, J. L. Virelizier, P. LeHoang, C. A. Bruggeman, and S. Michelson. 1999. Entry of human cytomegalovirus into retinal pigment epithelial and endothelial cells by endocytosis. Investig. Ophthalmol. Vis. Sci. 40:2598-2607. - PubMed
1. Booth, J. C., J. E. Beesley, and H. Stern. 1978. Syncytium formation caused by human cytomegalovirus in human embryonic lung fibroblasts. Arch. Virol. 57:143-152. - PubMed

Human cytomegalovirus UL131 open reading frame is required for epithelial cell tropism - PubMed (original) (raw)