Human cytomegalovirus glycoprotein variants governing viral tropism and syncytium formation in epithelial cells and macrophages

Abstract

Human cytomegalovirus (HCMV) displays a broad cell tropism, and the infection of biologically relevant cells such as epithelial, endothelial, and hematopoietic cells supports viral transmission, systemic spread, and pathogenesis in the human host. HCMV strains differ in their ability to infect and replicate in these cell types, but the genetic basis of these differences has remained incompletely understood. In this study, we investigated HCMV strain VR1814, which is highly infectious for epithelial cells and macrophages and induces cell-cell fusion in both cell types. A VR1814-derived bacterial artificial chromosome (BAC) clone, FIX-BAC, was generated many years ago but has fallen out of favor because of its modest infectivity. By sequence comparison and genetic engineering of FIX, we demonstrate that the high infectivity of VR1814 and its ability to induce syncytium formation in epithelial cells and macrophages depends on VR1814-specific variants of the envelope glycoproteins gB, UL128, and UL130. We also show that UL130-neutralizing antibodies inhibit syncytium formation, and a FIX-specific mutation in UL130 is responsible for its low infectivity by reducing the amount of the pentameric glycoprotein complex in viral particles. Moreover, we found that a VR1814-specific mutation in US28 further increases viral infectivity in macrophages, possibly by promoting lytic rather than latent infection of these cells. Our findings show that variants of gB and the pentameric complex are major determinants of infectivity and syncytium formation in epithelial cells and macrophages. Furthermore, the VR1814-adjusted FIX strains can serve as valuable tools to study HCMV infection of myeloid cells.IMPORTANCEHuman cytomegalovirus (HCMV) is a major cause of morbidity and mortality in transplant patients and the leading cause of congenital infections. HCMV infects various cell types, including epithelial cells and macrophages, and some strains induce the fusion of neighboring cells, leading to the formation of large multinucleated cells called syncytia. This process may limit the exposure of the virus to host immune factors and affect pathogenicity. However, the reason why some HCMV strains exhibit a broader cell tropism and why some induce cell fusion more than others is not well understood. We compared two closely related HCMV strains and provided evidence that small differences in viral envelope glycoproteins can massively increase or decrease the virus infectivity and its ability to induce syncytium formation. The results of the study suggest that natural strain variations may influence HCMV infection and pathogenesis in humans.

Keywords: cell fusion; entry; glycoproteins; human cytomegalovirus; infectivity; strain variation; syncytium.

Fig 1

HCMV infectivity and syncytium formation in different cell types. HFF, MRC-5, and ARPE-19 cells were infected with HCMV strains VR1814, TB40/E, and FIX at an MOI of 1. HUVEC cells, THP-1-derived macrophages, and M1 and M2 macrophages were infected at an MOI of 5. (A through F) The percentage of infected cells was determined by immunostaining with an antibody recognizing IE1 and IE2. Bars represent the mean ± SEM of three independent experiments. (G) Syncytium formation was analyzed in the indicated cell types at 5 dpi. Cell nuclei were counterstained with DAPI. Scale bar, 20 µm.

Fig 2

Replication and infectivity of recombinant FIX strains on ARPE-19 cells. (A) Schematic of the recombinant FIX strains generated by BAC mutagenesis. They contain VR1814_GF-specific variants of UL55/gB, the Pentamer proteins UL128 and UL130, and the US28 Chemokine receptor. (B and C) ARPE-19 cells were infected at an MOI of 0.5. Supernatants were harvested at different times post-infection and titered on HFF cells. Viral titers are shown as mean ± SEM of three biological replicates. (D and E) ARPE-19 cells were infected at an MOI of 1. Whole-cell lysates were harvested 1, 3, and 5 dpi, and the levels of the viral proteins IE1 and IE2, UL44, and pp65 were analyzed by immunoblotting. (F) ARPE-19 cells were infected at an MOI of 1. Relative infectivities of the recombinant FIX strains were determined at 1 dpi as percentage of IE1/IE2-positive nuclei. Mean ± SEM of three independent experiments. Recombinant FIX strains were compared to FIX. Significance was determined by one-way ANOVA with Dunnett’s multiple comparison test. *P < 0.05; ***P < 0.001; ns, not significant. (G) The infectivities of the virus stocks on ARPE-19 cells were determined by quantifying viral genome copies by quantitative PCR (qPCR) and infectious units (IU) by titration. Infectivity is shown as IU per 1,000 viral genomes. Mean values ± SEM are shown.

Fig 3

Epithelial cell fusion induced by VR1814_GF and recombinant FIX strains. (A) ARPE-19 cells were infected at an MOI of 1, fixed at 5 dpi, and stained with an anti-IE1/2 antibody (red). Nuclei were counterstained with DAPI (blue). Syncytium formation was analyzed by microscopic inspection. All FIX strains express GFP. Scale bar, 20 µm. (B) DSP-expressing ARPE-19 cells were infected as described above. Renilla luciferase activity was measured at 5 dpi. Mean ± SEM of three independent experiments. Recombinant FIX strains were compared to FIX. Significance was determined by one-way ANOVA with Dunnett’s multiple comparison test. ***P < 0.001; ns, not significant. (C and D) DSP-expressing ARPE-19 cells were infected with VR1814_GF or FIX-BP and incubated with dilutions of an anti-UL130 hybridoma supernatant. (C) Syncytium formation was observed microscopically on day 5 post-infection and (D) quantified by measuring Renilla luciferase activity. Syncytium formation (mean ± SEM of two independent experiments with three biological replicates) is shown relative to cells treated with a non-neutralizing anti-pp71 hybridoma supernatant.

Fig 4

Impact of VR1814_GF and FIX-specific variants of UL128L on infectivity and cell-cell fusion. (A) Schematic of the recombinant FIX strains. (B) ARPE-19 cells were infected at an MOI of 1. Relative infectivities of the recombinant FIX strains were determined at 1 dpi as percentage of IE1/IE2-positive nuclei. Mean ± SEM of three independent experiments. Recombinant FIX strains were compared to FIX. Significance was determined by one-way ANOVA with Dunnett’s multiple comparison test. *P < 0.05; ***P < 0.001; ns, not significant. (C and D) Syncytium formation in DSP-expressing ARPE-19 cells was observed at 5 dpi. (C) Cells were stained with an anti-IE1/2 antibody and nuclei were counterstained with DAPI. Scale bar, 20 µm. (D) Syncytium formation was quantified by measuring Renilla luciferase activity. Mean ± SEM of three independent experiments. Significance was determined as described above. (E) Lysates of purified virions were analyzed by immunoblot. The gH/UL128 disulfide-linked complex, separated on a non-denaturing gel, was detected with an anti-UL128 antibody. UL130 and pp150 (loading control) were separated on a denaturing gel and detected with specific antibodies.

Fig 5

Infectivity of recombinant FIX strains and ability to induce syncytium formation in THP-1-derived macrophages. (A and B) THP-1 cells were differentiated into macrophages by PMA treatment for 3 days and infected at a MOI of 5. Relative infectivities of the recombinant FIX strains were determined at 1 and 5 dpi as percentage of IE1/IE2-positive nuclei. Mean ± SEM of three independent experiments. Recombinant FIX strains were compared to FIX. Significance was determined by one-way ANOVA with Dunnett’s multiple comparison test. **P < 0.01; ***P < 0.001; ns, not significant. (C) THP-1-derived macrophages were infected at an MOI of 5, and syncytium formation was analyzed on day 5 post-infection. Cells were stained with an anti-IE1/2 antibody, and nuclei were counterstained with DAPI. Syncytia are marked by white circles. Scale bar, 20 µm. (D) Syncytium formation was quantified at 5 dpi by microscopic inspection and counting. Mean ± SEM of three independent experiments. Significance was determined as described above.

Fig 6

Infectivity of recombinant FIX strains and ability to induce syncytium formation in monocyte-derived macrophages. (A and B) M1 and M2 macrophages were infected at a MOI of 5. Relative infectivity of recombinant FIX strains was analyzed at 1 dpi in M1 and M2 macrophages, respectively, as percentages of IE1/IE2-positive nuclei. (C and D) Syncytium formation was quantified at 5 dpi by microscopic inspection and counting in M1 and M2 macrophages, respectively. Bars represent the mean ± SEM of four experiments with macrophages from different blood donors. Recombinant FIX strains were compared to FIX. Significance was determined by one-way ANOVA with Dunnett’s multiple comparison test. ***P < 0.001; ns, not significant.