A Virus Genetic System to Analyze the Fusogenicity of Human Cytomegalovirus Glycoprotein B Variants

Abstract

Viruses can induce the fusion of infected and neighboring cells, leading to the formation of syncytia. Cell-cell fusion is mediated by viral fusion proteins on the plasma membrane of infected cells that interact with cellular receptors on neighboring cells. Viruses use this mechanism to spread rapidly to adjacent cells or escape host immunity. For some viruses, syncytium formation is a hallmark of infection and a known pathogenicity factor. For others, the role of syncytium formation in viral dissemination and pathogenicity remains poorly understood. Human cytomegalovirus (HCMV) is an important cause of morbidity and mortality in transplant patients and the leading cause of congenital infections. Clinical HCMV isolates have broad cell tropism but differ in their ability to induce cell-cell fusions, and little is known about the molecular determinants. We developed a system to analyze HCMV glycoprotein B (gB) variants in a defined genetic background. HCMV strains TB40/E and TR were used as vectors to compare the fusogenicity of six gB variants from congenitally infected fetuses with those from three laboratory strains. Five of them conferred the ability to induce the fusion of MRC-5 human embryonic lung fibroblasts to one or both backbone strains, as determined by a split GFP-luciferase reporter system. The same gB variants were not sufficient to induce syncytia in infected ARPE-19 epithelial cells, suggesting that additional factors are involved. The system described here allows a systematic comparison of the fusogenicity of viral envelope glycoproteins and may help to clarify whether fusion-promoting variants are associated with increased pathogenicity.

Keywords: UL55; cell–cell fusion; entry; glycoprotein B; human herpesvirus 5; infectivity.

Figure 1

Amino acid sequence alignment of HCMV gBs discussed in this study. The D275Y and S585G variants are marked with an asterisk (*).

Figure 1

Amino acid sequence alignment of HCMV gBs discussed in this study. The D275Y and S585G variants are marked with an asterisk (*).

Figure 2

Construction of recombinant HCMV TB40 strains by BAC mutagenesis. (A) An optimized workflow was designed for the exchange of gB. (B) Restriction fragments of the parental TB40 BAC, TB40_ΔgB, and recombinant BACs carrying the gB of other strains. Expected differences in the restriction patterns are indicated by arrows.

Figure 3

Adaptation of the RLuc-GFP dual split protein (DSP) reporter system to detect HCMV-induced cell–cell fusion. (A) Fusion of cells expressing DSP 1–7 and DSP 8–11, respectively, leads to complementation of the split GFP and RLuc proteins. (B) Generation of DSP-expressing MRC-5 and ARPE-19 cells by lentiviral transduction. (C) Visualization of syncytia using the DSP system. Syncytia containing the complete RLuc-GFP are green fluorescent. The IE1 and IE2 proteins were detected by indirect immunofluorescence, and nuclei were stained with DAPI. Scale bar, 20 µm.

Figure 4

HCMV-induced cell–cell fusion of MRC-5 fibroblasts. Cells expressing the DSP system were infected at an MOI of 1. Cell–cell fusion was detected 3 days post-infection by measuring Renilla luciferase activity (A,B) or by the detection of GFP fluorescence (C). The bar diagrams show means ± SD of three biological replicates. Scale bar, 20 µm.

Figure 5

Cell–cell fusion and infectivity in ARPE-19 epithelial cells. (A,B) ARPE-19 cells expressing the DSP system were infected at an MOI of 1. Renilla luciferase activity was measured 3 days post-infection. (C,D) ARPE-19 cells were infected at an MOI of 1. The percentage of IE-antigen-positive cells was determined 3 days post-infection by immunofluorescence. The bar diagrams show means ± SD of three biological replicates.

Figure 6

Infectivity of recombinant TB40 strains. (A) MRC5 cells were HCMV-infected at an MOI of 0.5. The viral tegument protein pp150 and the IE1 and IE2 proteins were detected by immunoblot analysis. (B) For better comparison, selected samples shown in panel A were separated and analyzed on the same polyacrylamide gel. hpi, hours post-infection.

Figure 7

Inhibition of virus entry and IE gene expression by the macropinocytosis inhibitor EIPA. MRC-5 cells were pretreated for 30 min with 50 μM of EIPA and infected in the presence of EIPA at an MOI of 0.5. Two hours post-infection (hpi), EIPA was removed and fresh medium was added. Cells were harvested at the indicated times and IE1/IE2 expression was analyzed by immunoblot. IE1 expression levels at 16 and 24 hpi were measured by densitometry and normalized to β-actin levels. Numbers indicate IE protein levels relative to the levels at 16 hpi in untreated (−EIPA) cells. IntDen, integrated density.