Evidence for direct transfer of cytoplasmic material from infected to uninfected cells during cell-associated spread of human cytomegalovirus

Cell-associated spread is assumed to be the predominant mode of human cytomegalovirus (HCMV) dissemination in infected patients, however the underlying mechanisms are poorly understood. We tested the hypothesis that cell-to-cell spread of HCMV may be associated with direct transfer of cytoplasmic material by analyzing focal growth of green fluorescent HCMV Delta UL16GFP. In this recombinant virus, UL16 was partially replaced by the green fluorescent protein (EGFP). The resulting HCMV Delta UL16GFP showed unrestricted growth and expressed EGFP from the early UL16 promoter. EGFP transmission was then investigated in relation to viral spread from productively infected cells to cocultured uninfected cells. Alternatively, microinjection of fluorescent dextrane allowed for direct visualization of inter-cell-connections. Within 5 h of coculture, 8% of cells neighbouring productively infected cells had acquired EGFP. Detection of EGFP in the absence of IE antigen and during cycloheximide block excluded the possibility of de novo synthesis. Immediate distribution of microinjected fluorescent dyes from infected cells to adjacent cells proved the existence of cell-cell-fusions. These data, demonstrate that focal spread of HCMV is associated with direct transfer of cytoplasmic material, most likely through cell-cell-fusions. This would withdraw the virus from the control of neutralizing antibodies and thus provide an explanation for the limited antiviral effect of the humoral immune response.