Specialization for Cell-Free or Cell-to-Cell Spread of BAC-Cloned Human Cytomegalovirus Strains Is Determined by Factors beyond the UL128-131 and RL13 Loci

Abstract

It is widely held that clinical isolates of human cytomegalovirus (HCMV) are highly cell associated, and mutations affecting the UL128-131 and RL13 loci that arise in culture lead to the appearance of a cell-free spread phenotype. The bacterial artificial chromosome (BAC) clone Merlin (ME) expresses abundant UL128-131, is RL13 impaired, and produces low infectivity virions in fibroblasts, whereas TB40/e (TB) and TR are low in UL128-131, are RL13 intact, and produce virions of much higher infectivity. Despite these differences, quantification of spread by flow cytometry revealed remarkably similar spread efficiencies in fibroblasts. In epithelial cells, ME spread more efficiently, consistent with robust UL128-131 expression. Strikingly, ME spread far better than did TB or TR in the presence of neutralizing antibodies on both cell types, indicating that ME is not simply deficient at cell-free spread but is particularly efficient at cell-to-cell spread, whereas TB and TR cell-to-cell spread is poor. Sonically disrupted ME-infected cells contained scant infectivity, suggesting that the efficient cell-to-cell spread mechanism of ME depends on features of the intact cells such as junctions or intracellular trafficking processes. Even when UL128-131 was transcriptionally repressed, cell-to-cell spread of ME was still more efficient than that of TB or TR. Moreover, RL13 expression comparably reduced both cell-free and cell-to-cell spread of all three strains, suggesting that it acts at a stage of assembly and/or egress common to both routes of spread. Thus, HCMV strains can be highly specialized for either for cell-free or cell-to-cell spread, and these phenotypes are determined by factors beyond the UL128-131 or RL13 loci. IMPORTANCE Both cell-free and cell-to-cell spread are likely important for the natural biology of HCMV. In culture, strains clearly differ in their capacity for cell-free spread as a result of differences in the quantity and infectivity of extracellular released progeny. However, it has been unclear whether “cell-associated” phenotypes are simply the result of poor cell-free spread or are indicative of particularly efficient cell-to-cell spread mechanisms. By measuring the kinetics of spread at early time points, we were able to show that HCMV strains can be highly specialized to either cell-free or cell-to-cell mechanisms, and this was not strictly linked the efficiency of cell-free spread. Our results provide a conceptual approach to evaluating intervention strategies for their ability to limit cell-free or cell-to-cell spread as independent processes.

Neutralizing Antibodies Limit Cell-Associated Spread of Human Cytomegalovirus in Epithelial Cells and Fibroblasts

“…Our data demonstrate that mAbs against the essential viral glycoproteins B and H have the capacity to slow cell-associated HCMV spread. Interestingly and contrary to previous observations [38,75,76], we found that this inhibitory effect was independent of the target cell-type and the mAb concentrations as the respective anti-gB and anti-gH mAbs prevented HCMV spread equally efficiently in HFF and APRE-19 cells (Figure 4e,f). However, within the human host it seems possible that other cell types exist which may exhibit increased or reduced sensitivity to the mAbs that were tested in this study.…”

“…Fluorescence-based assays for quantitative measurement of HCMV spread have been described previously [46,49,76]. However, these assays have not been utilized to: (i) compare the viral spread in different target cell types of HCMV, (ii) have been performed with an only limited number of antigen target specificities or, (iii) not in a live cell imaging setting to allow for monitoring of plaque expansion in a timely manner.…”

Peptide Derivatives of Platelet-Derived Growth Factor Receptor Alpha Inhibit Cell-Associated Spread of Human Cytomegalovirus

“…Fibroblast-adapted HCMV strains that spread efficiently via the cell-free route also form small foci of infected cells in the presence of neutralizing antibodies, and this antibody-resistance is sometimes used to define cell-associated transmission of these strains [ 33 , 36 , 54 , 55 ]. This appears appropriate as other approaches to capture cell-free HCMV, e.g., by agarose or methyl cellulose overlays result in a similar focus size, cannot be further reduced by neutralizing antibodies [ 19 , 56 ].…”

The Human Cytomegalovirus UL116 Glycoprotein Is a Chaperone to Control gH-Based Complexes Levels on Virions

“…Trimer alone is sufficient for entry into fibroblasts ( Kabanova et al, 2016 ) whereas Pentamer, always required for infection in all other cell types ( Zhou et al, 2015 ; Kabanova et al, 2016 ), extends viral tropism through recognition of specific receptors recently identified ( Martinez-Martin et al, 2018 ; Xiaofei et al, 2019 ). Although cell type restricted receptors explain the tropism specificity, the molecular mechanism responsible for viral infectivity depends on several factors including glycoprotein isoforms, relative ratio of the complexes, RL13 locus and still non identified loci ( Stanton et al, 2010 ; Zhou et al, 2013 , 2015 ; Zhang et al, 2018 ; Schultz et al, 2020 ). In our system, we speculate that a 10-times reduction of infectivity, due to the absence of UL116, may be a consequence of the reduced levels of gH-based complexes on viral particles that cannot support priming of the whole population of gB impairing efficient membrane fusion in about 1/10 of the events.…”