Cytomegalovirus Strain TB40/E Restrictions and Adaptations to Growth in ARPE-19 Epithelial Cells

Abstract

Despite displaying broad tropism in vivo, human cytomegalovirus (CMV) contained in bodily fluids replicates inefficiently in most cultured cell types except fibroblasts. As propagation in fibroblasts leads to the accumulation of genomic changes, a number of strains were generated by serial passaging on endothelial cells. One of these, TB40/E, was shown to contain a mixture of genetically distinct virus variants, and to retain tropism for fibroblasts, endothelial and epithelial cells. Cloning of an endotheliotropic subpopulation produced the TB40-BAC4 variant, extensively used in CMV tropism studies. Because TB40-BAC4 represents only one of the different variants comprising TB40/E, we generated a series of epithelial-cell adapted stocks derived from a TB40/E mixed stock, rather than from TB40-BAC4. Within two passages on ARPE-19 cells, virus populations were produced with the ability to enter and initiate replication with similar efficiencies in both epithelial cells and fibroblasts. Although the ability to release progeny also increased, cell-free virus yields from ARPE-19 cells remained consistently two to three-logs lower than from fibroblasts, hinting at the existence of a post-entry and post-genome synthesis block in epithelial cells. Multinucleated syncytia also rapidly appeared exclusively in ARPE-19 cell cultures, where their numbers and dimensions increased with virus passage. Irrespective of the number of infected nuclei comprising each syncytium, however, only one cytoplasmic virion assembly compartment was consistently observed, leading us to speculate that improvements in entry efficiency associated with ARPE-19 cell adaptation lead to the development of syncytia, which may negatively affect progeny release by limiting the amount of resources available to maturing virions.

Genome sequences of human cytomegalovirus strain TB40/E variants propagated in fibroblasts and epithelial cells

“…In summary, whole genome sequencing identi ed variants impacting IE2 and PC subunits U128 and UL130 as being potentially selected during adaptation of HCMV strain TB40/E for growth in epithelial cells. Enrichment of viral genomes lacking disruptive mutations in UL128 and UL130 is consistent with the detected improvement in e ciency of epithelial cell entry [15], and, as the PC has been associated…”

“…The long length of PacBio reads connected not only the two loci in UL128, which are separated by107 bp, but also the UL130 locus 814 bp beyond; among the connecting reads, those containing one mutation did not contain the others. Thus, consistent with the low epithelial cell entry e ciency of the TB40/EF stock [15], these ndings suggest that cumulatively 83% of TB40/EF genomes contain mutations potentially impacting PC assembly or function. In contrast, and consistent with e cient epithelial cell entry [15], mutations impacting PC subunits were absent from TB40/EE.…”

“…Thus, consistent with the low epithelial cell entry e ciency of the TB40/EF stock [15], these ndings suggest that cumulatively 83% of TB40/EF genomes contain mutations potentially impacting PC assembly or function. In contrast, and consistent with e cient epithelial cell entry [15], mutations impacting PC subunits were absent from TB40/EE.…”

“…with increased cell-cell fusion [22][23][24][25], may also explain the reported increase in syncytium formation [15].…”

“…Given the low e ciency of epithelial cell entry observed for TB40/EF, mutations disrupting UL128, UL130, or UL131A (encoding the PC subunit proteins UL128, UL130, and UL131A, respectively), were anticipated. Indeed, targeted sequencing of TB40/EF had previously identi ed a suppressor substitution converting the UL128 stop codon (TGA) to TTA (encoding leucine), thereby extending UL128 by 19 residues [15]. Although the consensus genome sequence of TB40/EF did not re ect this mutation, examination of read frequencies revealed the existence of a subpopulation in which 30% of TB40/EF genomes contain this suppressor mutation.…”