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

The advent of whole genome sequencing has revealed that common laboratory strains of human cytomegalovirus (HCMV) have major genetic deficiencies resulting from serial passage in fibroblasts. In particular, tropism for epithelial and endothelial cells is lost due to mutations disrupting genes UL128, UL130, or UL131A, which encode subunits of a virion-associated pentameric complex (PC) important for viral entry into these cells but not for entry into fibroblasts. The endothelial cell-adapted strain TB40/E has a relatively intact genome and has emerged as a laboratory strain that closely resembles wild-type virus. However, several heterogeneous TB40/E stocks and cloned variants exist that display a range of sequence and tropism properties. Here, we report the use of PacBio sequencing to elucidate the genetic changes that occurred, both at the consensus level and within subpopulations, upon passaging a TB40/E stock on ARPE-19 epithelial cells. The long-read data also facilitated examination of the linkage between mutations. Consistent with inefficient ARPE-19 cell entry, at least 83% of viral genomes present before adaptation contained changes impacting PC subunits. In contrast, and consistent with the importance of the PC for entry into endothelial and epithelial cells, genomes after adaptation lacked these or additional mutations impacting PC subunits. The sequence data also revealed six single noncoding substitutions in the inverted repeat regions, single nonsynonymous substitutions in genes UL26, UL69, US28, and UL122, and a frameshift truncating gene UL141. Among the changes affecting protein-coding regions, only the one in UL122 was strongly selected. This change, resulting in a D390H substitution in the encoded protein IE2, has been previously implicated in rendering another viral protein, UL84, essential for viral replication in fibroblasts. This finding suggests that IE2, and perhaps its interactions with UL84, have important functions unique to HCMV replication in epithelial cells.