Developing a Human Cytomegalovirus strain for better in vitro research

Investigations into Human Cytomegalovirus (HCMV) pathogenesis should be based on strains that closely reflect the causative agent of disease, however HCMV invariably mutates in vitro, generating phenotypically distinct laboratory-adapted strains. In particular, mutations are selected in the HCMV genome UL128 locus (UL128L) that encodes sub-units of a virion envelope pentameric complex that impedes virus propagation in fibroblasts (the cell type most commonly used in vitro), but is required for infection of several naturally targeted cell-types (e.g. epithelial, endothelial, and myeloid cells). Addressing this issue, the genome of wildtype HCMV strain Merlin was cloned as a stable bacterial artificial chromosome (BAC), however similarly to clinical isolates, viruses reconstituted from the Merlin-BAC grow poorly and are prone to de novo mutation in cell culture. Direct comparison to viruses from the Merlin-BAC revealed that viruses from the BACcloned versions of strains TR (TR-BAC), TB40E (TB40-BAC4) and VR1814 (FIX-BAC) could be propagated more efficiently in fibroblasts, despite containing intact UL128L ORFs. Unique nucleotide variations identified in TB40-BAC4 and FIX-BAC UL128L ORFs were transferred into the Merlin-BAC, generating variants that produced greater titres of cell-free virus following reconstitution. Virions from these novel Merlin variants displayed reduced pentameric complex content, but remained able to infect epithelial cells, albeit with slightly compromised efficiency. The greater fitness of viruses from these novel Merlin-BAC variants alleviated the selective pressures for the selection of de novo UL128L mutations in fibroblasts. The Merlin virion proteome was determined, with up to 30 novel components identified to provide a more comprehensive picture of wildtype HCMV virion composition. Comparison of virions from several Merlin variants demonstrated that varying pentameric complex content impacted the incorporation of other components, however virions from the novel Merlin variants produced in this work closely matched those from the parental Merlin variant containing wildtype UL128L ORFs. Thus, the novel Merlin-BAC variants produced in this work represent valuable reagents for future HCMV research