The contribution of pUL74 to growth of human cytomegalovirus is masked in the presence of RL13 and UL128 expression

The glycoproteins gH and gL of human cytomegalovirus (HCMV) form a complex either with pUL74 (trimeric complex) or with proteins of the UL128 locus (pentameric complex). While the pentameric complex is dispensable for viral growth in fibroblasts, deletion of pUL74 causes a small plaque phenotype in HCMV lab strains, accompanied by greatly reduced cell-free infectivity. As HCMV isolates, shortly after cultivation from clinical specimens, do not release cell-free infectious viruses, we wondered whether deletion of pUL74 would also affect virus growth in this background. To address this question, we took advantage of the bacterial artificial chromosome (BAC)-cloned virus Merlin-RL13(tetO,) which grows cell associated due to the inducible expression of the viral RL13 gene, thereby resembling clinical isolates. Stop codons were introduced by seamless mutagenesis into UL74 and/or the UL128 locus to prevent expression of the trimeric or pentameric complex, respectively. Virus mutants were reconstituted by transfection of the respective genomes into cultured cells and analysed with respect to focal growth. When the UL128 locus was intact, deletion of pUL74 did not notably affect focal growth of Merlin, irrespective of RL13 expression. In the absence of UL128 expression, foci were increased compared with wild-type, and infectious cell-free virus was produced. Under these conditions, disruption of UL74 completely prevented virus spread from initially transfected cells to surrounding cells. In conclusion the contribution of pUL74 is masked when the UL128 locus is expressed at high levels, and its role in cell-free virus spread is only revealed when expression of the pentameric complex is inhibited.