Abstract
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-RL13tetO, 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.
Highlights
All herpesviruses depend on complexes of glycoproteins H and L for successful replication, but they vary with respect to the composition of these complexes (Connolly et al, 2011; Eisenberg et al, 2012)
Attempts to reconstitute a genetically complete version of the Merlin strain from a bacterial artificial chromosome (BAC) clone regularly resulted in the de-novo selection of mutations in both RL13 and the UL128 locus (UL128, UL130 and UL131A)
One major conclusion from our results is that disruption of UL74 has little, if any, effect on focal cell-associated spread of the genetically complete variant of human cytomegalovirus (HCMV) strain Merlin, in either fibroblasts or endothelial cells
Summary
All herpesviruses depend on complexes of glycoproteins H and L for successful replication, but they vary with respect to the composition of these complexes (Connolly et al, 2011; Eisenberg et al, 2012). GH and gL can form a pentameric complex with the three proteins encoded by the UL128 gene locus, and disruption of this complex restricts the cell tropism of the virus, greatly reducing infection efficiency of epithelial cells, endothelial cells and leukocytes (Adler et al, 2006; Hahn et al, 2004; Ryckman et al, 2008b; Wang & Shenk 2005). It was assumed that the trimeric and the pentameric gH/gL complex serve the same function on different cell types, e.g. triggering the proprietary fusion protein gB upon binding to the respective receptor (Ryckman et al, 2008a; Vanarsdall et al, 2011, 2012).
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