Abstract
The human cytomegalovirus (HCMV) virion envelope contains a complex consisting of glycoproteins gH and gL plus proteins encoded by the UL128 locus (UL128L): pUL128, pUL130, and pUL131A. UL128L is necessary for efficient infection of myeloid, epithelial, and endothelial cells but limits replication in fibroblasts. Consequently, disrupting mutations in UL128L are rapidly selected when clinical isolates are cultured in fibroblasts. In contrast, bacterial artificial chromosome (BAC)-cloned strains TB40-BAC4, FIX, and TR do not contain overt disruptions in UL128L, yet no virus reconstituted from them has been reported to acquire mutations in UL128L in vitro. We performed BAC mutagenesis and reconstitution experiments to test the hypothesis that these strains contain subtle mutations in UL128L that were acquired during passage prior to BAC cloning. Compared to strain Merlin containing wild-type UL128L, all three strains produced higher yields of cell-free virus. Moreover, TB40-BAC4 and FIX spread cell to cell more rapidly than wild-type Merlin in fibroblasts but more slowly in epithelial cells. The differential growth properties of TB40-BAC4 and FIX (but not TR) were mapped to single-nucleotide substitutions in UL128L. The substitution in TB40-BAC4 reduced the splicing efficiency of UL128, and that in FIX resulted in an amino acid substitution in UL130. Introduction of these substitutions into Merlin dramatically increased yields of cell-free virus and increased cell-to-cell spread in fibroblasts but reduced the abundance of pUL128 in the virion and the efficiency of epithelial cell infection. These substitutions appear to represent mutations in UL128L that permit virus to be propagated in fibroblasts while retaining epithelial cell tropism.
Highlights
The human cytomegalovirus (HCMV) virion envelope contains a complex consisting of glycoproteins gH and gL plus proteins encoded by the UL128 locus (UL128L): pUL128, pUL130, and pUL131A
We have previously described the cloning of the complete HCMV strain Merlin genome into a selfexcising bacterial artificial chromosome (BAC) following five passages in fibroblasts [38]
Because the FIX and TB40-BAC4 clones contain mutations in RL13 [38], which invariably mutates upon passage in fibroblast or epithelial cells [37, 38], a preexisting RL13 frameshift mutation present in these Merlin BACs was not repaired
Summary
The human cytomegalovirus (HCMV) virion envelope contains a complex consisting of glycoproteins gH and gL plus proteins encoded by the UL128 locus (UL128L): pUL128, pUL130, and pUL131A. The substitution in TB40-BAC4 reduced the splicing efficiency of UL128, and that in FIX resulted in an amino acid substitution in UL130 Introduction of these substitutions into Merlin dramatically increased yields of cell-free virus and increased cell-to-cell spread in fibroblasts but reduced the abundance of pUL128 in the virion and the efficiency of epithelial cell infection. 10489 –10500 jvi.asm.org 10489 insertion or deletion of one or more nucleotides, in-frame termination codons caused by single-nucleotide substitutions, or deletions [37,38,39,40,41,42,43,44] This results in the generation of laboratory-adapted viruses that display efficient growth in fibroblasts but limited growth in other cell types. As with clinical HCMV strains, the presence of wild-type UL128L in reconstituted virus results in the production of very low titers of cell-free virus in vitro, and the virus is prone to mutation when passaged in fibroblasts [38]
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