Impact of Sequence Variation in the UL128 Locus on Production of Human Cytomegalovirus in Fibroblast and Epithelial Cells

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.

Human Cytomegalovirus gH/gL/gO Promotes the Fusion Step of Entry into All Cell Types, whereas gH/gL/UL128-131 Broadens Virus Tropism through a Distinct Mechanism

“…Importantly, despite the overall small amount of gH/gL in ME, the amount of gH/gL/UL128-131 was dramatically more than was detected in TB or TR virions. While the HCMV virions analyzed here were produced in fibroblasts, Murrell et al produced virions in epithelial cells and found that TR contained amounts of gH/gL/ UL128-131 comparable to those in ME and that TB had noticeably less (59). These discrepancies suggest that factors of the host cell type might influence the assembly of HCMV gH/gL complexes.…”

Scanning Mutagenesis of Human Cytomegalovirus Glycoprotein gH/gL

“…Zhou et al demonstrated that the amount of these two complexes in the virion envelope varies dramatically among different strains of HCMV (17). This may be partly explained by the findings of Murrell et al indicating that nucleotide polymorphisms within the UL128-131 locus affect mRNA splicing and, thus, the amounts of the proteins available for assembly of the gH/gL/UL128-131 complex (18). Another factor may be the UL148 protein, which was recently characterized by Li et al as an endoplasmic reticulum (ER) resident protein that influences the assembly of gH/gL/gO and gH/gL/UL128-131 (19).…”

Viral Regulation of Cell Tropism in Human Cytomegalovirus

“…We suggest, however, that because trimer accounts for most of the gH/gL on TB40/E virions (17), a decrease in overall virion levels of gH/gL might be expected when a factor important for its efficient maturation is absent. In addition, TB40/E contains a mutation in intron 1 of UL128 that causes inefficient splicing of UL128 transcripts and hence, inefficient expression of the pentamer (11). Therefore, a strain-specific bottleneck in UL128 expression may have prevented an increase in pentamer levels from occurring when UL148 was disrupted.…”

“…It is now established that the pentameric complex is required for infection of epithelial cells and endothelial cells. During adaptation to culture in fibroblasts, natural isolates of HCMV quickly accumulate mutations in UL128, UL130, or UL131 that either prevent maturation of the pentamer or substantially diminish its expression on virions (10,11). Therefore, most laboratory-adapted HCMV strains express only gH/gL/gO (trimer).…”