Cytomegalovirus UL131-128 Products Promote gB Conformational Transition and gB-gH Interaction during Entry into Endothelial Cells

Abstract

Herpesviruses use gB and gH-gL glycoproteins to execute fusion. Other virus-specific glycoproteins are required for receptor binding and fusion activation. The human cytomegalovirus (HCMV) UL131-128 proteins are essential for the infection of leukocytes, endothelial cells (ECs), and many epithelial cell lines. Here we show that UL131-128 play a role in a chain of events involving gB and gH during HCMV entry into ECs. An HCMV strain bearing the wild-type (wt) UL131-128 locus exhibited a gB transition from a protease-resistant to protease-sensitive form, a conformational change that was suppressed by a thiourea inhibitor of fusion (WY1768); in contrast, gH was susceptible to proteolysis throughout entry. Moreover, gB and gH transiently interacted, and a lipid mixing assay showed that the wt strain had carried out fusion by 60 min postinfection. However, these events were greatly altered when UL131-128-defective strains were used for infection or when there was an excess of soluble pUL128 during wt infection: the gB conformational change became WY1768 resistant, the gB-gH complex was no longer observed, and fusion was prevented. Both gB and gH in this case showed late protease resistance, related to their endocytic uptake. Our data point to the involvement of UL131-128 proteins in driving gB through a WY1768-sensitive fold transition, thus promoting a short-lived gB-gH complex and fusion; they also suggest that HCMV fuses with the EC plasma membrane and that endocytosis ensues only when the virus cannot trigger UL131-128-dependent steps.Human cytomegalovirus (HCMV), the prototypical member of the Betaherpesvirinae subfamily, is the leading infectious cause of congenital defects, a major opportunist in transplant recipients and immunocompromised patients, and a suspected cofactor for cardiovascular diseases, systemic sclerosis, and gastrointestinal cancer (7,27,29,41,42,49). Clinical isolates of HCMV infect various cell types in vitro, including endothelial cells (ECs), thus replicating the broad cell type tropism observed in HCMV infections of immunocompromised subjects (EC-tropic strains). However, laboratory propagation in fibroblasts (FBs) restricts viral tropism (FB-tropic strains), and mutations causing the loss of tropism for ECs, epithelial cells, polymorphonuclear leukocytes, and dendritic cells (DCs) have been mapped to the contiguous UL131, UL130, and UL128 open reading frames (ORFs) (13,25,18,56).There is considerable indirect evidence indicating that UL131-128 proteins act as regulators of virus-cell fusion: (i) FB-tropic strains fail to transfer tegument pp65-UL83 protein to EC nuclei (18), which suggests that infection stops before virus uncoating; (ii) an HCMV deletion mutant lacking the UL128-UL150 ORFs infects retinal pigment epithelial cells after exposure to polyethylene glycol in order to force fusion between viral and cellular membranes (50); and (iii) EC-tropic strains are syncytiogenic at a high multiplicity of infection (18,56).The membrane-spanning glycoproteins gB and gH and the soluble gL...

Reconstruction of the complete human cytomegalovirus genome in a BAC reveals RL13 to be a potent inhibitor of replication

“…The UL128 locus is essential for the efficient infection of epithelial and endothelial cells by HCMV (37)(38)(39)(40)(41)(42)(43) but is detrimental to growth in fibroblasts. Consequently, mutants in this locus are selected when clinical isolates are passaged in fibroblasts (12,26,27,44,45,47,48).…”

“…However, the generation of laboratory strains of WT HCMV is problematic not only because clinical samples often contain multiple strains (27,(29)(30)(31)(32)(33)(34)(35)(36), but especially because genetic adaptations occur even during the early stages of passage in cell culture. Thus, most strains passaged in fibroblasts are mutated in 1 of 3 adjacent genes (UL128, UL130, and UL131A; collectively termed the UL128 locus, UL128L) whose products form a complex bound to glycoproteins gH and gL in the virion envelope (37)(38)(39)(40)(41)(42)(43). These mutations inhibit formation of the complex and thereby render the virus incapable of infecting epithelial, endothelial, and certain myeloid cell types (12,(44)(45)(46)(47)(48).…”

Human cytomegalovirus uses two distinct pathways to enter retinal pigmented epithelial cells

“…Alternatively, the ratio of the gH complex to one or more additional virion glycoprotein complexes might modify fusion activity. In this regard, Patrone et al (37) have identified a transient complex containing both gH and gB at an early stage of infection. Finally, it is conceivable that an unidentified cell protein, supplied to the virions when they are produced within epithelial cells or fibroblasts, might alter the complex.…”

The m74 Gene Product of Murine Cytomegalovirus (MCMV) Is a Functional Homolog of Human CMV gO and Determines the Entry Pathway of MCMV

“…It is a matter of current research whether fusion takes place at the plasma membrane or at membranes of endocytic vesicles after endocytosis, whether binding of differ-ent gH/gL complexes to different receptors initiates different entry pathways, and whether entry pathways vary between cell types (3,9,37,45,47,57). The role of alternative gH/gL complexes for cell-type-specific infection in vivo is a matter of speculation and is difficult to study in humans.…”

“…For gH/ gL, two alternative complexes have been described: a complex of gH (UL75), gL (UL115), and gO (UL74) (16,24) and a complex of gH, gL, and pUL128, pUL130, and pUL131A (1,56). HCMV gH/gL complexes are supposed to play a role in promoting fusion of the viral envelope and cellular membranes and probably act in concert with gB and potentially through binding to integrin receptors (11,22,36,37,52,58). Recently, the role of the gH/gL/gO complex was analyzed in more detail.…”