Specific Inhibition of Human Cytomegalovirus Glycoprotein B-Mediated Fusion by a Novel Thiourea Small Molecule

Jones, Thomas R.; Lee, Shi-Wu; Johann, S V; Razinkov, Vladimir I.; Visalli, Robert J.; Feld, Boris; Bloom, Jonathan D.; O’Connell, John F.

doi:10.1128/jvi.78.3.1289-1300.2004

Cited by 37 publications

(34 citation statements)

References 58 publications

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“…These proteins apparently pilot gB transition through a WY1768-sensitive, PrK-resistant intermediate, and so WY1768 would irreversibly trap VR1814 TM in a PrK-resistant form. This explanation is fully consistent with previous findings that thiourea inhibitors require virus attachment to be effective and that their viral target is gB, because the point mutations that confer WY1768 resistance map within TM pretransmembrane and transmembrane alpha-helical stretches (33).…”

Section: Vol 81 2007 Hcmv Ul131-128 Products and Gb-gh Interaction supporting

confidence: 80%

“…The bis-(aryl)thiourea inhibitor WY1768 is a potent inhibitor of HCMV fusion in FBs (3,33) and retains its activity on VR1814 strain infections of ECs (50% infective dose in ECs, 1.96 Ϯ 0.06 nM [mean Ϯ standard deviation {SD}; n ϭ 4] [data not shown]), suggesting that the affected mechanism is essential for HCMV entry in both cell types. When the lipid mixing assay was performed in the presence of 2 M WY1768, the VR1814 virus failed to transfer R18 fluorescence to either ECs or HELFs.…”

Section: Resultsmentioning

confidence: 99%

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

Patrone

Secchi

Bonaparte

et al. 2007

J Virol

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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...

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Section: Vol 81 2007 Hcmv Ul131-128 Products and Gb-gh Interaction supporting

confidence: 80%

Section: Resultsmentioning

confidence: 99%

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

Patrone

Secchi

Bonaparte

et al. 2007

J Virol

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“…It was shown previously that inhibition of HCMV virion fusion by CFI02 could be reversed by the treatment of virion-adsorbed cells with a fusion-inducing polymer, polyethylene glycol (PEG) (7). HFF cells were infected with HCMV in the absence or presence of a fusion inhibitor, CFI02, and then treated with PEG (Fig.…”

mentioning

confidence: 99%

“…To further determine if the inhibition of ISG induction by CFI02 was secondary to an inhibition of virion-cell fusion, a protocol adapted to study the effect of CFI02 on HCMV infectivity was utilized (7). It was shown previously that inhibition of HCMV virion fusion by CFI02 could be reversed by the treatment of virion-adsorbed cells with a fusion-inducing polymer, polyethylene glycol (PEG) (7).…”

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Postattachment Events Associated with Viral Entry Are Necessary for Induction of Interferon-Stimulated Genes by Human Cytomegalovirus

Netterwald

Jones

Britt

et al. 2004

J Virol

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Utilizing a human cytomegalovirus-specific fusion inhibitor and an antiglycoprotein H antibody, we studied the role of virion fusion in interferon-stimulated gene (ISG) induction. Our results indicate that ISG induction does not occur when virion-mediated, post-high-affinity attachment events are inhibited by either reagent. Thus, virion-mediated postattachment events, such as fusion, are required for ISG induction.A large number of interferon-stimulated genes (ISGs) are strongly activated following human cytomegalovirus (HCMV) infection (19). In early studies, UV-irradiated HCMV was shown to induce ISGs (19). Subsequent studies revealed that virion envelope proteins, specifically glycoprotein B (gB), were responsible for the induction of ISGs (3,17). Soluble gB applied to human fibroblasts resulted in the induction of ISGs. Because the latter experiment was done under nonphysiological conditions and because gB has been shown to have roles in both virion attachment and fusion, it was unclear whether virion attachment alone was sufficient to induce ISG expression. Alternatively, other HCMV envelope glycoproteins or perhaps the fusion process itself is required for ISG activation during natural infection, as is the case for ISG induction by herpes simplex virus (14). In this study, we examined the requirement of postattachment steps of HCMV entry for the induction of ISGs.To characterize the role of virion fusion in HCMV-mediated ISG induction, an HCMV-specific fusion inhibitor, CFI02, was used (2). It was previously shown that this inhibitor has no effect on the attachment of virus to cells, but that it efficiently blocks subsequent viral-cell fusion at a 2 M concentration (7). CFI02 was used to determine if the compound also blocks induction of ISGs by HCMV (Fig. 1A). Human foreskin fibroblast (HFF) cells were either mock infected or infected with HCMV in the presence of dimethyl sulfoxide (control) or CFI02 for 8 h. Induction of the ISGs isg54K and cig49 was determined by RNA blot analysis. A cellular pseudogene, 7SK (13), was used as an internal control. Induction of ISGs by HCMV was inhibited in the presence of CFI02 (Fig. 1A, compare lanes 2 and 3). These results indicated that a postattachment step of HCMV infection, presumably virion fusion with the cellular membrane, was required for the induction of ISG expression. The effect of CFI02 inhibition on HCMV fusion was specific because (i) it had no effect on CFI02-resistant, HCMV-mediated ISG induction (the resistance phenotype was mapped to gB) (7) (lanes 4 and 5); (ii) it did not block murine-CMV-mediated ISG induction (lanes 9 and 10); (iii) it had no effect on alpha interferon (IFN-␣)-induced ISG expression (lanes 6 and 7); and (iv) it was not able to block induction of ISGs when added 2 h after infection (lanes 3 and 8). Furthermore, inhibition of HCMV-induced ISG expression by CFI02 had no effect on IFN-␣-mediated induction of ISGs (lanes 11 and 12). In addition, as shown previously (19), HCMV is able to induce isg54K at 12 and 24 h postinfection (hpi)...

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Anti‐HCMV Compounds

Andrei¹,

Snoeck²

2011

Antiviral Drug Strategies

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Specific Inhibition of Human Cytomegalovirus Glycoprotein B-Mediated Fusion by a Novel Thiourea Small Molecule

Cited by 37 publications

References 58 publications

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

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

Postattachment Events Associated with Viral Entry Are Necessary for Induction of Interferon-Stimulated Genes by Human Cytomegalovirus

Anti‐HCMV Compounds

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