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Initiation of human cytomegalovirus infection requires initial interaction with cell surface heparan sulfate
T Compton1, D M Nowlin, N R Cooper
1Department of Immunology, Scripps Research Institute, La Jolla, California 92037.
Abstract:
In this report, we demonstrate that the initial event in human cytomegalovirus (HCMV) infection is attachment to extracellular heparan sulfate. Further, this interaction is important for initiation of infection in fibroblast cells. Using microbinding assays to specifically monitor virus attachment as well as plaque titration assays to measure infectivity, we found that heparin competition as well as enzymatic digestion of cells with heparinase blocked virus attachment, initiation of immediate-early gene expression and infectivity. Other major glycosaminoglycans were found not to be involved in HCMV attachment and infectivity. In addition, HCMV was unable to attach to mutant derivatives of Chinese hamster ovary cells deficient in synthesis of heparan sulfate proteoglycans. Basic fibroblast growth factor, which requires initial interaction with extracellular heparin prior to binding to its high affinity receptor, also inhibited HCMV attachment to cells. Time-course experiments revealed that the initial HCMV binding was sensitive to heparin competition (10 micrograms/ml) or 0.75 M salt washes. The initial heparin-dissociable binding converted rapidly to high affinity (heparin resistant) HCMV attachment. These data suggest that sequential receptor interactions may mediate HCMV adsorption to cells. Heparin affinity chromatography revealed that multiple HCMV envelope glycoproteins, including gB, are capable of binding to heparin.
Insights
Human cytomegalovirus (HCMV) infection begins with attachment to extracellular heparan sulfate, crucial for fibroblast cell infection. This interaction is essential for viral entry and gene expression.
Area of Science:
- Virology
- Cell Biology
- Glycobiology
Background:
- Human cytomegalovirus (HCMV) is a significant human pathogen.
- Understanding the initial steps of HCMV infection is critical for developing antiviral strategies.
- Cell surface molecules play a vital role in viral entry.
Purpose of the Study:
- To investigate the initial molecular interactions mediating HCMV attachment to host cells.
- To determine the role of heparan sulfate in HCMV infection.
- To elucidate the sequence of receptor engagements during HCMV adsorption.
Main Methods:
- Microbinding assays to quantify virus attachment.
- Plaque titration assays to measure viral infectivity.
- Enzymatic digestion of cell surface glycosaminoglycans (GAGs) with heparinase.
- Utilizing mutant Chinese hamster ovary (CHO) cells deficient in heparan sulfate proteoglycan synthesis.
- Heparin affinity chromatography to identify viral binding partners.
Main Results:
- HCMV attachment to fibroblast cells is mediated by initial binding to extracellular heparan sulfate.
- Heparin competition and heparinase treatment significantly blocked HCMV attachment, immediate-early gene expression, and infectivity.
- HCMV failed to attach to CHO cells lacking heparan sulfate proteoglycans.
- Basic fibroblast growth factor inhibited HCMV attachment, suggesting shared initial binding mechanisms.
- HCMV attachment involves a rapid transition from transient heparin-dissociable binding to stable, heparin-resistant interactions.
- HCMV envelope glycoproteins, including gB, bind to heparin, indicating their role in initial attachment.
Conclusions:
- Initial attachment to heparan sulfate is a critical step for HCMV infection of fibroblast cells.
- HCMV employs sequential receptor interactions for cell adsorption, starting with heparan sulfate.
- Multiple HCMV glycoproteins contribute to heparin binding, highlighting their involvement in the early stages of infection.