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Virus attachment at the cell surface often involves the establishment of multiple ligand-receptor interactions between viral capsid proteins and glycans in the glycocalyx. Multivalency likely contributes to strengthening and fine-tuning virus dynamics at the cell surface to optimize entry. Here, we present experimental and theoretical frameworks to describe how virus attachment, detachment, and diffusion at the cell surface are modulated by multivalency. We focus on human papillomavirus 16 (HPV16), a leading cause of cervical cancer and its multivalent interactions with heparan sulfate (HS), a ubiquitous cell-surface glycan. Using single-particle tracking microscopy, we investigate the dynamic behavior of HPV16 particles interacting with multiple HS chains through a glycocalyx mimic consisting of end-tethered heparin chains that were selectively desulfated. We further establish a theoretical framework to predict the dynamic behavior of individual virus particles interacting with multiple cellular receptors and apply it to the HPV16-HS binding system using previously published association and dissociation rates of the individual capsomer-glycan bonds. Our experiments reveal that N-sulfation is essential to ensure HPV16 association and validate our theoretical prediction that at biologically relevant timescales, the lifetime of monovalent interaction primarily influences the apparent particle attachment behavior. Conversely, the nature of the sulfate group had a marginal effect on particle dissociation in the experiments, due to the great influence of multivalency on the particle’s interaction lifetime. Experiments and simulations also indicate that the mobility of HPV16 particles on heparin surfaces is highly restricted due to the relatively high affinity of the monovalent interactions, suggesting that particle motion due to the creation and rupturing of individual bonds is unlikely key in HPV16 recruitment. Together, these findings provide quantitative mechanistic insights into how virus-glycan interactions are modulated. They highlight the importance of HS chemistry in modulating early HPV16 engagement and suggest new targets against viral binding.

The diffusion of viruses at the cell membrane is essential to reach a suitable entry site and initiate subsequent internalization. Although many viruses take advantage of glycosaminoglycans (GAG) to bind to the cell surface, little is known about the dynamics of the virus–GAG interactions. Here, single-particle tracking of the initial interaction of individual herpes simplex virus 1 (HSV-1) virions reveals a heterogeneous diffusive behavior, regulated by cell-surface GAGs with two main diffusion types: confined and normal free. This study reports that different GAGs can have competing influences in mediating diffusion on the cells used here: chondroitin sulfate (CS) enhances free diffusion but hinders virus attachment to cell surfaces, while heparan sulfate (HS) promotes virus confinement and increases entry efficiency. In addition, the role that the viral mucin-like domains (MLD) of the HSV-1 glycoprotein C plays in facilitating the diffusion of the virus and accelerating virus penetration into cells is demonstrated. Together, our results shed new light on the mechanisms of GAG-regulated virus diffusion at the cell surface for optimal internalization. These findings may be extendable to other GAG-binding viruses.

Chikungunya virus (CHIKV) is a re-emerging, mosquito-transmitted, enveloped positive stranded RNA virus. Chikungunya fever is characterized by acute and chronic debilitating arthritis. Although multiple host factors have been shown to enhance CHIKV infection, the molecular mechanisms of cell entry and entry factors remain poorly understood. The phosphatidylserine-dependent receptors, T-cell immunoglobulin and mucin domain 1 (TIM-1) and Axl receptor tyrosine kinase (Axl), are transmembrane proteins that can serve as entry factors for enveloped viruses. Previous studies used pseudoviruses to delineate the role of TIM-1 and Axl in CHIKV entry. Conversely, here, we use the authentic CHIKV and cells ectopically expressing TIM-1 or Axl and demonstrate a role for TIM-1 in CHIKV infection. To further characterize TIM-1-dependent CHIKV infection, we generated cells expressing domain mutants of TIM-1. We show that point mutations in the phosphatidylserine binding site of TIM-1 lead to reduced cell binding, entry, and infection of CHIKV. Ectopic expression of TIM-1 renders immortalized keratinocytes permissive to CHIKV, whereas silencing of endogenously expressed TIM-1 in human hepatoma cells reduces CHIKV infection. Altogether, our findings indicate that, unlike Axl, TIM-1 readily promotes the productive entry of authentic CHIKV into target cells.