GM1a functions as a coreceptor/ attachment factor for Dengue virus during infection in mammalian systems

Dengue virus (DENV) is a flavivirus causing an estimated 390 million infections per year around the world. Despite the immense global health and economic impact of this virus, its true receptor(s) for internalization into live cells has not yet been identified, and no successful antivirals or treatments have been isolated to this date. This study aims to improve our understanding of virus entry routs by exploring the sialic acid-based cell surface molecule GM1a and its role in DENV infection. The interaction of the virus with GM1a was studied using fluorescence correlation spectroscopy (FCS), fluorescence cross correlation spectroscopy (FCCS), imaging FCS (ImFCS) and amide hydrogen/deuterium exchange mass spectrometry (HDXMS), and the effect on infectivity and movement of the virus during infection was explored using plaque assays and fluorescence-based imaging and single particle tracking (SPT). GM1a was deemed to interact with DENV at domain I (DI) and domain II (DII) of the E protein of the protein coat at quaternary contacts of a fully assembled virus, leading to a ten-fold increase and seven-fold increase in infectivity for DENV1 and DENV2 in mammalian cell systems respectively. The interaction of virus with GM1a triggers a speeding up of virus movement on live cell surfaces, possibly resulting from a reduction in rigidity of cellular rafts during infection, and functions as a coreceptor/ attachment factor for DENV during infection in mammalian systems. Author Summary Dengue virus (DENV) is a flavivirus causing an estimated 390 million infections per year around the world. Despite the immense global health and economic impact of this virus, no successful antivirals or treatments have been isolated to this date. This may be due to the incomplete understanding of the virus infection mechanism, including a lack of an identified ‘true’ receptor and entry related attachment factors or co-receptors responsible for internalization of the virus. This work focuses on the early infection stage of DENV1 and DENV2 strains, to identify how the virus moves on cell surfaces in its search for its receptors, and identifies the critical role of the sialic acid ganglioside GM1a during internalization of the virus.


Mapping the binding hotspots of GM1a on DENV E-protein
Colocalization of GM1a glycosphingolipid with DENV2 particles was confirmed by FCCS. 171 We performed HDX-MS on free DENV2 and DENV2 in the presence GM1a sugar moiety to 172 identify the binding hotspot of GM1a on the DENV viral surface. 36 pepsin proteolyzed 173 peptides were obtained with high signal to noise ratios and covering 63% of the E protein sequence. Here, we used high molar ratio GM1a sugar moiety to that of E protein dimer (125: The interaction of DENV with GM1a was further studied using fluorescence based SPT, where Vero cells, and the diffusion coefficients were at 1.59 ± 0.77 m 2 /s (cells/curves = 6/11) and 272 1.70 ± 0.63 m 2 /s (cells/curves = 6/10) respectively. This indicates that the membrane disordered regions also do not undergo any organization changes due to the treatment with D-  DENV is known to enter mammalian cells using many different entry mechanisms, while 279 utilizing many different types of receptors/co-receptors/attachment factors. The range of 280 receptors and cells DENV is reported to infect, hints towards a more ubiquitous form of entry 281 which is available on many cells. Any virus when approaching the cell surface must find its 282 way to these entry points for successful access into the host system. Mammalian cell membrane 283 consists of a mosaic of lipids and proteins which are known to form liquid ordered (rafts) and 284 liquid disordered regions, and encompass various receptors both proteinaceous and others. The 285 cell surface is decorated with sugar antennas which form the glycocalyx, extending outward 286 towards the extracellular environment, to regions beyond the reach of cellular receptors, which 287 forms the first barrier that any virus will encounter as it approaches the cell surface. Thus, it is 288 important to note that any virus that internalizes into a living cell must travel past this first 289 barrier, and it is biologically important to identify how sugar-based molecules are involved in 290 the virus infection process.

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In this work we focus on GM1a, which is a glycosphingolipid with a sialic acid-based sugar 292 found ubiquitously on mammalian cell surfaces, with its sugar moiety located within the 293 glycocalyx. Sialic acid-based sugars have been widely reported to be involved in internalizing 294 many different types of viruses, including Influenza A and SARS-Cov-2. We explore the 295 involvement of GM1a and its sialic acid moiety in DENV internalization by using real-time with GM1a and its effect on infectivity of DENV1 and DENV2 was then explored by plaque 302 assay, where it was evident that the presence of GM1a on mammalian cell surfaces significantly 303 increases the infectivity of both DENV1 and DENV2 as compared to the GM1a depleted states.

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The association of DENV with GM1a shows a significant effect on the infection process of 305 DENV, and being a ubiquitously available molecule on mammalian cell surfaces, it acts as a 306 more universal interacting partner during virus internalization.

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The sialic acid receptor GM1a is reported to bind CTxB protein cargo via the two terminal 308 sugars (galactose and sialic acid) on the receptor, in the form of a two fingered grip, involving 309 ionic interactions between the positively charged protein cargo and the negatively charged  This increase in speed can allow the virus cargo to surf the cell surface more efficiently in 332 search of its final "true" receptor. Once the true receptor is found, the virus will attain an 333 immobile state and finally internalize. Thus, it can be interpreted that GM1a acts more as an

Fit models for confocal FCS measurements:
For FCS in the confocal, in the simplest case, fluorescent molecules moving in 3D diffusion in 520 the confocal volume is fitted using a single particle (1p) 3D diffusion fit model. where is the structure factor which defines the shape of the observation volume and is the 523 diffusion time.

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In the case of 2D diffusion on cell membranes, the fitting model can be simplified as Where is the ratio of the brightness of the ℎ species to that of species 1. is the diffusion 533 time and is the mole fraction of the ℎ species.

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The overall ACF is given as the product of all the individual dynamic processes that are present where is the fraction of the triplet state and is the relaxation time of the triplet state.

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The fitting models for 2D and 3D diffusion with triplet contribution is as below (     Green Beads shows no colocalization. Scale bar: 5 mm. Statistical data is given in S1 Table. 1230 1231 S1