Limits...
Structural and Functional Analysis of Murine Polyomavirus Capsid Proteins Establish the Determinants of Ligand Recognition and Pathogenicity.

Buch MH, Liaci AM, O'Hara SD, Garcea RL, Neu U, Stehle T - PLoS Pathog. (2015)

Bottom Line: By comparing electron density of the oligosaccharides within the binding pockets at various concentrations, we show that the [α-2,8]-linked sialic acid increases the strength of binding.Moreover, the amino acid exchanges have subtle effects on their affinity for the validated receptor GD1a.Our results indicate that both receptor specificity and affinity influence MuPyV pathogenesis.

View Article: PubMed Central - PubMed

Affiliation: Interfaculty Institute of Biochemistry, University of Tübingen, Tübingen, Germany.

ABSTRACT
Murine polyomavirus (MuPyV) causes tumors of various origins in newborn mice and hamsters. Infection is initiated by attachment of the virus to ganglioside receptors at the cell surface. Single amino acid exchanges in the receptor-binding pocket of the major capsid protein VP1 are known to drastically alter tumorigenicity and spread in closely related MuPyV strains. The virus represents a rare example of differential receptor recognition directly influencing viral pathogenicity, although the factors underlying these differences remain unclear. We performed structural and functional analyses of three MuPyV strains with strikingly different pathogenicities: the low-tumorigenicity strain RA, the high-pathogenicity strain PTA, and the rapidly growing, lethal laboratory isolate strain LID. Using ganglioside deficient mouse embryo fibroblasts, we show that addition of specific gangliosides restores infectability for all strains, and we uncover a complex relationship between virus attachment and infection. We identify a new infectious ganglioside receptor that carries an additional linear [α-2,8]-linked sialic acid. Crystal structures of all three strains complexed with representative oligosaccharides from the three main pathways of ganglioside biosynthesis provide the molecular basis of receptor recognition. All strains bind to a range of sialylated glycans featuring the central [α-2,3]-linked sialic acid present in the established receptors GD1a and GT1b, but the presence of additional sialic acids modulates binding. An extra [α-2,8]-linked sialic acid engages a protein pocket that is conserved among the three strains, while another, [α-2,6]-linked branching sialic acid lies near the strain-defining amino acids but can be accommodated by all strains. By comparing electron density of the oligosaccharides within the binding pockets at various concentrations, we show that the [α-2,8]-linked sialic acid increases the strength of binding. Moreover, the amino acid exchanges have subtle effects on their affinity for the validated receptor GD1a. Our results indicate that both receptor specificity and affinity influence MuPyV pathogenesis.

No MeSH data available.


Related in: MedlinePlus

GT1a, GT1b, and GD1a supplementation rescues MuPyV infection of Gang-/- MEFs.Ganglioside knock-out (Gang-/-) MEFs were completely resistant to infection of all strains of MuPyV as shown by the absence of T-antigen positive nuclei at 24 hours post infection (DMSO control). GD1a, GT1b, and GT1a ganglioside supplementation of Gang-/- MEFs restored RA (A), PTA (B), and LID MuPyV (C) infection, while GD1b and GM1 supplementation resulted in little to no infection by any virus strain. Infection levels were quantified at both 2 μM and 4 μM ganglioside supplementation (blue and green bars, respectively). Infection levels are normalized to MuPyV infection of WT MEFs, and error bars correspond to standard error.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4608799&req=5

ppat.1005104.g002: GT1a, GT1b, and GD1a supplementation rescues MuPyV infection of Gang-/- MEFs.Ganglioside knock-out (Gang-/-) MEFs were completely resistant to infection of all strains of MuPyV as shown by the absence of T-antigen positive nuclei at 24 hours post infection (DMSO control). GD1a, GT1b, and GT1a ganglioside supplementation of Gang-/- MEFs restored RA (A), PTA (B), and LID MuPyV (C) infection, while GD1b and GM1 supplementation resulted in little to no infection by any virus strain. Infection levels were quantified at both 2 μM and 4 μM ganglioside supplementation (blue and green bars, respectively). Infection levels are normalized to MuPyV infection of WT MEFs, and error bars correspond to standard error.

Mentions: Previous efforts to identify receptors for MuPyV used immortalized cell lines, such as Vero or C6 glioma cells that were supplemented with candidate gangliosides before infection [8,28]. We utilized a mouse embryo knock-out fibroblast cell line (Gang-/- MEFs) specifically deficient in ganglioside synthesis and completely resistant to MuPyV infection (S1A Fig and [29]) to test the ability of ganglioside receptors to rescue infection by different strains of MuPyV. Gang-/- MEFs were supplemented with individual gangliosides followed by infection with RA, PTA, and LID MuPyV (Fig 2). Importantly, it should be noted that the three MuPyV strains we used do not have the same particle to PFU ratio. The viruses have been normalized to similar MOIs, but they cannot be quantitatively compared to one another. However, each strain has been normalized to its own infection rate of WT MEFs; therefore, infection rates upon supplementation of gangliosides can be compared within a strain. The previously identified ganglioside receptors GD1a and GT1b [8] rescued RA, PTA, and LID infection of Gang-/- MEFs in a dose-responsive manner. We also analyzed the GT1a ganglioside that had not been previously investigated as a candidate infectious receptor for MuPyV. We found that GT1a, a member of the ganglio-series synthesized from GD1a (Fig 1), also rescued RA, PTA, and LID infection in a dose responsive manner (Fig 2). Moreover, GT1a supplementation of Gang-/- MEFs conferred higher levels of RA, PTA, and LID MuPyV infection than the previously identified receptors GD1a and GT1b. Finally, we tested the ability of the gangliosides GD1b and GM1 to rescue MuPyV infection of Gang-/- MEFs. GD1b and GT1b supplementation has previously been shown to restore BK polyomavirus infection of ganglioside deficient cells [30]; however, GD1b restored little to no MuPyV infection of Gang-/- MEFs. GM1 supplementation has previously been shown to restore infection by SV40 [8]; however, GM1 did not rescue MuPyV infection of Gang-/- MEFs. These data confirm that GT1a is an infectious receptor for all strains of MuPyV.


Structural and Functional Analysis of Murine Polyomavirus Capsid Proteins Establish the Determinants of Ligand Recognition and Pathogenicity.

Buch MH, Liaci AM, O'Hara SD, Garcea RL, Neu U, Stehle T - PLoS Pathog. (2015)

GT1a, GT1b, and GD1a supplementation rescues MuPyV infection of Gang-/- MEFs.Ganglioside knock-out (Gang-/-) MEFs were completely resistant to infection of all strains of MuPyV as shown by the absence of T-antigen positive nuclei at 24 hours post infection (DMSO control). GD1a, GT1b, and GT1a ganglioside supplementation of Gang-/- MEFs restored RA (A), PTA (B), and LID MuPyV (C) infection, while GD1b and GM1 supplementation resulted in little to no infection by any virus strain. Infection levels were quantified at both 2 μM and 4 μM ganglioside supplementation (blue and green bars, respectively). Infection levels are normalized to MuPyV infection of WT MEFs, and error bars correspond to standard error.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4608799&req=5

ppat.1005104.g002: GT1a, GT1b, and GD1a supplementation rescues MuPyV infection of Gang-/- MEFs.Ganglioside knock-out (Gang-/-) MEFs were completely resistant to infection of all strains of MuPyV as shown by the absence of T-antigen positive nuclei at 24 hours post infection (DMSO control). GD1a, GT1b, and GT1a ganglioside supplementation of Gang-/- MEFs restored RA (A), PTA (B), and LID MuPyV (C) infection, while GD1b and GM1 supplementation resulted in little to no infection by any virus strain. Infection levels were quantified at both 2 μM and 4 μM ganglioside supplementation (blue and green bars, respectively). Infection levels are normalized to MuPyV infection of WT MEFs, and error bars correspond to standard error.
Mentions: Previous efforts to identify receptors for MuPyV used immortalized cell lines, such as Vero or C6 glioma cells that were supplemented with candidate gangliosides before infection [8,28]. We utilized a mouse embryo knock-out fibroblast cell line (Gang-/- MEFs) specifically deficient in ganglioside synthesis and completely resistant to MuPyV infection (S1A Fig and [29]) to test the ability of ganglioside receptors to rescue infection by different strains of MuPyV. Gang-/- MEFs were supplemented with individual gangliosides followed by infection with RA, PTA, and LID MuPyV (Fig 2). Importantly, it should be noted that the three MuPyV strains we used do not have the same particle to PFU ratio. The viruses have been normalized to similar MOIs, but they cannot be quantitatively compared to one another. However, each strain has been normalized to its own infection rate of WT MEFs; therefore, infection rates upon supplementation of gangliosides can be compared within a strain. The previously identified ganglioside receptors GD1a and GT1b [8] rescued RA, PTA, and LID infection of Gang-/- MEFs in a dose-responsive manner. We also analyzed the GT1a ganglioside that had not been previously investigated as a candidate infectious receptor for MuPyV. We found that GT1a, a member of the ganglio-series synthesized from GD1a (Fig 1), also rescued RA, PTA, and LID infection in a dose responsive manner (Fig 2). Moreover, GT1a supplementation of Gang-/- MEFs conferred higher levels of RA, PTA, and LID MuPyV infection than the previously identified receptors GD1a and GT1b. Finally, we tested the ability of the gangliosides GD1b and GM1 to rescue MuPyV infection of Gang-/- MEFs. GD1b and GT1b supplementation has previously been shown to restore BK polyomavirus infection of ganglioside deficient cells [30]; however, GD1b restored little to no MuPyV infection of Gang-/- MEFs. GM1 supplementation has previously been shown to restore infection by SV40 [8]; however, GM1 did not rescue MuPyV infection of Gang-/- MEFs. These data confirm that GT1a is an infectious receptor for all strains of MuPyV.

Bottom Line: By comparing electron density of the oligosaccharides within the binding pockets at various concentrations, we show that the [α-2,8]-linked sialic acid increases the strength of binding.Moreover, the amino acid exchanges have subtle effects on their affinity for the validated receptor GD1a.Our results indicate that both receptor specificity and affinity influence MuPyV pathogenesis.

View Article: PubMed Central - PubMed

Affiliation: Interfaculty Institute of Biochemistry, University of Tübingen, Tübingen, Germany.

ABSTRACT
Murine polyomavirus (MuPyV) causes tumors of various origins in newborn mice and hamsters. Infection is initiated by attachment of the virus to ganglioside receptors at the cell surface. Single amino acid exchanges in the receptor-binding pocket of the major capsid protein VP1 are known to drastically alter tumorigenicity and spread in closely related MuPyV strains. The virus represents a rare example of differential receptor recognition directly influencing viral pathogenicity, although the factors underlying these differences remain unclear. We performed structural and functional analyses of three MuPyV strains with strikingly different pathogenicities: the low-tumorigenicity strain RA, the high-pathogenicity strain PTA, and the rapidly growing, lethal laboratory isolate strain LID. Using ganglioside deficient mouse embryo fibroblasts, we show that addition of specific gangliosides restores infectability for all strains, and we uncover a complex relationship between virus attachment and infection. We identify a new infectious ganglioside receptor that carries an additional linear [α-2,8]-linked sialic acid. Crystal structures of all three strains complexed with representative oligosaccharides from the three main pathways of ganglioside biosynthesis provide the molecular basis of receptor recognition. All strains bind to a range of sialylated glycans featuring the central [α-2,3]-linked sialic acid present in the established receptors GD1a and GT1b, but the presence of additional sialic acids modulates binding. An extra [α-2,8]-linked sialic acid engages a protein pocket that is conserved among the three strains, while another, [α-2,6]-linked branching sialic acid lies near the strain-defining amino acids but can be accommodated by all strains. By comparing electron density of the oligosaccharides within the binding pockets at various concentrations, we show that the [α-2,8]-linked sialic acid increases the strength of binding. Moreover, the amino acid exchanges have subtle effects on their affinity for the validated receptor GD1a. Our results indicate that both receptor specificity and affinity influence MuPyV pathogenesis.

No MeSH data available.


Related in: MedlinePlus