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. 2016 Sep 29;90(20):9420-32.
doi: 10.1128/JVI.00799-16. Print 2016 Oct 15.

B Virus (Macacine Herpesvirus 1) Divergence: Variations in Glycoprotein D from Clinical and Laboratory Isolates Diversify Virus Entry Strategies

Irina Patrusheva  1 , Ludmila Perelygina  1 , Ivan Torshin  2 , Julia LeCher  1 , Julia Hilliard  3
Affiliations

B Virus (Macacine Herpesvirus 1) Divergence: Variations in Glycoprotein D from Clinical and Laboratory Isolates Diversify Virus Entry Strategies

Irina Patrusheva et al. J Virol. .

Abstract

B virus (Macacine herpesvirus 1) can cause deadly zoonotic disease in humans. Molecular mechanisms of B virus cell entry are poorly understood for both macaques and humans. Here we investigated the abilities of clinical B virus isolates to use entry receptors of herpes simplex viruses (HSV). We showed that resistant B78H1 cells became susceptible to B virus clinical strains upon expression of either human nectin-2 or nectin-1. Antibody against glycoprotein D (gD) protected these nectin-bearing cells from B virus infection, and a gD-negative recombinant B virus failed to enter these cells, indicating that the nectin-mediated B virus entry depends on gD. We observed that the infectivity of B virus isolates with a single amino acid substitution (D122N) in the IgV-core of the gD ectodomain was impaired on nectin-1-bearing cells. Computational homology-based modeling of the B virus gD-nectin-1 complex revealed conformational differences between the structures of the gD-122N and gD-122D variants that affected the gD-nectin-1 protein-protein interface and binding affinity. Unlike HSV, B virus clinical strains were unable to use herpesvirus entry mediator (HVEM) as a receptor, regardless of conservation of the gD amino acid residues essential for HSV-1 entry via HVEM. Based on the model of the B virus gD-HVEM interface, we predict that residues R7, R11, and G15 are largely responsible for the inability of B virus to utilize HVEM for entry. The ability of B virus to enter cells of a human host by using a combination of receptors distinct from those for HSV-1 or HSV-2 suggests a possible mechanism of enhanced neuropathogenicity associated with zoonotic infections.

Importance: B virus causes brainstem destruction in infected humans in the absence of timely diagnosis and intervention. Nectins are cell adhesion molecules that are widely expressed in human tissues, including neurons and neuronal synapses. Here we report that human nectin-2 is a target receptor for B virus entry, in addition to the reported receptor human nectin-1. Similar to a B virus lab strain, B virus clinical strains can effectively use both nectin-1 and nectin-2 as cellular receptors for entry into human cells, but unlike HSV-1 and HSV-2, none of the clinical strains uses an HVEM-mediated entry pathway. Ultimately, these differences between B virus and HSV-1 and -2 may provide insight into the neuropathogenicity of B virus during zoonotic infections.

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Figures

FIG 1
FIG 1
Susceptibility of murine B78H1 cells expressing HSV entry receptors to B virus infection. B virus E2490 was titrated on confluent monolayers of Vero (A), B78-nectin-1 (B), B78-nectin-2 (C), B78-HVEM (D), and B78c (E) cells. At 48 hpi, the cells were fixed and immunostained with rhesus B virus antibody-positive serum. Panels D and E represent images captured from culture wells infected with 4.3 × 107 PFU/well of B virus. Only a few immunostained cells were observed in the B78c (E) and B78-HVEM (D) cells. Panels C, B, and A represent selected wells that best reflect the CPE produced by B virus on the nectin-2-expressing, nectin-1-expressing, and Vero cells, respectively. Magnification, ×10.
FIG 2
FIG 2
Blocking of virus infectivity by gD antibody. (A) B virus neutralization on B78-nectin-2 cells was performed using a CPE inhibition assay. The monolayers were fixed at 48 hpi and immunostained with rhesus B virus antibody-positive serum. Magnification, ×10. (B) B virus neutralization on B78-nectin-1 cells was assayed by a plaque reduction assay. The infected cells were fixed at 48 hpi and then stained with crystal violet to visualize viral plaques. The images represent culture wells of B78-nectin-1 cells infected with B virus-serum mixtures. (C) Neutralization curves for B virus and HSV-1 on Vero cells, representing negative and positive controls, respectively.
FIG 3
FIG 3
Infection of cells with a gD-negative recombinant B virus (BV-ΔgDZ). Nectin-2-expressing, nectin-1-expressing (negative control), and Vero (positive control) cell monolayers were infected with BV-ΔgDZ. Cells were fixed at 48 hpi and then stained with X-Gal as described in Materials and Methods. Magnification, ×5.
FIG 4
FIG 4
Essential differences in protein-protein interfaces involving HSV-1 and B virus gD proteins and HVEM. (A) Alignment of the gD ectodomain residues of HSV-1 (KOS), HSV-2 (HG52), and the B virus lab strain (E2490). Conserved amino acid residues are boxed. The red line above the gD sequences indicates HVEM contact residues. The residues essential for HVEM-mediated and nectin-1-mediated HSV-1 entry are shown in bold red and bold blue, respectively. Red shading indicates the M11 residue, which is involved in the interaction with the HVEM “hot spot” residue Y28. Blue shading indicates the only residue in the IgV-core, Q132, that directly interacts with the nectin-1 C″ strand. The arrows point to the residues that most probably distort the HVEM-B virus gD interface. Lollipops mark the predicted N-glycosylation sites. The black-filled lollipop marks an N-glycosylation site that is absent in the B virus lab strain. (B) Resolved structure of the HSV-1 gD and HVEM complex (PDB code 1JMA). Residues 1 to 50 of gD are shown. The residues A7, M11, and N15 (shown in space-filling format) are accurately packed at the molecular surface of the receptor. (C) Model of the HVEM and B virus gD complex. Severe steric clashes of R7 and R11 are predicted, with no interactions between G15 and the receptor.
FIG 5
FIG 5
Computational modeling and comparison of the structures of B virus gD-122N and gD-122D variants liganded with nectin-1. (A) Computational modeling of B virus N122-carrying gD and nectin-1. The solved crystal structure of HSV-1 gD bound to human nectin-1 (PDB code 3U82) was used as a template. B virus gD and nectin-1 are represented as a stereo ribbon and van der Waals surfaces, respectively. The N122 residue is located more than 15 Å from the protein-protein nectin-1–gD interface and thus is not involved in the direct interaction between gD and nectin-1. (B) Overlay of the B virus gD-122N and gD-122D structures. The arrows mark N122 (gray) and D122 (orange). Note the considerable conformational changes of the globule in the area of the nectin-1 binding domain. These changes lead to a reduction in the energy of binding of the gD-122D variant to nectin-1, by ∼0.16 kcal/mol, compared to that of the gD-122N variant.
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