Mutagenesis of varicella-zoster virus glycoprotein B: putative fusion loop residues are essential for viral replication, and the furin cleavage motif contributes to pathogenesis in skin tissue in vivo

Abstract

Glycoprotein B (gB), the most conserved protein in the family Herpesviridae, is essential for the fusion of viral and cellular membranes. Information about varicella-zoster virus (VZV) gB is limited, but homology modeling showed that the structure of VZV gB was similar to that of herpes simplex virus (HSV) gB, including the putative fusion loops. In contrast to HSV gB, VZV gB had a furin recognition motif ([R]-X-[KR]-R-|-X, where | indicates the position at which the polypeptide is cleaved) at residues 491 to 494, thought to be required for gB cleavage into two polypeptides. To investigate their contribution, the putative primary fusion loop or the furin recognition motif was mutated in expression constructs and in the context of the VZV genome. Substitutions in the primary loop, W180G and Y185G, plus the deletion mutation Delta491RSRR494 and point mutation 491GSGG494 in the furin recognition motif did not affect gB expression or cellular localization in transfected cells. Infectious VZV was recovered from parental Oka (pOka)-bacterial artificial chromosomes that had either the Delta491RSRR494 or 491GSGG494 mutation but not the point mutations W180G and Y185G, demonstrating that residues in the primary loop of gB were essential but gB cleavage was not required for VZV replication in vitro. Virion morphology, protein localization, plaque size, and replication were unaffected for the pOka-gBDelta491RSRR494 or pOka-gB491GSGG494 virus compared to pOka in vitro. However, deletion of the furin recognition motif caused attenuation of VZV replication in human skin xenografts in vivo. This is the first evidence that cleavage of a herpesvirus fusion protein contributes to viral pathogenesis in vivo, as seen for fusion proteins in other virus families.

FIG. 1.

Location of the amino acids deleted or substituted in gB of VZV pOka. (A) Homology model of the ectodomain of VZV gB showing the location of the furin recognition motif (⁴⁹¹RSRR⁴⁹⁴) (red space fill). Domains are indicated by colors used by Heldwein et al. (28) as follows: blue (domain I), green (domain II), yellow (domain III), orange (domain IV), and red (domain V). (B) Homology model of VZV gB domain I showing the location of residues W180 and Y185 in the putative primary fusion loop. (C) Amino acid alignment of the complete gBs from VZV[pOka] and HSV-1[KOS] showing the location of the amino acid substitutions (solid underline and pink shading) at W180 and Y185 plus the deletion/substitution of arginine at residues 491 to 494. Domains are indicated by the same colors used in panel A except for the transmembrane domain (gray). Dots in the alignment represent identical amino acids, and dashes represent gaps in the alignment. The dotted underline at residues 833 to 846 shows the location of the epitope in VZV gB used to generate the polyclonal rabbit serum 746-868.

FIG. 2.

Confocal microscopy showing the localization of VZV gB in melanoma cells transiently transfected with pCDNA3.1 constructs expressing wild-type or mutated gBs. (A) Cells were stained for gB (the conformation-dependent MAb SG2) (blue), early endosomes (rabbit polyclonal to EEA-1) (red), multivesicular bodies (goat anti-VPS4) (green), and nuclei (Hoechst 33342) (yellow). (B) Cells were stained for gB (MAb SG2) (blue), early endosomes (rabbit polyclonal to EEA) (red), trans-Golgi (sheep anti-TGN46) (green), and nuclei (Hoechst 33342) (yellow). Bar, 50 μm.

FIG. 3.

Western blot of VZV gB immunoprecipitated from HEK 293, melanoma, or LoVo cells transiently transfected with pCDNA3.1 constructs expressing wild-type or mutated gBs. gB was immunoprecipitated with the conformation-dependent MAb 158. The numbered arrowheads highlight the four polypeptides detected by the rabbit polyclonal antiserum 746-868 developed to the peptide ⁸³³PEGMDPFAEKPNAT⁸⁴⁶ located in the cytoplasmic domain of gB. The molecular masses (in kilodaltons) shown next to the arrowheads for each of the four polypeptides were calculated from protein standards. The bottom panel of LoVo cells was from a longer exposure to show the presence of the 60-kDa protein. The vector-only lane for the LoVo cells was from the same blot but was placed on the right side for consistency.

FIG. 4.

PCR analysis to confirm that the MiniF⁻ plasmid was excised from the self-excisable pPOka-DX BAC upon passage in HELFs. SopA and Cat are genes in the MiniF plasmid. Mk, DNA ladder in 100-bp increments from 400 to 800 bp; WT, wild-type pOka; Δ, pOka-gBΔ⁴⁹¹RSRR⁴⁹⁴; G, pOka-gB⁴⁹¹GSGG⁴⁹⁴; −ve, no-template PCR-negative control; +ve, pP-Oka-DX BAC DNA (10 ng/μl).

FIG. 5.

Immunoprecipitation of gB from melanoma cells infected with pOka, pOka-gBΔ⁴⁹¹RSRR⁴⁹⁴, and pOka-gB⁴⁹¹GSGG⁴⁹⁴. gB was immunoprecipitated using MAb 158, and either the gels were silver stained (A) or Western blotting was performed using the rabbit anti-gB peptide serum 746-868 (B). The numbers next to the arrowheads are the molecular masses calculated for each of the proteins. Molecular masses were calculated from standard curves derived from the protein molecular mass markers.

FIG. 6.

Replication of wild-type pOka and the two gB mutant viruses pOka-gBΔ⁴⁹¹RSRR⁴⁹⁴ and pOka-gB⁴⁹¹GSGG⁴⁹⁴ in melanoma cells. (A) Replication kinetics of pOka, pOka-gBΔ⁴⁹¹RSRR⁴⁹⁴, and pOka-gB⁴⁹¹GSGG⁴⁹⁴ over 6 days. (B) Plaque morphologies of pOka, pOka-gBΔ⁴⁹¹RSRR⁴⁹⁴, and pOka-gB⁴⁹¹GSGG⁴⁹⁴ in melanoma cells at 4 days postinoculation.

FIG. 7.

Confocal microscopy showing the cellular localization of the viral proteins gB and IE63 in melanoma cells infected with wild-type pOka and the two gB mutant viruses pOka-gBΔ⁴⁹¹RSRR⁴⁹⁴ and pOka-gB⁴⁹¹GSGG⁴⁹⁴. Images were captured at 24, 48, and 72 h postinfection. Melanoma cells were stained with rabbit anti-IE63 (green), MAb to gB (SG2) (red), and Hoechst 33342 to stain the nuclei (blue). Bar, 50 μm.

FIG. 8.

Confocal microscopy showing the localization of VZV gB in melanoma cells infected with wild-type pOka, pOka-gBΔ⁴⁹¹RSRR⁴⁹⁴, or pOka-gB⁴⁹¹GSGG⁴⁹⁴ at 48 h postinfection. (A) Cells were stained for gB (MAb SG2) (blue), early endosomes (rabbit polyclonal to EEA-1) (red), multivesicular bodies (goat anti-VPS4) (green), and nuclei (Hoechst 33342) (yellow). (B) Cells were stained for gB (MAb SG2) (blue), early endosomes (rabbit polyclonal to EEA-1) (red), trans-Golgi (sheep anti-TGN46) (green), and nuclei (Hoechst 33342) (yellow). Bar, 50 μm.

FIG. 9.

Electron microscopy of virus particles in HELFs infected with the wild-type pOka or mutant gB viruses pOka-gBΔ⁴⁹¹RSRR⁴⁹⁴ and pOka-gB⁴⁹¹GSGG⁴⁹⁴. Images were captured using a Philips CM-12 transmission electron microscope at a magnification of ×3,000 (3K) (large image), ×10K (left), ×22K (middle), or ×75K (right). White arrows indicate the location of the virus particle shown in the images taken at a magnification of ×75K.