Varicella-zoster Virus gB and gE coexpression, but not gB or gE alone, leads to abundant fusion and syncytium formation equivalent to those from gH and gL coexpression

Abstract

Varicella-zoster virus (VZV) is distinguished from herpes simplex virus type 1 (HSV-1) by the fact that cell-to-cell fusion and syncytium formation require only gH and gL within a transient-expression system. In the HSV system, four glycoproteins, namely, gH, gL, gB, and gD, are required to induce a similar fusogenic event. VZV lacks a gD homologous protein. In this report, the role of VZV gB as a fusogen was investigated and compared to the gH-gL complex. First of all, the VZV gH-gL experiment was repeated under a different set of conditions; namely, gH and gL were cloned into the same vaccinia virus (VV) genome. Surprisingly, the new expression system demonstrated that a recombinant VV-gH+gL construct was even more fusogenic than seen in the prior experiment with two individual expression plasmids containing gH and gL (K. M. Duus and C. Grose, J. Virol. 70:8961-8971, 1996). Recombinant VV expressing VZV gB by itself, however, effected the formation of only small syncytia. When VZV gE and gB genes were cloned into one recombinant VV genome and another fusion assay was performed, extensive syncytium formation was observed. The degree of fusion with VZV gE-gB coexpression was comparable to that observed with VZV gH-gL: in both cases, >80% of the cells in a monolayer were fused. Thus, these studies established that VZV gE-gB coexpression greatly enhanced the fusogenic properties of gB. Control experiments documented that the fusion assay required a balance between the fusogenic potential of the VZV glycoproteins and the fusion-inhibitory effect of the VV infection itself.

FIG. 1

Confocal microscopic imaging following infection with VZV recombinant VVs. (A) HeLa cells were infected by VV-gH+gL double recombinant virus and labeled with anti-gH MAb 258 plus Texas Red-conjugated secondary antibody as well as anti-gH MAb V₃ plus Alexa 488-conjugated secondary antibody. Yellow represents colocalization of the two fluoroprobes within syncytia. Blue represents TOTO-3 staining of nuclei. (B) HeLa cells were infected by VV-gH and labeled as described above; no antibody attached to gH. (C) HeLa cells were infected by VV-gB and labeled with anti-gB MAb 158 plus Texas Red-conjugated secondary antibody as well as anti-gB MAb V₁ plus Alexa 488-conjugated secondary antibody. Yellow indicates colocalization of gB forms within syncytia. (D) HeLa cells were infected by VV-gE and labeled with anti-gE MAb 3B3 plus Alexa 488-conjugated secondary antibody.

FIG. 2

Confocal microscopic imaging of VZV gB and gE expressed by single recombinant VVs. HeLa cells were coinfected by single recombinant viruses VV-gE and VV-gB and labeled with anti-gB MAb V₁ plus Alexa 488-conjugated secondary antibody as well as anti-gE MAb 3B3 plus Texas Red-conjugated secondary antibody. (A) VZV gB-specific staining. (B) VZV gE-specific staining. (C) Colocalization of VZV gB and gE (yellow) produced by merging the fluoroprobes in panels A and B.

FIG. 3

Confocal microscopic imaging of a polykaryon induced by VV-gE+gB. HeLa cells were infected by VV-gE+gB double recombinant virus, and the two glycoproteins were labeled as described in the legend to Fig. 2. Colocalization of VZV gE together with gB is indicated by the orange pseudocolor produced from merging of the two fluoroprobes. Cell nuclei were pseudocolored blue with TOTO-3. Note that this micrograph represents one horizontal plane; more nuclei would be detectable below and above that represented in the figure.

FIG. 4

Analysis of VZV gE as well as gB biosynthesis and cell surface expression. HeLa cells were infected with either VV-gE (A and E) or VV-gB (C and G) recombinant virus alone or double recombinant virus VV-gE+gB (B, D, F, and H). The cultures were pulse-labeled with [³⁵S]methionine-cysteine for 45 min (A to D), after which the radioactive medium was replaced with regular minimum essential medium-fetal bovine serum for increasing time intervals indicated in the figure. Cell lysates were immunoprecipitated with either MAb 3B3 (A and B) or MAb V₁ (C, D, and A and B, Pulse). After elution, the samples were analyzed by SDS-PAGE. Molecular mass markers are on the right. The infected cultures (E to H) were examined for surface expression of gE and gB by laser scanning microscopy. Unpermeabilized cell cultures were labeled with anti-gE MAb 3B3 and Alexa 488-conjugated secondary antibody (E and F) as well as anti-gB MAb 158 and Alexa 488-conjugated secondary antibody (G and H). In the micrographs (20× magnification), the green color is represented by white; quantification of fluorescent pixels was carried out in an Adobe Photoshop software program.

FIG. 5

Confocal microscopic imaging of VZV gB and gE expressed by the VV T7-pTM1 transfection system. HeLa cells were cotransfected by plasmids containing the VZV gB and gE genes, and the two glycoproteins were labeled as described in the legend to Fig. 2 to verify that both VZV products were coexpressed. In this micrograph, the yellow color demonstrating colocalization within a syncytium is represented by white.

FIG. 6

Polykaryocyte formation induced by different VZV glycoproteins. HeLa cells were infected by the following recombinant VVs: VV-gE (A), VV-gB (B), VV-gE+gB (C), VV-gH (D), VV-gE and VV-gH (E), and VV-gH+gL (F). The infected cell monolayers were processed as described in Materials and Methods. Bar, 100 μm.

FIG. 7

Inhibition of VZV gE+gB-mediated fusion by VV. HeLa cell monolayers were infected with a recombinant VV as described in the text; in addition, some monolayers were coinfected with wild-type VV. (A) VV-gE+gB at an MOI of 5 PFU per cell; (B) VV-gE+gB at an MOI of 5 as well as VV strain Praha at an MOI of 15; (C) VV strain Praha at an MOI of 15; (D) VV-gE+gB at an MOI of 5 as well as VV strain WR at an MOI of 15; (E) VV strain WR at an MOI of 15; (F) VV-gE+gB double recombinant virus at an MOI of 15. The infected cell monolayers were processed as described in Materials and Methods. Bar, 100 μm.