Novel mutations in gB and gH circumvent the requirement for known gD Receptors in herpes simplex virus 1 entry and cell-to-cell spread

Abstract

Both entry and cell-to-cell spread of herpes simplex virus (HSV) involve a cascade of cooperative interactions among the essential glycoproteins D, B, and H/L (gD, gB, and gH/gL, respectively) initiated by the binding of gD to a cognate HSV entry receptor. We previously reported that a variant (D285N/A549T) of glycoprotein B (gB:NT) enabled primary virus entry into cells that were devoid of typical HSV entry receptors. Here, we compared the activities of the gB:NT variant with those of a newly selected variant of glycoprotein H (gH:KV) and a frequently coselected gB variant (gB:S668N). In combination, gH:KV and gB:S668N enabled primary virus entry into cells that lacked established HSV entry receptors as efficiently as did gB:NT, but separately, each variant enabled only limited entry. Remarkably, gH:KV uniquely facilitated secondary virus spread between cells that lacked canonical entry receptors. Transient expression of the four essential entry glycoproteins revealed that gH:KV, but not gB:NT, induced fusion between cells lacking the standard receptors. Because the involvement of gD remained essential for virus spread and cell fusion, we propose that gH:KV mimics a transition state of gH that responds efficiently to weak signals from gD to reach the active state. Computational modeling of the structures of wild-type gH and gH:KV revealed relatively subtle differences that may have accounted for our experimental findings. Our study shows that (i) the dependence of HSV-1 entry and spread on specific gD receptors can be reduced by sequence changes in the downstream effectors gB and gH, and (ii) the relative roles of gB and gH are different in entry and spread.

Fig 1

Selective characterization of virus isolates. A total of 46 isolates were initially characterized by complete sequencing of their gD genes and grouped according to the results; the number of isolates in each group is shown. Sequencing of the gB genes of selected isolates identified the existence of 2 alleles, wt and S668N. Additional isolates were screened for the presence of the S668N-diagnostic HincII restriction site, and the number of isolates scoring positive [Hinc(+)] or negative [Hinc(-)] is indicated. Several isolates representing similar or different combinations of gD/gB alleles were further characterized by nearly complete or localized (*) gH sequencing, and the complete sequence of the gL gene of one isolate was determined.

Fig 2

Effects of gB and gH mutations on virus entry into gD receptor-deficient cells. CHO-K1 and Vero cells (A) and B78H1 cells (B) were infected for 6 h at the MOIs shown at the top of the columns and immunostained for ICP4. Images are representative of 3 independent experiments.

Fig 3

Effects of gB and gH mutations on cell-to-cell spread to and between gD receptor-deficient cells. (A) Vero cells were infected with the viruses indicated above the panels (MOI of 10). Extracellular virus was inactivated by acidic wash, and equal numbers of infected (donor) cells were added onto monolayers of the uninfected cells indicated at the left (acceptor cells). The mixed cultures were overlaid with methylcellulose-containing medium and immunostained for VP16 at 48 hpi. (B) B78/0G cells cultured for 48 h with the donor cells as described for panel A were observed under a fluorescence microscope. Magnifications are indicated at the left. Images are representative of 2 independent experiments.

Fig 4

Effects of gD on cell-to-cell spread by gH:KV mutant viruses. VD60 cells were infected at an MOI of 10 with K-gH:KV, K-gB:668N-gH:KV [both gD(+)], or their gD knockout derivatives prepared on VD60 cells [gD(-/+)]. Extracellular virus was inactivated by acidic wash, and equal numbers of infected (donor) cells were added onto monolayers of uninfected B78H1 or Vero (acceptor) cells. The mixed cultures were overlaid with methylcellulose-containing medium and immunostained for VP16 at 72 hpi. Images are representative of 2 independent experiments.

Fig 5

Effects of the gH:KV mutations on viral replication and egress. Vero (A) or B78/C (B) cells were infected at an MOI of 3 for 1 h followed by acid treatment. The titers of the cell lysates and media were determined following additional incubation at 37°C for 4, 8, or 24 h.

Fig 6

Effects of the gH:KV mutations on cell-cell fusion. (A) B78H1 cells were cotransfected with plasmids for the indicated glycoproteins, Giemsa staining was performed at 72 h posttransfection, and syncytia were counted. The gH plasmids containing either of the KV mutations alone are indicated as K (for N753K) and V (for A778V). Shown are the averages ± the SEM of data from two experiments performed in triplicate. (B) Representative images of syncytium formation by B78H1 (upper panels) and B78/C (lower panels) cells after cotransfection of different combinations of expression plasmids for wild-type gD, gB, and gL, indicated at the top, with plasmids expressing gH:wt or gH:KV, as indicated at the left. Giemsa-stained cells were photographed at either 20 h posttransfection (B78/C, first column) or 72 h posttransfection (all other panels).

Fig 7

Global and local effects of the gH:KV mutations. (A) Ribbon diagram of the H2-H3 domains of HSV-1 gH:wt with the positions of the mutant residues in gH:KV (N753K, A778V) highlighted as space-filling models in magenta and the two halves of the flap represented as dark- and light-blue sticks. (B) Cumulative root mean square deviation (RMSD) from the respective starting structures for the H2-H3 domain of HSV-1 gH:wt and gH:KV. (C) RMSDs of two sides of the flap region, E707-K716 and R717-D726.

Fig 8

Ribbon diagrams showing the location of residues whose pairwise interactions were monitored. (A) Pairs of residues in the H3 domain; (B) pairs of residues across the H2-H3 interface. Magenta spheres, positions of the KV mutations (753 and 778); dark- and light-blue sticks, flap residues. Monitored interactions are labeled and circled or indicated by two-sided arrows.

Fig 9

Time evolution of the interaction between pairs of residues in the H3 domain for gH:wt (A) and gH:KV (B). Top, T700_L680 and T700_G685; middle, S745_R736 and A/V778_L790; bottom, M735_V675 and G654_G708. Interactions are defined as the minimum distance between any two atoms of the pairs at a given time along the simulation trajectory.

Fig 10

Time evolution of the interaction between pairs of residues at the H2-H3 interface and structure representations. (A) Interactions L593_L664 (black), M744_S556 (light blue), and D596_R662 (dark blue) for gH:wt (left) and gH:KV (right). (B) Ribbon diagrams of the H2-H3 domains of gH:wt (left) and gH:KV (right) at 45 ns. The pairs of interacting residues monitored in the diagrams at the top are highlighted in space-filling representation and are color coded as in panel A.