Herpes simplex virus glycoproteins gB and gH function in fusion between the virion envelope and the outer nuclear membrane

Abstract

Herpesviruses must traverse the nuclear envelope to gain access to the cytoplasm and, ultimately, to exit cells. It is believed that herpesvirus nucleocapsids enter the perinuclear space by budding through the inner nuclear membrane (NM). To reach the cytoplasm these enveloped particles must fuse with the outer NM and the unenveloped capsids then acquire a second envelope in the trans-Golgi network. Little is known about the process by which herpesviruses virions fuse with the outer NM. Here we show that a herpes simplex virus (HSV) mutant lacking both the two putative fusion glycoproteins gB and gH failed to cross the nuclear envelope. Enveloped virions accumulated in the perinuclear space or in membrane vesicles that bulged into the nucleoplasm (herniations). By contrast, mutants lacking just gB or gH showed only minor or no defects in nuclear egress. We concluded that either HSV gB or gH can promote fusion between the virion envelope and the outer NM. It is noteworthy that fusion associated with HSV entry requires the cooperative action of both gB and gH, suggesting that the two types of fusion (egress versus entry) are dissimilar processes.

Fig. 1.

EM of HaCaT cells infected with F-BAC gB−/gH− for 18 h. The arrow in B points to a virion within the perinuclear space and protruding into the cytoplasm. The arrow in C points to a continuity between the inner NE and the membrane surrounding a herniation.

Fig. 2.

EM of Vero cells infected with F-BAC gB−/gH− for 18 h. N, nucleus; C, cytoplasm.

Fig. 3.

HSV gB and gH localize to the nuclear envelope. (A) Immunofluorescence studies of HaCaT cells infected with TsProtA at 39°C for 12 h then stained with chicken anti-UL34 or mouse anti-emerin antibodies and, simultaneously, with rabbit gB− or gH− antibodies. Secondary Texas red-conjugated anti-chicken IgY or anti-mouse IgG and FITC-conjugated anti-rabbit IgG secondary antibodies were used. (B and C) Immunoelectron microscopy of wild-type HSV-infected HaCaT cells stained with rabbit anti-gH antibodies and donkey anti-rabbit IgG conjugated onto 18-nm gold particles. Arrows point to perinuclear particles. N, nucleus; C, cytoplasm.

Fig. 4.

Immunoelectron microscopy of HaCaT cells infected with F-BAC gB−/gH− for 18 h then stained with chicken UL34-specific antibodies followed by donkey anti-chicken IgY conjugated onto 6-nm gold particles (A and B) or rabbit anti-gD antibodies followed by donkey anti-rabbit IgG conjugated onto 18-nm gold particles (C).