Abstract
The late stages of assembly of herpes simplex virus (HSV) and other herpesviruses are not well understood. Acquisition of the final virion envelope apparently involves interactions between viral nucleocapsids coated with tegument proteins and the cytoplasmic domains of membrane glycoproteins. This promotes budding of virus particles into cytoplasmic vesicles derived from the trans-Golgi network or endosomes. The identities of viral membrane glycoproteins and tegument proteins involved in these processes are not well known. Here, we report that HSV mutants lacking two viral glycoproteins, gD and gE, accumulated large numbers of unenveloped nucleocapsids in the cytoplasm. These aggregated capsids were immersed in an electron-dense layer that appeared to be tegument. Few or no enveloped virions were observed. More subtle defects were observed with an HSV unable to express gD and gI. A triple mutant lacking gD, gE, and gI exhibited more severe defects in envelopment. We concluded that HSV gD and the gE/gI heterodimeric complex act in a redundant fashion to anchor the virion envelope onto tegument-coated capsids. In the absence of either one of these HSV glycoproteins, envelopment proceeds; however, without both gD and gE, or gE/gI, there is profound inhibition of cytoplasmic envelopment.
Highlights
Herpes simplex virus (HSV) is in many respects the best studied of the herpesviruses and the paradigm for ␣-herpesvirus replication and assembly
In the case of cytoplasmic envelopment, it is clear that viral membrane glycoproteins accumulate extensively in the trans-Golgi network (TGN) or endosomes and this likely promotes incorporation into the virion envelope
Wild-type herpes simplex virus (HSV)-1 strain F and the HSV type 1 (HSV-1) gEϪ recombinants derived from F: F-gE [14] and F-gE-green fluorescent protein (GFP) were propagated and their titers were determined on Vero cells. gDϪ recombinants derived from F, F-US6kan [21, 25], F-gD, which lacks gD and gI sequences [31], and vRR1097, which has much of the gD coding sequences replaced by GFP sequences [41], as well as the gDϪ/gEϪ recombinants described below, were propagated and their titers were determined on gD-expressing VD60 cells [31]
Summary
Herpes simplex virus (HSV) is in many respects the best studied of the herpesviruses and the paradigm for ␣-herpesvirus replication and assembly. The cytosolic domains of these membrane glycoproteins probably provide a surface on which the last stage of assembly, i.e., acquisition of the virion envelope, occurs It is not clear which of the viral membrane proteins are important for tethering of tegument-coated capsids onto the envelope. Large numbers of nucleocapsids accumulated in the cytoplasm of cells infected with PRV double mutants lacking gE, or just the cytoplasmic (CT) domain of gE, and a second glycoprotein gM [6, 7] These cytoplasmic capsids were immersed in an electron-dense array that appeared to be tegument and which stained with anti-UL49 antibodies [7]. HSV VP16 was found to associate with gD in mild detergent extracts of purified preparations of HSV virions [28] It appears that several tegument proteins may interact with more than a single ␣-herpesvirus membrane protein to promote cytoplasmic envelopment. This was consistent with subtle effects of the gD CT domain in virus replication or cell-to-cell spread
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