Contribution of Endocytic Motifs in the Cytoplasmic Tail of Herpes Simplex Virus Type 1 Glycoprotein B to Virus Replication and Cell-Cell Fusion

Abstract

The use of endocytic pathways by viral glycoproteins is thought to play various functions during viral infection. We previously showed in transfection assays that herpes simplex virus type 1 (HSV-1) glycoprotein B (gB) is transported from the cell surface back to the trans-Golgi network (TGN) and that two motifs of gB cytoplasmic tail, YTQV and LL, function distinctly in this process. To investigate the role of each of these gB trafficking signals in HSV-1 infection, we constructed recombinant viruses in which each motif was rendered nonfunctional by alanine mutagenesis. In infected cells, wild-type gB was internalized from the cell surface and concentrated in the TGN. Disruption of YTQV abolished internalization of gB during infection, whereas disruption of LL induced accumulation of internalized gB in early recycling endosomes and impaired its return to the TGN. The growth of both recombinants was moderately diminished. Moreover, the fusion phenotype of cells infected with the gB recombinants differed from that of cells infected with the wild-type virus. Cells infected with the YTQV-mutated virus displayed reduced cell-cell fusion, whereas giant syncytia were observed in cells infected with the LL-mutated virus. Furthermore, blocking gB internalization or impairing gB recycling to the cell surface, using drugs or a transdominant negative form of Rab11, significantly reduced cell-cell fusion. These results favor a role for endocytosis in virus replication and suggest that gB intracellular trafficking is involved in the regulation of cell-cell fusion.Herpes simplex virus type 1 (HSV-1), a member of the herpesviruses family, is a ubiquitous human pathogen mainly responsible for infections of mucocutaneous epithelia that may recur due to the virus latency-reactivation cycle. HSV-1 occasionally spreads to the central nervous system, causing severe encephalitis. HSV-1 envelope glycoprotein B (gB), one of the most conserved glycoproteins among herpesviruses, is a major determinant of virus infectivity, in vitro and in vivo. The essential functions of this type 1 transmembrane protein at various steps of viral replication such as entry, fusion, and cell-cell spread, have been extensively studied and in part assigned to specific domains of the protein (11-13, 16, 24, 27, 32, 33, 43, 53, 59, 79), although a precise picture of the mechanisms by which gB fulfills these functions is still missing. Various studies have also illustrated the role of gB in experimental pathogenesis, in particular in HSV-1 neuroinvasion (27,41,78,83). How glycoproteins might be related to neuroinvasion has not been completely elucidated. However, transneuronal spread might be functionally related to the mechanisms of intracellular assembly and the formation of viral mature particles (25,50,69).It is well established that endocytosis pathways are used by numerous viral glycoproteins in infected cells, in a strategy supposedly aimed to help successful replication and to promote pathogenesis (18,45). Endocytosis of herpesviruses glycoproteins has...

Optimal Replication of Human Cytomegalovirus Correlates with Endocytosis of Glycoprotein gpUL132

“…84, 2010 HCMV GLYCOPROTEIN ENDOCYTOSIS 7049 necessary for efficient secondary envelopment was the explanation for the defect in virus replication in this mutant virus (19,24). The finding that the triple or quadruple gpUL132 mutant proteins are not incorporated into the virus particle is in contrast to findings with regard to other herpesvirus glycoproteins such as gE and Us9 of PRV or gB of HSV-1, for which endocytosis is not a prerequisite for incorporation into the virion (3,9,55). Regardless of the underlying mechanism, our data show, to our knowledge for the first time, that endocytosis of an HCMV glycoprotein is important for optimal virus replication.…”

“…Another layer of complexity with respect to intracellular transport is the nature of the trafficking motif that is analyzed. It was reported, for example, that for gB of herpes simplex virus type 1 (HSV-1), mutation of the LL motif induces formation of giant syncytia, whereas mutation of a YXXØ motif reduced cell-cell fusion (3).…”

“…For example, endocytosis of gE of varicellazoster virus (VZV) has been shown to be essential for viral replication, whereas it is irrelevant for replication of pseudorabies virus (PRV) (37,55). For gB of several herpesviruses, endocytosis seems to be dispensable for virion formation since its inhibition has little effect on replication kinetics (3,40). In the case of HCMV little information is available on endocytosis of envelope glycoproteins.…”

Preventing Cleavage of the Respiratory Syncytial Virus Attachment Protein in Vero Cells Rescues the Infectivity of Progeny Virus for Primary Human Airway Cultures

“…All enveloped viruses have hijacked this system to deliver their glycoproteins to the correct membranes to envelop their capsid structures (52)(53)(54)(55)(56)(57)(58)(59)(60)(61)(62)(63). In at least one instance, the Nipah virus F protein requires endocytic recycling to activate its F protein in the late endosome/lysosome by either cathepsin B or cathepsin L (26,64,65).…”

An immunoreceptor tyrosine-based inhibition motif in varicella-zoster virus glycoprotein B regulates cell fusion and skin pathogenesis

“…Whereas syncytia formation is typical of VZV, engineered or spontaneous substitutions in the gBcyt in other herpesviruses lead to syncytial herpesvirus phenotypes (15)(16)(17). The cytoplasmic domains of gB homologs contain α-helices and endocytosis motifs (18)(19)(20). Endocytosis, linked to dileucine and YXXΦ motifs (X, any amino acid; Φ, a large hydrophobic residue), is necessary for gB cellsurface retrieval, trans-Golgi network (TGN) localization, and incorporation into the virion envelope (21).…”