Herpes simplex virus type 1 (HSV-1) is a human pathogen of the alphaherpesvirus family which infects and spreads in the nervous system. Glycoproteins play a key role in the process of assembly and maturation of herpesviruses, which is essential for neuroinvasion and transneuronal spread. Glycoprotein B (gB) is a main component of the HSV-1 envelope and is necessary for the production of infectious particles. The cytoplasmic domain of gB, the longest one among HSV-1 glycoproteins, contains several highly conserved peptide sequences homologous to motifs involved in intracellular sorting. To determine the specific roles of these motifs in processing, subcellular localization, and the capacity of HSV-1 gB to complement a gB-null virus, we generated truncated or point mutated forms of a green fluorescent protein (GFP)-tagged gB. GFP-gB with a deletion in the acidic cluster DGDADEDDL (amino acids [aa] 896 to 904) behaved the same as the parental form. Deletion or disruption of the YTQV motif (aa 889 to 892) abolished internalization and reduced complementation by 60%. Disruption of the LL motif (aa 871 to 872) impaired the return of the protein to the trans-Golgi network (TGN) while enhancing its recycling to the plasma membrane. Truncations from residue E 857 abolished transport and processing of the truncated proteins, which had null complementation activity, through the Golgi complex. Altogether, our results favor a model in which HSV-1 gets its final envelope in the TGN, and they suggest that endocytosis, albeit not necessary, might play a role in infectivity.Herpes simplex virus type 1 (HSV-1) is a human alphaherpesvirus which generally causes benign infections of the skin and mucosa. The virus also infects neurons of the sensory ganglia, where it establishes latency. HSV-1 periodically reactivates to cause peripheral recurrences and occasionally spreads transneuronally to cause severe central nervous system infections. Although the mechanisms of neuroinvasion and transneuronal spread of alphaherpesviruses are not fully understood, they seem to be linked to the process of assembly and maturation of virions in infected cells, in which glycoproteins play a major role (33, 56).How HSV-1 particles are assembled and enveloped in infected cells is not completely known. The current view is that after primary envelopment at the inner nuclear membrane, capsids are de-enveloped through their exit from the nucleus and acquire their final envelope at the trans-Golgi network (TGN), where mature glycoproteins are incorporated (reviewed in reference 32). In support of this model, it has been shown that retention of glycoprotein D (gD) or gH in the endoplasmic reticulum (ER) of infected cells prevents their insertion into virions (6, 52, 61). Thus, proper intracellular transport of glycoproteins is necessary for successful incorporation into virions.gB, one of the most conserved envelope glycoproteins among herpesviruses, is essential at many steps of the viral replication cycle. In vitro, gB is essential for attachment and entr...