Productive entry of HSV into host cells proceeds following endocytosis  or by direct penetration at the cell surface . The viral and cellular factors that determine which pathway is utilized are not clear. The viral envelope glycoproteins gB, gD, and gH-gL are required for entry by both endocytic and non-endocytic routes [3–7]. Expression of a cellular entry receptor is required for both penetration at the plasma membrane and for penetration following endocytosis [1, 7–9]. Such receptors function individually and can mediate entry into non-permissive cells, such as Chinese hamster ovary (CHO) cells . The viral ligand for HSV entry receptors is gD [11–17]. In the absence of a gD-receptor, HSV is still endocytosed by CHO cells, but fails to penetrate the endosomal membrane and is degraded .
The known gD-receptors include nectins, which belong to a subgroup of the immunoglobulin (Ig) superfamily [17–20]. They are broadly distributed cell-cell adhesion molecules that are components of cadherin-based adherens junctions . Nectin-1 and nectin-2 are ~40% identical, and their N-terminal Ig-like variable (V) domains are critical for gD-binding [11, 22–26] and for viral entry [11, 23–28]. All HSV strains tested to date [11, 17, 29] are able to utilize nectin-1 as an entry receptor. Nectin-2 mediates entry of several laboratory strains and clinical isolates of HSV-1 and HSV-2, including HSV-1 isolates from the CNS of patients with herpes simplex encephalitis [19, 29]. Amino acid changes in gD at residues 25, 27, or 28 confer the ability to utilize nectin-2 [19, 24, 30, 31]. Additional gD-receptors include HVEM, a member of the TNF-receptor superfamily  and heparan sulfate that has been modified by 3-O-sulfotransferase-3 . Nectin-3  and B5  also mediate HSV entry, but their viral ligand(s) is not clear.
Following endocytosis from the cell surface, HSV entry into a subset of cell types also requires intracellular low pH [1, 7, 9, 35, 36]. CHO cells expressing gD-receptors are a widely used, well-characterized model system to study pH-dependent, endocytic entry. Inhibitors of endosomal acidification block HSV entry at a step subsequent to endocytic uptake but prior to penetration of the capsid into the cytosol . It has been proposed that HSV utilizes distinct cellular pathways to enter its relevant target cells . Alphaherpesviruses undergo pH-dependent, endocytic entry into certain epithelial cells [1, 9, 35], including primary human epidermal keratinocytes , yet utilize a pH-independent entry pathway into neurons [35, 37, 38]. Recently, Whitbeck et al. showed that in vitro binding of HSV to liposomes could be triggered by a combination of receptor-binding and low pH .
Direct study of the membrane fusion activity of herpesvirions has proven difficult. Fusion-from-without (FFWO) is the induction of target cell fusion by addition of intact virions to the monolayer surface in the absence of viral protein expression. Virus-cell fusion during entry and virion-induced FFWO are analogous inasmuch as both involve similar effector (virion) membranes and target membranes. Several syncytial strains of HSV-1, such as ANG path, are capable of triggering FFWO . HSV-induced FFWO is cell type-dependent , but the receptor requirements of FFWO are not known. In the present study, ANG path is used as a tool to investigate the influence of viral and cellular proteins on the route that HSV takes into cells. The ANG path-CHO cell model system allows examination of the inter-relatedness of gD-receptor usage, HSV-induced fusion, and selection of entry pathway.