HSV-1 utilizes multiple receptors to attach and enter into a variety of cells including neurons. Recently, it was shown that gB binds to cellular receptors that are required for gB-mediated membrane fusion during virus entry and virus-induced cell fusion. We show here that the HSV-1 McKrae strain utilizes the gB receptor PIRLα more efficienty than HSV-1(F). DNA sequencing of all viral glycoprotein genes involved in membrane fusion indicates that there are a number of aa differences between HSV-1(F) and McKrae in gB, gH and gL that may affect PIRLα- mediated virus entry.
Initial entry experiments revealed that PIRL-α did not function as efficiently in facilitating virion entry in comparison to the gD receptors nectin-1 and HVEM when strains HSV-1(F) and KOS were used (not shown). Recently, we cloned the HSV-1(McKrae) strain as a bacterial articifical chromosome (bac) that has enabled us to rapidly produce mutant viruses, as we have previously done with HSV-1(F) bac [42, 43]. We have found that this viral strain efficiently enters into a variety of cells including CHO cells constitutively expressing PIRLα. PIRLα binds gB and membrane fusion can be affected by interactions of gB with viral glycoproteins gD, gH/gL and gK/UL20 [18, 40, 41]. Therefore, we compared the predicted aa sequences between F and McKrae strains and found a number of aa that could contribute to the observed increased efficiency of entry of McKrae over F and other strains. Of particular importance were aa changes between F and McKrae located within the amino terminus of gB, known to bind to PIRLα and gK [41, 44]. Specifically, gB binds to PIRLα via O-linked glycans located at aa positions 53 and 480 . However, gB binding to PIRLα is also dependent on the conformation of the amino terminus of gB, since aa insertions in gB could reduce this binding . Insertional mutagenesis has revealed that the structure and function of gB is not particularly flexible in tolerating aa insertions . The McKrae gB contains additional proline residues at aa positions 67 and 77, while other proline residues have been re-arranged. Specifically, the F gB has a proline at aa 61, which is an alanine for the McKrae strain, but the McKrae gB contains a proline at aa 62 instead of alanine. The structure of the amino terminus of gB is not known, although the x-ray structure of gB (aa 111-726) was obtained . The additional proline residues suggest that the amino terminus of gB assumes a conformation that may affect binding to PIRLα [44, 47–49]. In addition, this altered conformation of gB may affect interactions with gK, which binds to the amino terminus of gB and regulates gB-mediated membrane fusion . Interestingly, six aa changes seen in McKrae versus F gB were conserved in the gB specified by the neurovirulent strain ANG  suggesting that these aa may contribute to neurovirulence.
Viral glycoproteins gD and gH have been shown to bind gB and modulate its ability to cause membrane fusion . Therefore, mutations within the extracellular portions of gD and gH, as well as gL may affect the ability of gB to utilize the PIRL-α receptor. In addition, since gH forms a functional heterodimer with gL , it is possible that the observed aa differences between McKrae and F within gH domain H1 may affect interaction with gL, known to bind exclusively to this domain . The carboxyl terminus of gH has been shown to be important for virus-induced cell fusion . Therefore, the observed aa changes N670S and R720C may alter virus entry kinetics. Multiple aa changes in gL between McKrae and F (S22P, K90R, V100G, N115D) are within the gL domain known to interact with gH and may affect gH/gL cell-surface expression, cell fusion and virus entry.
Positively charged aa residues are known to be critical for interaction with negatively charged sulfate/carboxylate groups of the HS chain . Moreover, basic aa residues are known to be critical for HS-binding activity . Therefore, the gC Q75K and H306R aa changes in McKrae compared to F may cause increased HS binding. Previous studies have delineated the HS-binding domain that interacts with gC amino terminal residues to be located between aa 33 and 123 , and between 129 and 247 . Therefore amino acid changes in McKrae gC (aa 75, 116 and 132) may result in increased initial binding of McKrae gC.
The PILR alpha gene is expressed on cells of the immune system (monocytes, dendritic cells, NK cells, B cells, macrophages, neutrophils, eosinophils, mast cells), as well as neurons [13, 56]. Moreover, HSV-1 enters into corneal epithelial cells (HCE) via the nectin1, HVEM and PILRα receptors . Additional experiments are needed to determine the biological and pathogenetic implications of increased utilization of the PIRLα receptor by the HSV-1(McKrae). The availability of the McKrae strain as a bacterial artificial chromosome will enable the rapid construction of mutant viruses that could be used to elucidate the role of each viral glycoprotein in PIRLα mediated virion entry.