Role of Nectin-1, HVEM, and PILR-α in HSV-2 Entry into Human Retinal Pigment Epithelial Cells

Abstract

Purpose Herpes simplex virus-type 2 (HSV-2) can cause Acute Retinal Necrosis (ARN), which can lead to exudative and rhegmatogenous retinal detachment; yet, little is know about the cellular and molecular mechanisms of HSV-2 entry into retinal pigment epithelial (RPE) cells. The goal of this study was to establish the identity of the critical receptors utilized by the virus for infection. Methods A reporter HSV-2 virus, which expresses beta-galactosidase, was used to quantify entry into RPE cells and viral replication was ascertained using a plaque assay. Flow cytometry and immunocytochemistry were used to determine cellular expression of entry receptors. Localization of these receptors to either the apical or basal surface of RPE cells was determined using immunocytochemistry. The necessity of these receptors, both individually and in combination, for viral entry was established using both receptor-specific antibodies and siRNAs. Results RPE are highly susceptible to HSV-2 entry and replication. Several assays demonstrated the expression of the entry receptors nectin-1, HVEM, and PILR-alpha and their localization primarily to the apical surface of RPE cells. Receptor-specific antibodies and siRNA knockdown of receptors significantly reduced viral entry and implicated nectin-1 as an important receptor with HVEM and PILR-alpha potentially also contributing to entry. Conclusions HSV-2 is capable of developing a productive infection in RPE cells by utilizing nectin-1 as an important entry receptor. To lesser degrees, HVEM and PILR-alpha may also contribute to HSV-2 entry into RPE cells.

Syndecan-1 and syndecan-2 play key roles in herpes simplex virus type-1 infection

“…However, high affinity interactions (accompanied by conformation changes) may not be blocked by antibodies and therefore, the net effect by combining the antibodies may not be significantly higher than individual effects. This may be a reason why a near complete blocking of HSV-1 infection by antibodies has been extremely rare even when all gD receptors were blocked (Akhtar et al, 2008;Shukla et al 2009). Our results, nevertheless, highlight that syndecan-1 and syndecan-2 play a critical role during HSV-1 entry and that the two HSPGs show detectable differences in their abilities to facilitate infection.…”

“…Many known protein receptors for HSV-1 entry can be blocked by antibodies, which in turn, blocks viral entry (Akhtar et al, 2008;Shukla et al 2009). Similar to those receptors, we also found that pAbs against synedecan-1 and syndecan-2 block entry.…”

“…of 10), syndecan-1 and syndecan-2 protein expression was determined by Western blot analysis. The Western blot assay was performed according to the protocol described previously (Burbach et al, 2003;Shukla et al, 2009). Briefly, approximately 150-200 mg of total cell protein in lysis buffer was incubated with 2.5 volumes of 220 uC 100 % methanol overnight at 220 uC.…”

Investigations on molecular mechanisms of epithelial cell stress responses

“…Many known protein receptors for HSV-1 entry can be blocked by antibodies, which in turn, blocks viral entry [157,169]. Similar to those receptors, we also found that pAbs against synedecan-1 and syndecan-2 block entry.…”

“…However, high affinity interactions (accompanied by conformation changes) may not be blocked by antibodies and therefore, the net effect by combining the antibodies may not be significantly higher than the individual effects. This may be a reason why a near complete blocking of HSV-1 infection by antibodies has been extremely rare even when all gD receptors were blocked [157,169]. Our results, nevertheless, highlight that syndecan-1 and syndecan-2 both play a critical role during HSV-1 entry and that the two HSPGs show detectable differences in their abilities to facilitate infection.…”