Equine herpesvirus type 1 (EHV-1) and equine herpesvirus type 4 (EHV-4) are closely related viruses that have important differences in the key molecular aspects of infection. EHV-1 unlike EHV-4, can induce signaling cascade inside the host cell following gH-integrin interaction, that leads to exposure of phosphatidylserine (PS) in the outer leaflet of the plasma membrane, and is required for fusion at the plasma membrane. To evaluate what role exposure of PS to the outer leaflet of the plasma membrane plays in case of EHV-1 infection we investigated what effects lipids with positive, natural or negative charge had on EHV-1 infection in cell culture. We found that liposomes containing negatively charged PS or positively charged DOTAP inhibited EHV-1 infection, while neutral phosphatidylcholine (PC) had no effect. Inhibition of infection with PS was dose dependent, decreased with time, and transient. Reduction in infection can be attributed to the interaction between the lipid vesicles and the virus particle itself. The strong virus interaction was identified with both DOTAP and PS, as measured with microscopy with large unilamellar vesicles (LUVs) and giant unilamellar vesicles (GUVs) as well as Surface plasmon resonance (SPR) analysis of EHV-1 interactions with model membrane composed of neutral, cationic, or anionic phospholipids, in both cases interaction with cationic DOTAP was stronger that with PS. This suggests that PS induced inhibition of infection is more specific, than that of DOTAP. Perhaps PS inhibition is mediated through specific interaction with viral glycoproteins gH/gL and gB, while DOTAP can act in a less specific manner, by surrounding and shielding the viral particle. Small GTPases are ubiquitous signaling proteins, that could be activated downstream of EHV-1 induced calcium release. To investigate whether small GTPases play role in EHV-1 infection we infected cell treated with small GTPase inhibitors, we found that activation of small GTPases is required for EHV-1 infection at the post entry steps, using fluorescence resonance energy transfer (FRET) we identified that EHV-1 is activating Rac1 and Cdc42 in a few minutes after contact with the cell, although due to technical limitations this hypothesis could not be confirmed with alternative experiments. The finding that EHV-1 can penetrate inhibitor treated cells and that infection can be rescued through dilution of the inhibitor media, indicates that small GTPases are not essential for the virus entry itself. It was identified that small GTPases Rac1 and Cdc42 are required for EHV-1 induced acetylation of tubulin, which is consistent with the finding that Rac1 or Cdc42 inhibitor treated cells are worse at delivering the virus to the nucleus, and have reduced rates of cell-to-cell fusion, as measured by plaque diameter assay, and luciferase fusion assay. In order to examine if the small GTPases are being activated downstream from gH integrin induced pathway, number of non-integrin interacting viruses were employed, such as EHV-4, EHV-1 gH4 and EHV-1 gHS440A, were affected by small GTPase inhibitors, suggesting that small GTPases Rac1 and Cdc42 are activated in some way orthogonal from previously described gH integrin pathway. Taken together, the data suggests that PS could play a specific role in the EHV-1 infection, and that activation of small GTPases Rac1 and Cdc42 is important for infection of both EHV-1 and EHV-4. In EHV-1 Rac1 and Cdc42 play role in intracellular transport of the virus particle through acetylation of tubulin, and facilitate cell to cell spread, while not affecting the virus entry.
