Arriving at an in-depth understanding of equine herpesvirus type 1 and 4 (EHV-1 & EHV-4) disease: The role of mononuclear cells in viral pathogenesis
Virology
Final Report Abstract
Cell entry of most alphaherpesviruses is mediated by the binding of glycoprotein D (gD) to different cell surface receptors. Equine herpesvirus 1 (EHV-1) and EHV-4 gDs interact with equine major histocompatibility complex I (MHC-I) as an initial step to enter quine cells. We have characterized gD-MHC-I interaction by solving the crystal structures of EHV-1 and EHV-4 gDs (gD1, gD4), performing protein-protein docking simulation, surface plasmon resonance (SPR) analysis, and viral assays. The structures of gD1 and gD4 revealed the existence of common V-set immunoglobulin-like (IgV-like) core comparable to that of other gD homologs. Molecular modeling yielded plausible binding hypotheses and identified key residues (F213 and D261) that are important for virus binding. Mutating these key residues impaired virus growth in cells, which highlights the important role of these residues in gD-MHC-I interaction. These results added to our understanding of herpesvirus-cell interactions and will contribute to the targeted design of antiviral drugs and vaccine development. Viruses utilize host cell signaling to facilitate productive infection. Equine herpesvirus type 1 (EHV-1) has been shown to activate Ca2+ release and phospholipase C upon contact with α4β1 integrins on the cell surface. Signaling molecules including small GTPases have been shown to be activated downstream of Ca2+ release and modulate virus entry, membrane remodeling and intracellular transport. The work conducted in this project could show that EHV-1 activates the small GTPases Rac1 and Cdc42 during infection. Activation of Rac1 and Cdc42 is necessary for virus-induced acetylation of tubulin, effective viral transport to the nucleus, and cell-to-cell spread. We also could show that inhibitors of Rac1 and Cdc42 did not block virus entry but inhibited overall virus infection. Exposure of phosphatidylserine (PS) in the outer leaflet of the plasma membrane is induced by infection with several members of the Alphaherpesvirinae subfamily. There is evidence that PS is used by the equine herpesvirus type 1 (EHV-1) during entry, but the exact role of PS and other phospholipids in the entry process remains unknown. We have investigated the interaction of differently charged phospholipids with EHV particles and determined their influence on infection. We could show that liposomes containing negatively charged PS or positively charged DOTAP (N-[1-(2,3-Dioleoyloxy)propyl]- N,N,N-trimethylammonium) inhibited EHV-1 infection, while neutral phosphatidylcholine (PC) had no effect. Inhibition of infection with PS was transient, decreased with time, and was dose dependent. Our findings indicate that both cationic and anionic phospholipids can interact with the virus and reduce infectivity, while, presumably, acting through different mechanisms. Charged phospholipids were found to have antiviral effects and may be used to inhibit EHV-1 infection of horses. EHV-1 causes encephalomyelopathy and abortion, for which cell-associated viremia and subsequent virus transfer to and replication in endothelial cells (EC) are responsible and prerequisites. Viral and cellular molecules responsible for efficient cell-to-cell spread of EHV-1 between peripheral blood mononuclear cells (PBMC) and EC remain unclear. We could identify three genes (ORF1, ORF2 and ORF17) that significantly reduced cell-to-cell virus transfer from virus-infected PBMC to EC and modulated chemokine signaling and MAPK pathways in infected PBMC, which may explain their role in virus spread between PBMC and EC. This study uncovered cellular proteins and pathways influenced by EHV-1 after PBMC infection and provide an important resource for EHV-1 pathogenesis. EHV-1-immunomodulatory genes could be potential targets for the development of live attenuated vaccines or therapeutics against virus infection. We further unraveled unique mechanisms of how equid herpesvirus-1 manipulates PBMC to travel further in the body. (i) “PBMC-hitching”: at the initial contact, herpesviruses lurk in the extracellular matrix (ECM) of PBMC without entering the cells. The virus exploits the components of the ECM to bind, transport and then egress to infect other cells. (ii) “Intracellular delivery”: transendothelial migration is a physiological mechanism where mononuclear cells can transmigrate through the endothelial cells. The virus was intangible and probably did not interfere with such a mechanism where the infected PBMC can probably deliver the virus inside the endothelium. (iii) “Classical-fusion”: this process is well mastered by herpesviruses due to a set of envelope glycoproteins that facilitate cell-cell fusion and virus spread.
Publications
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Differentially-Charged Liposomes Interact with Alphaherpesviruses and Interfere with Virus Entry. Pathogens. 2020;9(5):E359
Kolyvushko O, Latzke J, Dahmani I, Osterrieder N, Chiantia S, Azab W
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Equine alphaherpesviruses require activation of the small GTPases Rac1 and Cdc42 for intracellular transport. Microorganisms. 2020 Jul 7;8(7):1013
Kolyvushko O, Kelch M, Osterrieder N, Azab W
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Equine Herpesvirus Type 1 Modulates Cytokine and Chemokine Profiles of Mononuclear Cells for Efficient Dissemination to Target Organs. Viruses 2020 Sep 8;12(9):E999
Pavulraj S, Kamel M, Stephanowitz H, Liu F, Plendl J, Osterrieder N, and Azab W
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Herpesviruses exploit the extracellular matrix of mononuclear cells to ensure transport to target cells. iScience 2020 Oct 23;23(10):101615
Kamel M, Pavulraj S, Fauler B, Mielke T, and Azab W
