Glycerophospholipids are major components of cell membranes. Phosphatidylethanolamine (PE) is a glycerophospholipid and is involved in multiple cellular processes, such as membrane fusion, cell cycle, autophagy and apoptosis. In this study, we investigated the role of PE biosynthesis in herpes simplex virus 1 (HSV-1) infection by knocking out the host cell gene encoding phosphate cytidylyltransferase 2, ethanolamine (Pcyt2), which is a key rate-limiting enzyme in one of the two major pathways for PE biosynthesis. Pcyt2 knockout reduced HSV-1 replication and caused an accumulation of unenveloped and partially enveloped nucleocapsids in the cytoplasm of an HSV-1-infected cell culture. A similar phenotype was observed by treating infected cells with meclizine, which is an inhibitor of Pcyt2. In addition, treatment of HSV-1-infected mice with meclizine significantly reduced HSV-1 replication in the mouse brains and improved their survival. These results indicated that PE biosynthesis mediated by Pcyt2 was required for efficient HSV-1 envelopment in the cytoplasm of infected cells and for viral replication and pathogenicity in vivo. The results also identified the PE biosynthetic pathway as a possible novel target for antiviral therapy of HSV-associated diseases and raised an interesting possibility for meclizine repositioning for treatment of these diseases, since it is an over-the-counter drug used for decades against nausea and vertigo in motion sickness. IMPORTANCE Glycerophospholipids in cell membranes and virus envelopes often affects viral entry and budding. However, the role of glycerophospholipids in herpesvirus infected cells on membrane-associated events in viral replication has not been reported thus far. In this study, we have presented data showing that cellular phosphatidylethanolamine (PE) biosynthesis mediated by Pcyt2 is important for HSV-1 envelopment in the cytoplasm as well as for viral replication and pathogenicity in vivo. This is the first report showing the importance of PE biosynthesis in herpesvirus infections. Our results showed that inhibition of Pcyt2, a key cell enzyme for PE synthesis, significantly inhibited HSV-1 replication and pathogenicity in mice. This suggested that the PE biosynthetic pathway, as well as the HSV-1 virion maturation pathway, can be targets for the development of novel anti-HSV drugs.
Identification of the mechanisms of viral evasion from human antibodies is crucial both for understanding viral pathogenesis and for designing effective vaccines. However, the in vivo efficacy of the mechanisms of viral evasion from human antibodies has not been well documented. Here we show in cell cultures that an N-glycan shield on the HSV-1 envelope glycoprotein B (gB) mediated evasion from neutralization and antibody-dependent cellular cytotoxicity due to pooled γ-globulins derived from human blood. We also demonstrated that the presence of human γ-globulins in mice and HSV-1 immunity induced by viral infection in mice significantly reduced the replication of a mutant virus lacking the glycosylation site in a peripheral organ but had little effect on the replication of its repaired virus. These results suggest that the glycan shield on the HSV-1 envelope gB mediated evasion from human antibodies in vivo and from HSV-1 immunity induced by viral infection in vivo. Notably, we also found that the glycan shield on HSV-1 gB was significant for HSV-1 neurovirulence and replication in the central nervous system (CNS) of naïve mice. Thus, we have identified a critical glycan shield on HSV-1 gB that has dual impacts, namely evasion from human antibodies in vivo and viral neurovirulence.
Viral cell-to-cell spread, a method employed by several viral families for entrance via cell junctions, is highly relevant to the pathogenesis of various viral infections. Cell-to-cell spread of herpes simplex virus 1 (HSV-1) is known to depend greatly on envelope glycoprotein E (gE). However, the molecular mechanism by which gE acts in HSV-1 cell-to-cell spread and the mechanisms of cell-to-cell spread by other herpesviruses remain poorly understood. Here, we describe our identification of prohibitin-1 as a novel gE-interacting host cell protein. The ectopic expression of prohibitin-1 increased gE-dependent HSV-1 cell-to-cell spread. As observed with the gE-null mutation, the decreased expression or pharmacological inhibition of prohibitin-1 reduced HSV-1 cell-to-cell spread without affecting the yield of virus progeny. Similar effects were produced by pharmacological inhibition of the mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) pathway, wherein prohibitin-1 acts as a protein scaffold and is required for induction of this pathway. Furthermore, artificial activation of the MAPK/ERK pathway restored HSV-1 cell-to-cell spread impaired by the gE-null mutation. Notably, pharmacological inhibition of prohibitins or the MAPK/ERK pathway reduced viral cell-to-cell spread of representative members in all herpesvirus subfamilies. Our results suggest that prohibitin-1 contributes to gE-dependent HSV-1 cell-to-cell spread via the MAPK/ERK pathway and that this mechanism is conserved throughout the Herpesviridae, whereas gE is only conserved in the Alphaherpesvirinae subfamily. Importance Herpesviruses are ubiquitous pathogens of various animals, including humans. These viruses primarily pass through cell junctions to spread to uninfected cells. This method of cell-to-cell spread is an important pathogenic characteristic of these viruses. Here, we show that the host cell protein prohibitin-1 contributes to HSV-1 cell-to-cell spread via the downstream intracellular signaling cascade, the MAPK/ERK pathway. We also demonstrate that the role of the prohibitin-1-mediated MAPK/ERK pathway in viral cell-to-cell spread is conserved in representative members of every herpesvirus subfamily. This study has revealed a common molecular mechanism of the cell-to-cell spread of herpesviruses.
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