Equine Herpesvirus Type 4 UL56 and UL49.5 Proteins Downregulate Cell Surface Major Histocompatibility Complex Class I Expression Independently of Each Other

Self Cite

Infected peripheral blood mononuclear cells (PBMC) effectively transport equine herpesvirus type 1 (EHV-1), but not EHV-4, to endothelial cells (EC) lining the blood vessels of the pregnant uterus or central nervous system, a process that can result in abortion or myeloencephalopathy. We examined, using a dynamic in vitro model, the differences between EHV-1 and EHV-4 infection of PBMC and PBMC-EC interactions. In order to evaluate viral transfer between infected PBMC and EC, cocultivation assays were performed. Only EHV-1 was transferred from PBMC to EC, and viral glycoprotein B (gB) was shown to be mainly responsible for this form of cell-to-cell transfer. For addressing the more dynamic aspects of PBMC-EC interaction, infected PBMC were perfused through a flow channel containing EC in the presence of neutralizing antibodies. By simulating capillary blood flow and analyzing the behavior of infected PBMC through live fluorescence imaging and automated cell tracking, we observed that EHV-1 was able to maintain tethering and rolling of infected PBMC on EC more effectively than EHV-4. Deletion of US3 reduced the ability of infected PBMC to tether and roll compared to that of cells infected with parental virus, which resulted in a significant reduction in virus transfer from PBMC to EC. Taking the results together, we conclude that systemic spread and EC infection by EHV-1, but not EHV-4, is caused by its ability to infect and/or reprogram mononuclear cells with respect to their tethering and rolling behavior on EC and consequent virus transfer. IMPORTANCEEHV-1 is widespread throughout the world and causes substantial economic losses through outbreaks of respiratory disease, abortion, and myeloencephalopathy. Despite many years of research, no fully protective vaccines have been developed, and several aspects of viral pathogenesis still need to be uncovered. In the current study, we investigated the molecular mechanisms that facilitate the cell-associated viremia, which is arguably the most important aspect of EHV-1 pathogenesis. The newly discovered functions of gB and pUS3 add new facets to their previously reported roles. Due to the conserved nature of cell-associated viremia among numerous herpesviruses, these results are also very relevant for viruses such as varicella-zoster virus, pseudorabies virus, human cytomegalovirus, and others. In addition, the constructed mutant and recombinant viruses exhibit potent in vitro replication but have significant defects in certain stages of the disease course. These viruses therefore show much promise as candidates for future live vaccines. E quine herpesvirus type 1 (EHV-1) and EHV-4 are members of the Herpesviridae family and Alphaherpesvirinae subfamily (1, 2). After initial replication in the upper respiratory tract, EHV-1 infects immune cells and migrates past the epithelial basement membrane to the lymph nodes and bloodstream (1-4). As a result, EHV-1 is able to spread throughout the body, where it infects endothelial cells (EC), causing vascular lesions an...

Section: Figsupporting

confidence: 77%

Section: Importancementioning

confidence: 99%

Role of gB and pUS3 in Equine Herpesvirus 1 Transfer between Peripheral Blood Mononuclear Cells and Endothelial Cells: a DynamicIn VitroModel

Spiesschaert

Goldenbogen

Taferner

et al. 2015

Self Cite

“…EHV-1 encodes pUL49.5, a type I transmembrane protein that blocks TAP-mediated peptide transport into the ER lumen, where the MHC-I complex is retained as a consequence of the absence of antigenic peptides. With respect to MHC-I downregulation, pUL49.5 and pUL56 are functionally independent (14), suggesting that pUL56 likely targets an alternative pathway to prevent MHC-I presentation. To examine whether pUL56 facilitates lysosomal proteolysis, we recorded the colocalization of MHC-I with lysosomes in a time course experiment by confocal laser scanning microscopy (CLSM).…”

Section: Resultsmentioning

confidence: 99%

“…Recently, we identified pUL56 of EHV-1 as an early viral protein that plays a dominant role in this process (13). A similar function of the pUL56 homologue of EHV-4 was also reported (14), suggesting that MHC-I downregulation caused by pUL56 might be evolutionarily conserved in the genus. However, the mechanism of pUL56 in downregulating cell surface MHC-I remains unknown.…”

mentioning

confidence: 90%

Major Histocompatibility Complex Class I Downregulation Induced by Equine Herpesvirus Type 1 pUL56 Is through Dynamin-Dependent Endocytosis

Huang

Lehmann

Said

et al. 2014

Self Cite

Here, we report that cell surface MHC-I in EHV-1-infected cells is internalized and degraded in the lysosomal compartment in a pUL56-dependent fashion. pUL56-induced MHC-I endocytosis required dynamin and tyrosine kinase but was independent of clathrin and caveolin-1, the main constituents of the clathrin-and raft/caveola-mediated endocytosis pathways, respectively. Downregulation of cell surface MHC-I was significantly inhibited by the ubiquitin-activating enzyme E1 inhibitor PYR41, indicating that ubiquitination is essential for the process. Finally, we show that downregulation is not specific for MHC-I and that other molecules, including CD46 and CD63, are also removed from the cell surface in a pUL56-dependent fashion. IMPORTANCEWe show that alphaherpesvirus induces MHC-I downregulation through endocytosis, which is mediated by pUL56. The dynamin-dependent endocytic pathway is responsible for MHC-I internalization in infected cells. Furthermore, we discovered that this endocytic process can be disrupted by the inhibiting ubiquitin-activating E1 enzyme, which is indispensable for ubiquitination. Finally, pUL56 action extends to a number of cell surface molecules that are significant for host immunity. Therefore, the protein may exert a more general immunomodulatory effect.

“…However, despite its presence on nuclear membranes (see below), gM is seemingly not involved in the release of herpesviruses from the nucleus, where newly made viral capsids are initially assembled (17). In contrast, with the conserved gN viral protein, gM alters immunity against the virus by downregulating the transport and peptide loading of major histocompatibility complex class I in the endoplasmic reticulum (ER) (25)(26)(27)(28)(29)(30). Finally, and perhaps most interestingly, gM has been reported to modulate virulence in animal models (31,32).…”

mentioning

confidence: 99%

Herpes Simplex Virus 1 gN Partners with gM To Modulate the Viral Fusion Machinery

Kasmi

Lippé

2015

Herpes simplex virus 1 (HSV-1) capsids are assembled in the nucleus, where they incorporate the viral genome. They then transit through the two nuclear membranes and are wrapped by a host-derived envelope. In the process, several HSV-1 proteins are targeted to the nuclear membranes, but their roles in viral nuclear egress are unclear. Among them, glycoprotein M (gM), a known modulator of virus-induced membrane fusion, is distributed on both the inner and outer nuclear membranes at the early stages of the infection, when no other viral glycoproteins are yet present there. Later on, it is found on perinuclear virions and ultimately redirected to the trans-Golgi network (TGN), where it cycles with the cell surface. In contrast, transfected gM is found only at the TGN and cell surface, hinting at an interaction with other viral proteins. Interestingly, many herpesvirus gM analogs interact with their gN counterparts, which typically alters their intracellular localization. To better understand how HSV-1 gM localization is regulated, we evaluated its ability to bind gN and discovered it does so in both transfected and infected cells, an interaction strongly weakened by the deletion of the gM amino terminus. Functionally, while gN had no impact on gM localization, gM redirected gN from the endoplasmic reticulum (ER) to the TGN. Most interestingly, gN overexpression stimulated the formation of syncytia in the context of an infection by a nonsyncytial strain, indicating that gM and gN not only physically but also functionally interact and that gN modulates gM's activity on membrane fusion. H erpesviridae are among the most complex human viruses from the point of view of their large genomes and viral particle composition. Among them, herpes simplex virus 1 (HSV-1), the prototype of human alphaherpesviruses, incorporates its 152-kb genome into an icosahedral capsid surrounded by a multiprotein tegument layer and a cell-derived lipid layer containing over a dozen viral proteins (1, 2). Of the latter, those mediating viral entry, namely, gB, gD, and the gH/gL complex, are essential for the propagation of the virus (3). In contrast, the viral glycoprotein M (gM) is conserved throughout the family and typically critical for beta-and gammaherpesviruses but is not essential for most alphaherpesviruses, including HSV-1 (4-16). Consequently, when gM is depleted from HSV-1 or the related alphaherpesvirus pseudorabies virus, viral yields are minimally reduced by 3-to 50-fold. However, its impact is substantially increased when U L 11 and gE/gI are codepleted in combination with gM, likely due to overlapping functions between these viral proteins (5,(17)(18)(19). IMPORTANCE HSV-1 gM is an important modulator of virally induced cell-cell fusion and viral entry, a process that is likely finely modulatedDespite its nonessential status in tissue culture, gM of several alphaherpesviruses has been associated with a number of functions throughout the viral life cycle (7,10,(19)(20)(21)(22). The glycoprotein is thus known to downregu...