Structure based functional analyses of pseudorabies virus glycoprotein H
Entry and spread of enveloped viruses can be used as basic model for membrane fusion in eukaryotic cells, since the viral fusion machineries consist of only few proteins. Whereas influenza virus uses only one protein for attachment and penetration, alphaherpesviruses require four different envelope glycoproteins for these processes: core fusion protein gB, heterodimer gH/gL, and gD. After attachment to specific receptors like nectin-1/-2 gD is considered to submit a signal to gH/gL, leading to conformational changes in gH. These are assumed to activate the fusogen gB, resulting in merger of the viral envelope with the host cell membrane. However, experimental studies are required to elucidate this process in detail.This thesis is focused on gH and its role in herpesvirus induced membrane fusion. Based on the recently resolved crystal structures of a core fragment of PrV gH, and of the gH/gL complexes of HSV-2 and EBV, targeted amino acid substitutions were introduced into domains II and III of PrV gH. Appropriately located cyteine pairs can lead to formation of artificial disulfide bonds, immobilizing e.g. the "syntaxin-like-bundle" (SLB) of three a-helices in domain II of gH, which are supposed to undergo structural changes like similar helices in eukaryotic fusogens (Syntaxin-1A). The introduced alterations clamped the SLB helices (a2-a4) to each other (A250C-A276C: a2-a3; A253C-A269C: a2-a3; A284C-S291C: a3-a4), to a proximate b-sheet designated as "fence" (V174C-A270C: b3-a3), or to domain III (H252C-A419C: a2-a10). Prolines were inserted to disrupt either of the three a-helices, which resulted in inhibition of maturation, transport, and virion incorporation of gH, almost complete loss of function in membrane fusion and entry. The effects of the proline mutation in helix a3 were shown to be partly corrected by a compensatory mutation within the same helix (V271A). Structure predictions indicate, that helix distortion by the proline mutation affects packing of the SLB to the fence, and that the substitution of valiine by the smaller amino acid alanine at an adjacent postion enables a closer packing, which is necessary for gH maturation and/or function.After synthesis at the rER, gH is transported for final processing to the Golgi apparatus and either incorporated into virions or forwarded to the cell surface. In western blots analyses, several of the generated disulfide bond mutants showed maturation defects (H251C-L432C; A250C-A276C), whereas others did not (e.g. A284C-S291C). Indirect immunofluorescence investigations further revealed that transport of maturation deficient gH to the cell surface was inhibited. Nevertheless, several gH mutants (H251C and A250C-A276C) exhibited wild-type like in vitro fusion activities when co-expressed with PrV gB, gD, and gL. Thus, minimal amounts of mature gH on the cell surface are sufficient for function. Also flexibility of the SLB seems to be crucial for effective function, since clamping of two of the a-helices in gH mutant A284C-S291C led to strongly reduced fusion activity, inefficient cell-to-cell spread and a very slow penetration, although maturation, cell surface expression and virion incorporation of gH were not affected. The defects were partly corrected after treatment with DTT, which reduces disulfide bonds. Transient in vitro fusion assays include the same viral glycoproteins which are also involved in virus entry, but the present studies indicated some mechanistic differences between these two fusion events. This was demonstrated by gH mutation A250C-A276C which barely affected fusion but entailed significantly delayed penetration kinetics of a corresponding virus recombinant. Conversely, neither of the above mentioned gH mutants V275P and V271A-V275P showed any in vitro fusion activity but nevertheless the compensatory mutation V271A increased plaque sizes (about 200 fold), and infectious virus titres of the A275P mutant about 200-fold, and 10.000-fold, respectively. Thus different elements of gH might be relevant for efficient transport to the cell surface and virion incorporation, and different amounts or structural properties of gH might be required for fusion between two plasma membranes or viral envelope and plasma membrane.gH chimeras composed of gH domains originating from two different alphaherpesviruses, HSV-1 and PrV, were constructed to test functional conservation and independence of structurally conserved domains. The studies revealed that replacement of the best conserved domain IV in PrV gH by the corresponding part of HSV-1 gH leads to a functional protein which is able to activate the core fusion proteins gB of PrV and HSV-1 in transient fusion assays. The least conserved domain I of HSV-1 gH could substitute for the corresponding domain in PrV gH, but only when coexpressed with HSV gL. This finding demonstrated that gH domain I is required and sufficient for interaction with the corresponding gL. Separate exchange of domains II and III between PrV and HSV-1 generally resulted in complete loss of function indicating species-specific protein interactions and a structural interdependence of these domains. In vitro assays further confirmed that the core fusion machinery of PrV, unlike that of HSV-1, exhibits activity in the absence of gD or gL. Interestingly, gD-independent fusion activity of the PrV set of glycoproteins was blocked by addition of HSV-1 gD, indicating that this protein might possess not only activating, but also inhibitory functions to prevent unspecific fusion reactions, gD-independent membrane fusion was also not observed with the two gH chimeras, which both required PrV gD for function.To determine whether in vitro fusion activity of these gH chimeras correlated with function during virus infection, stably expressing cell lines were prepared and tested for trans-complementation of gH-deleted PrV or HSV-1. The gH chimera consisting of HSV-1 domain I and PrV domains II to IV did not support replication of either of the virus mutants. However, the gH chimera containing domains I to III from PrV and domain IV from HSV-1 enabled cell-to-cell spread and formation of infectious, phenotypically complemented, virus particles of gH-deleted PrV at high titres. This finding represents the first example for function of a chimeric form of the essential herpesvirus glycoprotein H in the viral context.
Böhm, Sebastian: Structure based functional analyses of pseudorabies virus glycoprotein H. Ernst-Moritz-Arndt-Universität Greifswald, Mathematisch-Naturwissenschaftliche Fakultät 2016.
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