Insights into the interaction and nuclear capsid-association of herpes simplex virus type 1 tegument proteins UL36 and UL37.

摘要

Herpesvirus virions are composed of three morphologically distinct structures: the capsid, tegument and envelope. The icosahedral capsid encloses the viral DNA genome. Virions are bounded by a host-derived lipid envelope embedded with viral glycoproteins and other transmembrane proteins. The tegument is the proteinaceous region that lies between the capsid and envelope. More than 25 virus-encoded proteins, as well as host proteins, are packaged into the tegument region. Incorporation of tegument proteins may occur at multiple sites within the cell and is governed by a complex series of protein interactions. Tegument proteins may associate with capsids within the nucleus, as the capsid exits the nucleus through budding and fusion with the nuclear membrane, during microtubule-mediated transport in the cytoplasm and during final envelopment at vesicles derived from the trans-Golgi network (TGN). The studies described in this dissertation were initiated to identify tegument proteins associated with capsids isolated from the nucleus of herpes simplex virus type 1 (HSV-1)-infected cells.;Several lines of evidence led us to investigate if the conserved, essential tegument protein, UL36, is associated with capsids in the nucleus. UL36 is tightly associated with capsids, coimmunoprecipitates with the major capsid protein, VP5, and is present in the nucleus of HSV-1-infected cells. Furthermore, viruses lacking UL36 exhibit a severe defect in tegumentation and do not undergo final envelopment, suggesting that incorporation of UL36 may be required for subsequent acquisition of other tegument proteins. To address this issue, capsids were isolated and purified from the nuclear fraction of HSV-1-infected cells and analyzed for the presence of UL36 by Western blotting. As hypothesized, detectable amounts of UL36 associate with capsids from the nuclear fraction. Interestingly, UL36 associates only with DNA-filled C capsids and is not detected in association with A capsids or B capsids, which do not mature into infectious virions. Further analysis of nuclear capsids revealed that the UL36 binding partner, UL37, also associates with capsids isolated from the nuclear fraction. These results suggest that UL36 and UL37 are among the initial tegument proteins incorporated into virions and therefore may function to facilitate the incorporation of other tegument proteins into the virus particle.;The interaction of UL36 and UL37 homologues is conserved across the herpesvirus family. The region and individual residues of UL36 critical for interaction with UL37 have been identified. In contrast, the region of UL37 necessary for binding UL36 was unknown. To fill this gap of knowledge, coimmunoprecipitation assays were used to identify the region of UL37 necessary for binding UL36. Deletion of amino acids 568-809 of UL37 reduced coimmunoprecipitation of UL36 to near background levels. The carboxy-terminal half of UL37, residues 568-1123, was sufficient for interaction with UL36. Further deletion of this region of UL37 caused deleterious effects on UL36 binding. These studies revealed that amino acids 568-809 are necessary and the carboxy-terminal half of UL37 is sufficient for interaction with UL36.;To expand the identification of interaction domains of UL37, studies were also performed to identify the regions of UL37 involved in self-association. Coimmunoprecipitation experiments revealed that two regions of UL37, amino acids 1- 300 and 568-1123, are independently capable of self-association. Because the carboxy-terminal half of UL37 serves dual roles of self-association and UL36 binding, studies were performed to determine if this region is capable of interacting with both proteins simultaneously, thus forming a UL37-UL37-UL36 complex. Full length UL37 appears to self-associate only under conditions when UL36 is not present, or is present in low amounts. This result suggests that UL36 competes with UL37 for interaction with another molecule of UL37. Therefore UL36 appears to regulate the self-association of UL37.;Expression of UL37 is necessary for tegumentation and envelopment of infectious HSV-1 virions. The functional role(s) of UL37 that is critical during infection is unknown. To identify regions of UL37 necessary for production of infectious virions, a trans-complementation assay was utilized. The carboxy-terminal region of UL37, amino acids 568-1123, partially rescues the UL37 deletion virus, KAUL37. These results suggest that the carboxy-terminus of UL37 may contribute to its essential functional role within the virus-infected cell. In conclusion, the carboxy-terminal half of UL37, amino acids 568-1123, is involved in UL37 self-association, interaction with UL36 and plays a critical role during virus assembly.;Collectively, the studies described in this dissertation provide novel insights regarding the interaction of tegument proteins UL36 and UL37 with capsids and the regions of UL37 involved in the conserved interaction with UL36. These studies may provide the foundation for future investigations to elucidate the functional role(s) of UL36 and UL37 critical for assembly of infectious virions.