Role of the Herpes Simplex Virus CVSC Proteins at the Capsid Portal Vertex

Abstract

The packaging of DNA into preformed capsids is a critical step during herpesvirus infection. For herpes simplex virus, this process requires the products of seven viral genes: the terminase proteins pUL15, pUL28 and pUL33; the capsid vertex specific component (CVSC) proteins pUL17 and pUL25; the portal protein pUL6 and pUL32. The pUL6 portal dodecamer is anchored at one vertex of the capsid by interactions with the adjacent triplexes as well as helical density attributed to the pUL17 and pUL25 subunits of the CVSC. To define the roles and structures of the CVSC proteins in virus assembly and DNA packaging we isolated a number of recombinant viruses expressing pUL25, pUL17 and pUL36 fused with green or red fluorescent proteins as well as viruses with specific deletions in the CVSC genes. Biochemical and structural studies of these mutants demonstrated that: (i) four of the helices in the CVSC helix bundle can be attributed to two copies each of pUL36 and pUL25; (ii) pUL17 and pUL6 are required for capsid binding of the terminase complex in the nucleus; (iii) pUL17 is important for determining the site of the first cleavage reaction generating replicated genomes with termini derived from the long-arm component of the HSV-1 genome; (iv) pUL36 serves no direct role in cleavage/packaging; (v) cleavage and stable packaging of the viral genome involves an ordered interaction of the terminase complex and pUL25 with pUL17 at the portal vertex; and (vi) packaging of the viral genome results in a dramatic displacement of the portal. Importance Herpes simplex virus type I (HSV-1) is the causative agent of several pathologies ranging in severity from the common cold sore to life-threatening encephalitic infection. A critical step during productive HSV-1 infection is the cleavage and packaging of replicated, concatemeric viral DNA into preformed capsids. A key knowledge gap is how the capsid engages the replicated viral genome and the subsequent packaging of a unit length HSV genome. Here biochemical and structural studies focused on the unique portal vertex of wild type HSV and packaging mutants provide insights into the mechanism of HSV genome packaging. The significance of our research is in identifying the portal protein pUL6 and pUL17 as key viral factors for engaging the terminase complex with the capsid and the subsequent cleavage, packaging, and stable incorporation of the viral genome in the HSV-1 capsid.

Cryo-Electron Tomography of the Herpesvirus Procapsid Reveals Interactions of the Portal with the Scaffold and a Shift on Maturation

“…This led to placing the portal either at the level of capsomers (28) or inside the capsid shell (29). Subsequent analysis employing cryo-electron microscopy (cryoEM) and single-particle analysis (SPA), indicated that the portal is located inside the capsid shell (30,31). One of the difficulties in the SPA approach is that the overlap of one vertex with the large mass of the capsid (;100-fold ratio) makes it hard to distinguish a portal vertex from the penton vertices.…”

Cryo-electron tomography of the herpesvirus procapsid reveals interactions of the portal with the scaffold and a shift on maturation

“…Fig. 3B) (for the assignment of this density as a triplex, see Huet et al(31)). The widest part of the portal, the "wing domain" extends towards the floors of the neighboring capsomers (blue arrow inFig.…”

“…Subsequent analysis employing cryo-electron microscopy (cryoEM) and single particle analysis (SPA), indicated that the portal is located inside the capsid shell (30,31). One of the difficulties in the SPA approach is that the overlap of one vertex with the large mass of the capsid (~100 fold ratio) makes it hard to distinguish a portal vertex from the penton vertices.…”

“…Nevertheless, the extensive averaging of structural information means that correct assignments would dominate incorrect assignments distributed over the eleven other possibilities and reveal the location of the portal. The recent technological advances in cryoEM enabled the high resolution reconstruction of various herpesvirus capsids showing the portal on the inside of one vertex (18,(31)(32)(33)(34).…”

Structures and Divergent Mechanisms in Capsid Maturation and Stabilization Following Genome Packaging of Human Cytomegalovirus and Herpesviruses

“…Recently, it was shown that pUL17 (pUL93 in HCMV) anchors the terminase complex to the capsid and is involved in determining the site of the first cleavage reaction. In addition, pUL17 interacts with the asymmetric dimer of pUL25 (pUL77 in HCMV) [58].…”