Capsid Maturation

During the herpesvirus cleavage/packaging reaction, DNA is inserted into preformed capsids. Cleaved viral genomes are not detected in cells infected with viruses that fail to assemble capsids, suggesting that capsids contain essential parts of the cleavage/packaging machinery.32 There are four morphologic types of capsids that can be distinguished by electron microscopic examination of thin sections of herpesvirus-infected cells, and they are designated procapsids and capsid types A, B, and C.33'34 The capsid shell of procapsids is morphologically distinct from all other capsid types and appears porous and roughly spherical by electron microscopy.33'35 Procapsids contain internal scaffold proteins. Capsid types A, B and C are more angularized icosahedrons of approximately 120 nm in diameter. Type A capsids consist of only the icosahedral shell, type B capsids contain the angular shell and a roughly spherical internal scaffold, and type C capsids contain DNA and lack the scaffold.34'36"38 The fact that type C capsids contain only the capsid shell and DNA indicates that the scaffold is lost during the packaging reaction (see Fig. 4).

The internal scaffold is comprised of the scaffold protein encoded by Ul26.5 and the viral protease encoded by Ul26.39"43 The Ul26 and Ul26.5 proteins are encoded by overlapping transcripts and the encoded proteins share identical C-termini (see Fig. 3). The amount of Ul26.5 gene product far exceeds that of Ul26, presumably because of the strong Ul26.5 promoter that lies within the Ul26 open reading frame. The protease domain resides in the N-terminus of Ul26, and is absent from the UL26.5-encoded scaffold. The unprocessed forms of Ul26 and Ul26.5 proteins are initially incorporated into capsids (Fig. 4).44'45 The C-termini of the Ul26 and Ul26.5 gene products can interact with the major capsid protein, VP5, and such associations are believed to play critical roles in the assembly of the procapsid.46"49

In one widely accepted model of capsid maturation,33 the procapsid is the precursor of all capsid types. Upon activation, the protease cleaves both (i) itself to release protease (VP24), scaffold (VP21), and a 25 amino acid C-terminal peptide, and (ii) the C-terminus of Ul26.5 protein to release the major scaffold protein (VP22a) and the 25 amino acid peptide42'43'50 (Fig. 3). The protease domain (VP24) remains within the capsid while the C termini of the Ul26 and Ul26.5 scaffold proteins (VP21 and VP22a, respectively) are extruded. Presumably, canb t-ŒD-

3) CapükL binding

5) Second Cleavage c u ZM7{1 bp)

Packaged Genome

Unpacked Contatemer

Figure 2. Proposed events in cleavage and packaging of HSV-1 DNA. Schematic diagram of concatemeric DNA, and a detailed view of the a sequence are shown in the first two lines. The first cleavage occurs within the DR1 immediately adjacent to I'ac 2, such that the DNA that is to be packaged contains DR1 with 18 of its 20 base pairs and a single base 3' overhang (not indicated, step 1). The other (Us) end, terminated by a single bp of DR1 plus a single base 3' overhang (not indicated), is presumably degraded (step 2). After association of the cleaved DNA end with the capsid portal (step 3), Ul is packaged, followed by Us (step 4). The second cleavage is diagrammed in the last 2 lines as occurring within the DR1 shared between two a sequences (step 5), although the presence of 2 tandem a sequences is not necessary for successful cleavage/ packaging. This second cleavage is dependent on Pac 1 sequences, and occurs such that the DNA to be packaged is terminated by a single base pair of DR1. As a result, the terminus of the unpackaged concatemeric DNA (bottom right) is identical to the end to be packaging after the first DNA cleavage (step 1), and should therefore be able to initiate a subsequent round of packaging.

| UL26 ORF

1

UL2G mRNA

UL26.5 mRNA

UL26 protein (protease)

VP24 VP21

25 aa

(protease) (scaffold)

peptide

UL26.5 protein (scaifold)

VP22a

25 aa

(major

peptide

scaffold)

Figure 3. The Ul26 gene and the Ul26 and Ul26.5 mRNA transcripts and protein products are depicted at the top. Primary translation products are represented by black boxes. Proteolysis products are depicted in gray. The proteolytic products are VP24, which contains the protease domain, two scaffold proteins (VP21 and VP22a) and a 25 amino acid terminal peptide.

Figure 3. The Ul26 gene and the Ul26 and Ul26.5 mRNA transcripts and protein products are depicted at the top. Primary translation products are represented by black boxes. Proteolysis products are depicted in gray. The proteolytic products are VP24, which contains the protease domain, two scaffold proteins (VP21 and VP22a) and a 25 amino acid terminal peptide.

the capsid shell is thereby released from constraints that preclude maturation into the more angularized form of capsids. Supporting this proposition are observations that the timing of protease activation correlates with the morphological transformation of the procapsid to the mature angularized form.51"53

There are 3 possible outcomes of the proposed maturation pathway, but only one that leads to productive nucleocapsid assembly, (i) Type A capsids arise when DNA is not inserted or retained (i.e., packaging is incomplete or aborted) but the internal scaifold is expelled, (ii) Type B capsids result when DNA is not inserted and the internal scaffold is trapped within the angularized capsid shell, or (iii) C capsids arise when the packaging machinery is properly engaged, genomic DNA is inserted, the internal scaffold is expelled, and the capsid shell undergoes the conformational change, sealing the DNA inside. This model is supported by the observations that procapsids spontaneously form in vitro upon mixture of capsid components produced in heterologous expression systems,54 and that such procapsids transform into type B capsids over time.33

The model is appealing because it incorporates the procapsid into the assembly pathway and explains the origin of all capsid types from a single reaction. Because types A and B capsids are dead ends in this model, the remarkable implication is that the packaging reaction must be tighdy coordinated with capsid maturation to favor production of type C capsids over types A and B capsids. It might therefore be envisioned that in order to prevent premature angularization and sealing of the capsid, the viral protease packaged within the internal capsid shell must somehow be restrained from cleavage of the internal scaffold until the precise time that DNA and the packaging machinery engage the portal vertex.

Herpesvirus Assembly

Figure 4. Model of HSV-1 DNA cleavage and packaging and capsid maturation. Based on published reports on capsid assembly, scaffold, and DNA packaging proteins associated with wild-type and mutant virus capsids, we propose a model for the maturation of capsids in herpesvirus infected cells (described in the text). The unprocessed form of Ul26 protease/scaffold protein is depicted in blue, and the Ul26.5 scaffold is depicted in red. The Ul6 protein is depicted as a gray cylinder and the putative two subunit terminase composed of Ul15 and Ul28 is depicted as green and purple ovals. Note, Ui.33 has recently been shown to associate with Ul 15 and U[28 and may be a subunit of the teminase. Ul25 is depicted as a green triangle. Note that although Ul25 is drawn as being located on the outside of the capsid, it is possible that some or all of Ul25 is actually located inside the capsid. This figure is adapted from the model presented in Dr. Amy Sheafier's thesis (University of Connecticut Health Center). A color version of this figure is available online at http://www.Eurekah.com.

Figure 4. Model of HSV-1 DNA cleavage and packaging and capsid maturation. Based on published reports on capsid assembly, scaffold, and DNA packaging proteins associated with wild-type and mutant virus capsids, we propose a model for the maturation of capsids in herpesvirus infected cells (described in the text). The unprocessed form of Ul26 protease/scaffold protein is depicted in blue, and the Ul26.5 scaffold is depicted in red. The Ul6 protein is depicted as a gray cylinder and the putative two subunit terminase composed of Ul15 and Ul28 is depicted as green and purple ovals. Note, Ui.33 has recently been shown to associate with Ul 15 and U[28 and may be a subunit of the teminase. Ul25 is depicted as a green triangle. Note that although Ul25 is drawn as being located on the outside of the capsid, it is possible that some or all of Ul25 is actually located inside the capsid. This figure is adapted from the model presented in Dr. Amy Sheafier's thesis (University of Connecticut Health Center). A color version of this figure is available online at http://www.Eurekah.com.

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