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. 2005 Aug;79(16):10540-6.
doi: 10.1128/JVI.79.16.10540-10546.2005.

Involvement of the portal at an early step in herpes simplex virus capsid assembly

William W Newcomb  1 , Fred L Homa, Jay C Brown
Affiliations

Involvement of the portal at an early step in herpes simplex virus capsid assembly

William W Newcomb et al. J Virol. 2005 Aug.

Abstract

DNA enters the herpes simplex virus capsid by way of a ring-shaped structure called the portal. Each capsid contains a single portal, located at a unique capsid vertex, that is composed of 12 UL6 protein molecules. The position of the portal requires that capsid formation take place in such a way that a portal is incorporated into one of the 12 capsid vertices and excluded from all other locations, including the remaining 11 vertices. Since initiation or nucleation of capsid formation is a unique step in the overall assembly process, involvement of the portal in initiation has the potential to cause its incorporation into a unique vertex. In such a mode of assembly, the portal would need to be involved in initiation but not able to be inserted in subsequent assembly steps. We have used an in vitro capsid assembly system to test whether the portal is involved selectively in initiation. Portal incorporation was compared in capsids assembled from reactions in which (i) portals were present at the beginning of the assembly process and (ii) portals were added after assembly was under way. The results showed that portal-containing capsids were formed only if portals were present at the outset of assembly. A delay caused formation of capsids lacking portals. The findings indicate that if portals are present in reaction mixtures, a portal is incorporated during initiation or another early step in assembly. If no portals are present, assembly is initiated in another, possibly related, way that does not involve a portal.

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Figures

FIG. 1.
FIG. 1.
Electron micrographs showing the products of in vitro capsid assembly reactions. Samples of reaction mixtures were examined after 15 min (a, c, and d) and 20 h (b) of incubation. Note that partial procapsids predominate in 15-min reactions (arrows in panel a), while completed capsids predominate after 20 h (arrows in panel b). Partial procapsids (arrows) are shown at higher magnification in panels c and d, with more mature capsids close by. Bars = 500 nm (a and b) and 100 nm (c and d).
FIG. 2.
FIG. 2.
Time course for appearance of HSV-1 capsids and capsid assembly intermediates during in vitro capsid formation. The total number of completed capsids and capsid assembly intermediates were counted in electron micrographs, such as those shown in Fig. 1. Counts for each time point were made in one ×10,000 electron microscope negative. Note that the rate of appearance of assembly products was not affected by the presence of the portal in reaction mixtures.
FIG. 3.
FIG. 3.
Determination of the UL6 contents in capsids assembled in vitro. Analyses were carried out with capsids formed in incubations in which the time of portal addition varied, as described in the text. After incubation, the capsids were purified by sucrose gradient ultracentrifugation and analyzed in two ways: by SDS-PAGE, followed by Western immunoblotting (a), and by agarose gel electrophoresis, followed by SDS-PAGE and immunoblotting (b). Note that the amount of UL6 incorporated into capsids was greater when UL6 was present at the outset of incubation than when its addition was delayed.
FIG. 4.
FIG. 4.
Effect of input UL6 on the amount of UL6 incorporated into HSV-1 capsids assembled in vitro. Capsids were formed in vitro and analyzed for the presence of UL6 as described in Materials and Methods. The bar indicates the amount of UL6 present in HSV-1 B capsids isolated from infected cells.
FIG. 5.
FIG. 5.
Electron micrograph showing HSV-1 capsids that were assembled in vitro and stained to identify the location of UL6 protein. The capsids were stained with antibody specific for UL6, followed by a gold-labeled secondary antibody. The gold beads (black dots; ∼15 nm in diameter) identify the locations of UL6.
FIG. 6.
FIG. 6.
Schematic diagram illustrating HSV-1 procapsid formation in the absence (a) and presence (b) of the portal. The diagram illustrates the observation that procapsids are assembled normally in the absence of the portal (a). If the portal is present, however, it is suggested to be involved in the initiation of procapsid assembly and to be donated by way of a complex with the scaffolding protein, as shown in panel b.
FIG. 7.
FIG. 7.
Schematic diagram illustrating the proposed mechanism by which portal incorporation is limited to the initiation step of capsid assembly. The model postulates that (i) the scaffolding protein, UL26.5, is found in the form of a head-to-tail polymer and (ii) the portal attaches only at one end of the polymer, as illustrated in step 1, with major capsid protein molecules bound to all other scaffold molecules. Procapsid formation is suggested to occur by successive steps in which one major capsid protein molecule is delivered to a growing edge of the nascent procapsid (steps 2 and 3). Note that the proposed mechanism restricts portal incorporation to the initiation step.
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