Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2003 Sep;77(18):9862-71.
doi: 10.1128/jvi.77.18.9862-9871.2003.

Assembly of the herpes simplex virus capsid: identification of soluble scaffold-portal complexes and their role in formation of portal-containing capsids

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

Assembly of the herpes simplex virus capsid: identification of soluble scaffold-portal complexes and their role in formation of portal-containing capsids

William W Newcomb et al. J Virol. 2003 Sep.

Abstract

The herpes simplex virus type 1 (HSV-1) portal complex is a ring-shaped structure located at a single vertex in the viral capsid. Composed of 12 U(L)6 protein molecules, the portal functions as a channel through which DNA passes as it enters the capsid. The studies described here were undertaken to clarify how the portal becomes incorporated as the capsid is assembled. We tested the idea that an intact portal may be donated to the growing capsid by way of a complex with the major scaffolding protein, U(L)26.5. Soluble U(L)26.5-portal complexes were found to assemble when purified portals were mixed in vitro with U(L)26.5. The complexes, called scaffold-portal particles, were stable during purification by agarose gel electrophoresis or sucrose density gradient ultracentrifugation. Examination of the scaffold-portal particles by electron microscopy showed that they resemble the 50- to 60-nm-diameter "scaffold particles" formed from purified U(L)26.5. They differed, however, in that intact portals were observed on the surface. Analysis of the protein composition by sodium dodecyl sulfate-polyacrylamide gel electrophoresis demonstrated that portals and U(L)26.5 combine in various proportions, with the highest observed U(L)6 content corresponding to two or three portals per scaffold particle. Association between the portal and U(L)26.5 was antagonized by WAY-150138, a small-molecule inhibitor of HSV-1 replication. Soluble scaffold-portal particles were found to function in an in vitro capsid assembly system that also contained the major capsid (VP5) and triplex (VP19C and VP23) proteins. Capsids that formed in this system had the structure and protein composition expected of mature HSV-1 capsids, including U(L)6, at a level corresponding to approximately 1 portal complex per capsid. The results support the view that U(L)6 becomes incorporated into nascent HSV-1 capsids by way of a complex with U(L)26.5 and suggest further that U(L)6 may be introduced into the growing capsid as an intact portal.

PubMed Disclaimer

Figures

FIG. 1.
FIG. 1.
Agarose gel electrophoresis of complexes containing the portal (UL6). Reaction components were mixed and subjected to electrophoresis as described in Materials and Methods. The gel was then blotted electrophoretically onto nitrocellulose paper, which was stained with Ponceau S, destained, and stained with antibody specific for UL6. (a) Note that UL6 is soluble in the presence of UL26.5 or VP22a. (b) Note that UL6 is solubilized by UL26.5 but not by bovine serum albumin (BSA).
FIG. 2.
FIG. 2.
Determination of the UL6/UL26.5 ratio in scaffold-portal particles. Scaffold-portal particles formed in vitro with different amounts of UL6 were isolated by sedimentation on sucrose density gradients as described in Materials and Methods. The gradient fractions were then analyzed by SDS-polyacrylamide gel electrophoresis (a), followed by densitometric scanning of the stained gel. The gels were all similar to the one shown in panel a, where 7.5 μg of UL6 was mixed with 38 μg of UL26.5. (a) Note that UL6 and UL26.5 migrated coincidentally during centrifugation, suggesting they are part of the same complex. (b) The UL6/UL26.5 ratio in scaffold-portal particles was determined by summing the UL6 and UL26.5 amounts over all positive fractions, and the UL6/UL26.5 ratio was plotted as a function of the input UL6 amount. Note the nonlinear binding of UL6 at a high input UL6 concentration.
FIG. 3.
FIG. 3.
Electron microscopic analysis of scaffold particles (top) and scaffold-portal particles (bottom). Note the presence of intact portals (arrows) in scaffold-portal particles.
FIG. 4.
FIG. 4.
Histograms showing the diameters of scaffold particles (top) and scaffold-portal particles (bottom) as measured from electron micrographs such as those shown in Fig. 3. The largest dimension across the particle was considered the diameter, as described in Materials and Methods. Note that the particle diameter is larger in scaffold-portal (bottom) than in scaffold (top) particles.
FIG. 5.
FIG. 5.
Effect of WAY-150138 on formation of scaffold-portal particles in vitro. Portals and scaffold particles were mixed in vitro in the presence of WAY-150138 and subjected to electrophoresis on an agarose gel. The gel was then blotted electrophoretically onto nitrocellulose paper, which was stained with Ponceau S (a), destained, and stained with antibody specific for UL6 (b). The amount of UL6 binding was then determined by densitometric scanning of the UL6-stained bands, with a correction applied to account for small differences in the amount of UL26.5 present (c). Note that the amount of UL6 bound to scaffold particles decreased with increasing WAY-150138 concentration.
FIG. 6.
FIG. 6.
Characterization of capsids formed in the in vitro capsid assembly system. Capsids assembled in vitro were compared to HSV-1 B capsids in sedimentation rate on sucrose density gradients (a), protein composition as determined by SDS-polyacrylamide gel electrophoresis followed by Coomassie staining (b), and electron microscopy (c and d). Note the similarity of capsids formed in vitro to B capsids in all three parameters.
FIG. 7.
FIG. 7.
UL6 content of capsids formed in the in vitro capsid assembly system. The reaction mixtures contained scaffold-portal particles prepared from UL26.5 and UL6. Capsids were assembled in vitro and purified by two steps of sucrose density gradient centrifugation as described in Materials and Methods. After the second sucrose gradient step, capsid-containing fractions were analyzed by SDS-polyacrylamide gel electrophoresis followed by Coomassie staining (top) or immunoblot staining for UL6 (bottom). Note that capsids contained UL6.
FIG. 8.
FIG. 8.
Effects of GuHCl on the UL6 contents of capsids isolated from infected cells (in vivo capsids) and capsids formed in the in vitro assembly system (in vitro capsids). Procedures described in Materials and Methods were used for isolating capsids by sucrose density gradient centrifugation, treating capsids in vitro with 2.0 M GuHCl, and reisolating them by sucrose gradient centrifugation. Capsid-containing fractions were then analyzed by SDS-polyacrylamide gel electrophoresis followed by Coomassie staining (top) or immunoblot staining for UL6 (bottom). Untreated and treated capsids are called B capsids and G capsids, respectively. Note that UL6 was resistant to extraction with 2.0 M GuHCl in capsids formed in vivo and in vitro. Since the number of capsids was different in each of the four specimens, quantitative determination of the amount of UL6 present required that the UL6 signal be scaled to that of VP5. The results of the quantitative analysis are shown in Table 1.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Bazinet, C., J. Benbasat, J. King, J. M. Carazo, and J. L. Carrascosa. 1988. Purification and organization of the gene 1 portal protein required for phage P22 DNA packaging. Biochemistry 27:1849-1856. - PubMed
    1. Bazinet, C., and J. King. 1988. Initiation of P22 procapsid assembly in vivo. J. Mol. Biol. 202:77-86. - PubMed
    1. Brown, J. C., M. A. McVoy, and F. L. Homa. 2001. Packaging DNA into herpesvirus capsids, p. 111-153. In A. Holzenburg and E. Bogner (ed.), Structure-function relationships of human pathogenic viruses. Kluwer Academic Publishers, London, United Kingdom.
    1. Eiserling, F. A., and L. W. Black. 1994. Pathways in T4 morphogenesis, p. 209-212. In J. D. Karam (ed.), Molecular biology of bacteriophage T4. ASM Press, Washington, D.C.
    1. Engvall, E., and P. Perlman. 1971. Enzyme-linked immunosorbent assay (ELISA): quantitative assay for immunoglobulin. Immunochemistry 8:871-874. - PubMed

Publication types

LinkOut - more resources