The UL6 Gene Product Forms the Portal for Entry of DNA into the Herpes Simplex Virus Capsid

Abstract

During replication of herpes simplex virus type 1 (HSV-1), viral DNA is synthesized in the infected cell nucleus, where DNA-free capsids are also assembled. Genome-length DNA molecules are then cut out of a larger, multigenome concatemer and packaged into capsids. Here we report the results of experiments carried out to test the idea that the HSV-1 UL6 gene product (pUL6) forms the portal through which viral DNA passes as it enters the capsid. Since DNA must enter at a unique site, immunoelectron microscopy experiments were undertaken to determine the location of pUL6. After specific immunogold staining of HSV-1 B capsids, pUL6 was found, by its attached gold label, at one of the 12 capsid vertices. Label was not observed at multiple vertices, at nonvertex sites, or in capsids lacking pUL6. In immunoblot experiments, the pUL6 copy number in purified B capsids was found to be 14.8 +/- 2.6. Biochemical experiments to isolate pUL6 were carried out, beginning with insect cells infected with a recombinant baculovirus expressing the UL6 gene. After purification, pUL6 was found in the form of rings, which were observed in electron micrographs to have outside and inside diameters of 16.4 +/- 1.1 and 5.0 +/- 0.7 nm, respectively, and a height of 19.5 +/- 1.9 nm. The particle weights of individual rings as determined by scanning transmission electron microscopy showed a majority population with a mass corresponding to an oligomeric state of 12. The results are interpreted to support the view that pUL6 forms the DNA entry portal, since it exists at a unique site in the capsid and forms a channel through which DNA can pass. The HSV-1 portal is the first identified in a virus infecting a eukaryote. In its dimensions and oligomeric state, the pUL6 portal resembles the connector or portal complexes employed for DNA encapsidation in double-stranded DNA bacteriophages such as phi29, T4, and P22. This similarity supports the proposed evolutionary relationship between herpesviruses and double-stranded DNA phages and suggests the basic mechanism of DNA packaging is conserved.

Disulfide Bond Formation in the Herpes Simplex Virus 1 UL6 Protein Is Required for Portal Ring Formation and Genome Encapsidation

“…The UL6 protein plays multiple roles during viral assembly and encapsidation, including ring formation, interaction with scaffold and VP5 to nucleate the assembly of the procapsid shell, the docking of the terminase, and acting as a conduit for the uptake of viral DNA into capsids (8,9,20,25,26,28,31,32,40,42,46). It also is likely that the UL6 portal plays a role during subsequent infections.…”

“…UL6 expressed in insect cells infected with recombinant baculovirus forms rings that can be observed by electron microscopy (25,28). To address whether disulfide bonds contribute to ring stability, UL6 rings were treated with DTT followed by the addition of sodium bisulfite to prevent free sulfhydryl groups from reforming disulfide bonds.…”

“…These results also suggest that capsid assembly is regulated such that capsids lacking UL6 do not assemble efficiently in infected cells. UL6 is known to self assemble into a dodecameric ring in lysates from insect cells infected with recombinant UL6-expressing baculovirus (28). Interestingly, two UL6 mutant proteins, L429E L436E and D-LZ, bearing mutations in the leucine zipper region, are unable to produce rings and form polymorphic aggregates instead (25).…”

“…Bacteriophage and herpesviruses share an important structural element, a dodecameric portal ring located at a unique capsid vertex (8,9,28,40). During HSV genome encapsidation, the portal ring provides a docking site for the terminase, an ATPdriven molecular motor that facilitates the uptake of viral DNA (34,42,45,46).…”

Autophagy interaction with herpes simplex virus type-1 infection

“…While viral-host interactions occur in the cytosol, the nucleocapsid attaches to the nuclear pore via the capsid portal created by UL6 portal proteins. 8 Viral DNA is then injected into the nucleus. When this occurs, host sensors alert innate immune responses, which will be discussed below.…”