A Putative Leucine Zipper within the Herpes Simplex Virus Type 1 UL6 Protein Is Required for Portal Ring Formation

Abstract

The herpes simplex virus type 1 UL6 protein forms a 12-subunit ring structure at a unique capsid vertex which functions as a conduit for encapsidation of the viral genome. To characterize UL6 protein domains that are involved in intersubunit interactions and interactions with other capsid proteins, we engineered a set of deletion mutants spanning the entire gene. Three deletion constructs, D-5 (⌬198-295), D-6 (⌬322-416), and D-LZ (⌬409-473, in which a putative leucine zipper was removed), were introduced into the viral genome. All three mutant viruses produced only B capsids, indicating a defect in encapsidation. Western blot analysis showed that the UL6 protein was present in the capsids isolated from two mutants, D-6 and D-LZ. The protein encoded by D-5, on the other hand, was not associated with capsids and was instead localized in the cytoplasm of the infected cells, indicating that this deletion affected the nuclear transport of the portal protein. The UL6 protein from the KOS strain (wild type) and the D-6 mutant were purified from insect cells infected with recombinant baculoviruses and shown to form ring structures as assessed by sucrose gradient centrifugation and electron microscopy. In contrast, the D-LZ mutant protein formed aggregates that sedimented throughout the sucrose gradient as a heterogeneous mixture and did not yield stable ring structures. A mutant (L429E L436E) in which two of the heptad leucines of the putative zipper were replaced with glutamate residues also failed to form stable rings. Our results suggest that the integrity of the leucine zipper region is important for oligomer interactions and stable ring formation, which in turn are required for genome encapsidation.The formation of mature capsids containing the linear double-stranded herpes simplex virus type 1 (HSV-1) genome is a complex process involving a preassembled protein shell (procapsid), concatemeric viral DNA, and seven other viral proteins believed to play a role in cleavage and packaging of the genome (1, 10). The procapsid contains three shell proteins, VP5, VP19C, and VP23, and the viral scaffolding proteins (encoded by UL26 and UL26.5). During wild-type (WT) infection, three capsid forms are observed: "A" capsids, which have initiated the encapsidation process and have lost both scaffold and DNA; "B" capsids, which contain the scaffold but no DNA; and DNA-containing mature "C" capsids, which have lost scaffold during the process of taking up DNA. The cleavage and packaging machinery consists of the UL6 portal ring, a two-subunit terminase (UL15 and UL28), a protein which seals the capsid after DNA uptake has occurred (UL25), and three other proteins which play less well defined roles in the process (UL17, UL32, and UL33). Defects in any of these genes except the UL25 gene result in the accumulation of B capsids.The protein encoded by the UL6 gene is present in all three forms of viral capsids (19) and is believed to form a ring structure at a unique vertex which serves as the portal for the entry of viral DNA into t...

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

“…Proteins were electrotransferred to polyvinylidene difluoride (PVDF), blocked with 5% fat-free milk in TBST (150 mM NaCl, 20 mM Tris, pH 7.5, and 0.1% Tween), and incubated with antibodies against VP5 or UL6. Monoclonal antibody IC9 or 4G9 (anti-UL6) (25) was used at a dilution of 1:10,000 or 3E8 (anti-VP5) (5) at a dilution of 1:2,000. Membranes were washed with TBST and incubated with secondary antibodies conjugated to either horseradish peroxidase or alkaline phosphatase and washed again with TBST, and proteins were detected as described by the manufacturer's protocol.…”

“…For electron microscopy (EM) analysis, treated and untreated ring samples were adsorbed onto Formvar-carbon-coated grids, stained with uranyl acetate, and visualized in a Philips CM10 transmission electron microscope operating at 60 kV (52,000ϫ magnification). Wild-type and mutant rings also were analyzed by EM as previously described (25).…”

“…African green monkey kidney fibroblast cells (Vero) were obtained from the ATCC and used to propagate the WT type virus. The UL6 complementing cell line, UL6-31 (20,25), was used to propagate hr74, C166A, and C254A recombinant viruses. Jay C. Brown (University of Virginia Health System) provided anti-UL6 monoclonal antibodies 1C9 and 4G9 (7).…”

“…Wild-type protein and C166A and C254A recombinant proteins were expressed in Sf9 insect cells and purified as described previously (25). Briefly, 3.2 ϫ 10 8 insect cells were infected with recombinant baculoviruses at a multiplicity of infection (MOI) of 5 PFU per cell and incubated at 28°C for 48 h. Inclusion bodies containing UL6 were isolated and resuspended in 0.5 ml of arginine (1.0 M, pH 7.5), kept on ice for 15 min with occasional mixing, and centrifuged at 30,000 ϫ g in a Beckman TL-100 ultracentrifuge at 4°C for 15 min to remove insoluble UL6 as described in reference 25.…”

“…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). Moreover, these mutant viruses assemble B capsids that are defective for virus growth and encapsidation.…”