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A putative leucine zipper within the herpes simplex virus type 1 UL6 protein is required for portal ring formation - PubMed

A putative leucine zipper within the herpes simplex virus type 1 UL6 protein is required for portal ring formation

Jacob K Nellissery et al. J Virol. 2007 Sep.

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 (Delta 198-295), D-6 (Delta 322-416), and D-LZ (Delta 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.

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Figures

FIG. 1.
FIG. 1.

UL6 gene deletion mutants generated for this study. Schematic representation of the UL6 gene showing the predicted leucine zipper region (amino acids 422 to 445) and the various deletion mutants engineered for this study. The numbers correspond to the positions of the amino acids within the UL6 protein; -RGD, -NP, and -LZ denote deletions of a putative RGD motif, a predicted nuclear pore binding region, and a leucine zipper motif, respectively.

FIG. 2.
FIG. 2.

Transient complementation assay. Vero cells were transfected with plasmid DNAs of deletion mutants and superinfected with hr74 virus, and the resulting progeny virus was titered on 31 cells. The percent complementation was calculated by dividing the titer obtained for the mutant plasmid with the titer of the WT plasmid and multiplying by 100.

FIG. 3.
FIG. 3.

Mutant viruses show reduced virus yields. Graph showing the growth curves of UL6 WT and mutant viruses obtained following infection of Vero cells with the WT (KOS), the UL6 null (hr74), or one of the three mutant (D-5, D-6, or D-LZ) viruses harvested at different time points (h) after infection (hpi).

FIG. 4.
FIG. 4.

UL6 mutant viruses are defective in DNA cleavage. Southern blot of DNA from cells infected with WT (KOS), hr74 (null), or UL6 mutant virus (D-5, D-6, or D-LZ) probed with a terminal “b” repeat sequence. The probe was derived from the pNAR plasmid by digestion with NcoI and DraI, and it contains the “b” repeat sequences that detect the S termini and the internal SQ junction (16). The left panel shows DNA obtained from infected Vero cells, and the right panel shows DNA samples from the infected 31 cell line. The positions of the molecular weight markers are indicated on the right.

FIG. 5.
FIG. 5.

The D-6 and D-LZ mutant proteins are associated with capsids. Western blot of fractions (1, top; 18, bottom) obtained by sucrose density gradient sedimentation of capsids from cell lysates of Vero cells infected with KOS (WT), hr74 (null), or the D-6, D-LZ, or D-5 mutant. Each blot was divided into two strips and probed separately with anti-UL6 and anti-VP5 antibodies. A, B, and C represent the positions of the three types of capsids in the sucrose gradient. The last lane, CL, contained a sample of the infected-cell lysate, used as a control to monitor the expression of the mutant proteins.

FIG. 6.
FIG. 6.

The D-5 protein localizes to the cytoplasm of infected cells. Immunofluorescence images of Vero cells infected with KOS, hr74 (null), or D-6, D-LZ, or D-5 mutant UL6 viruses double labeled with anti-UL6 and ant-ICP8 antibodies and detected by Alexa Fluor 488- and 594-conjugated secondary antibodies, respectively. The panels in each row show an image from the green channel (UL6) and the red channel (ICP8) and the merged image. The yellow color denotes colocalization of UL6 with ICP8 in the replication compartments.

FIG. 7.
FIG. 7.

A putative leucine zipper domain is present within the UL6 gene. Multiple sequence alignment of the predicted leucine zipper regions of the following alphaherpesvirus portal proteins: human herpesvirus 1 (HHV-1) gp011, human herpesvirus 2 (HHV-2) gp08, simian herpesvirus 1 (CeHV-1) gp07, equine herpesvirus 4 (EHV-4) gp55, chicken herpesvirus 2 (GaHV-2) gp018, equine herpesvirus 1 (EHV-1) orf56, human herpesvirus 3 (HHV-3) gp55, simian herpesvirus 7 (CeHV-7) gp55, chicken herpesvirus 3 (GaHV-3) gp019, meleagrid herpesvirus 1(MeHV-1) gp013, pseudorabies virus (SuHV-1) gp52, bovine herpesvirus 1 (BoHV-1) gp30, and bovine herpesvirus 5 (BoHV-5) gp51. The numbering on the left denotes the amino acid position of the first leucine of the zipper motif. The conserved heptad leucines are underlined. The stars in the bottom row represent identical residues, and the dots denote amino acid similarities at the respective positions. The boxed residues of the HSV-1 sequence were changed to glutamic acid to generate the L429E L436E mutant. The sequences were obtained from the NCBI viral genome database, accession numbers NC_001806, NC_001798, NC_004812, NC_001844, NC_002229, NC_001491, NC_001348, NC_002686, NC_002577, NC_002641, NC_006151, NC_001847, and NC_005261, respectively. Panel B shows a helical wheel diagram of the HSV1-LZ motif generated using the GCG Wisconsin package (31). The hydrophobic side chains, most of which face one side of the helix, are shaded gray.

FIG. 8.
FIG. 8.

The L429E L436E mutant virus does not grow on Vero cells and produces B capsids containing reduced amounts of the UL6 protein. (A) The WT (pCD6-FL) and the mutant (pCD6-L429E/L436E) plasmids were transfected into Vero cells, which were incubated for 18 h and then superinfected with the null virus (hr74), and the yield of resulting progeny viruses was determined by plating on the complementing cell line. The graph shows the percent complementation relative to results for the WT, normalized to 100%. (B) The Vero and 31 cell lines were infected with KOS (WT), hr74 (null), or the L429E L436E mutant (leucine zipper mutant) virus at an MOI of 0.5 PFU/cell; 24 h postinfection, progeny viruses were collected and assayed for yield on the complementing cell line. (C) B-capsid fractions from the sucrose density gradient-purified L429E L436E mutant (leucine zipper point mutant), D-LZ (leucine zipper deletion), and KOS (WT) viruses were serially diluted (1:1, 1:2, and 1:4) and analyzed by Western blotting using anti-VP5 and anti-UL6 antibodies. The images were quantified using Image J software, and results were expressed as a ratio of the relative intensities of the UL6 and VP5 protein bands.

FIG. 9.
FIG. 9.

The D-LZ mutant protein does not form rings in vitro. Electron micrographs show purified preparations of the WT and three mutant (D-6 and D-LZ deletions and the site-specific L429E L436E mutant) UL6 proteins made in insect cells using a recombinant baculovirus expression system. The photographs show negatively stained rings with dark central channels in the WT and the D-6 mutant. The D-LZ and L429 L436E mutant proteins show amorphous particles of variable dimensions and without a distinct central channel. The scale bar in each panel corresponds to 100 nm.

FIG. 10.
FIG. 10.

Sedimentation profile of purified WT, D-6, D-LZ, and L429 436E recombinant UL6 proteins. Western blot image of the fractions (1, top; 13, bottom) from sucrose gradient centrifugation of the WT, D-6, D-LZ, and L429E L436E recombinant UL6 proteins probed with anti-UL6 antibody. The WT protein forms a sharp peak in the middle of the gradient, while the D-6 mutant migrates in a similar mode; the D-LZ and L429E L436E mutant proteins are distributed in a range of sizes throughout the gradient.

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