Role of the UL25 protein in herpes simplex virus DNA encapsidation

Abstract

The herpes simplex virus protein UL25 attaches to the external vertices of herpes simplex virus type 1 capsids and is required for the stable packaging of viral DNA. To define regions of the protein important for viral replication and capsid attachment, the 580-amino-acid UL25 open reading frame was disrupted by transposon mutagenesis. The UL25 mutants were assayed for complementation of a UL25 deletion virus, and in vitro-synthesized protein was tested for binding to UL25-deficient capsids. Of the 11 mutants analyzed, 4 did not complement growth of the UL25 deletion mutant, and analysis of these and additional mutants in the capsid-binding assay demonstrated that UL25 amino acids 1 to 50 were sufficient for capsid binding. Several UL25 mutations were transferred into recombinant viruses to analyze the effect of the mutations on UL25 capsid binding and on DNA cleavage and packaging. Studies of these mutants demonstrated that amino acids 1 to 50 of UL25 are essential for its stable interaction with capsids and that the C terminus is essential for DNA packaging and the production of infectious virus through its interactions with other viral packaging or tegument proteins. Analysis of viral DNA cleavage demonstrated that in the absence of a functional UL25 protein, aberrant cleavage takes place at the unique short end of the viral genome, resulting in truncated viral genomes that are not retained in capsids. Based on these observations, we propose a model where UL25 is required for the formation of DNA-containing capsids by acting to stabilize capsids that contain full-length viral genomes.

FIG. 1.

(Top) Physical map of the HSV-1 genome showing the location of the UL25 gene. U_L and U_S refer to the long and short unique region sequences, respectively, and TR_L and TR_S and IR_L and IR_S refer to the terminal and internal repeat sequences, respectively. (Middle) Schematic representation of the 580-amino-acid UL25 protein. Dashed regions are the folded protein core as determined by characterization of the UL25 crystal structure (5). Numbers above the line indicate the amino acid location where 15 bp were inserted into the UL25 ORF, resulting in either the insertion (i) of five amino acids or an in-frame stop codon (s). (Bottom) The locations of the insertions and the predicted amino acid changes are shown along with the predicted molecular mass (MM) for each mutant. *, stop codon.

FIG. 2.

Complementation assays for the UL25 transposon mutants. The UL25 transposon mutants were examined for their ability to complement the growth of the UL25-null virus. (A) Western blots for UL25 protein and the major capsid protein, UL19. (B) The progeny virus was titrated on the UL25-complementing cell line, 8-1, and the results shown are averages of at least six experiments. Error bars represent the standard deviation. The data listed at the bottom are the percentages of complementation relative to the wild-type (WT) UL25 protein.

FIG. 3.

Capsid binding by UL25 transposon mutants. (A) Autoradiograph of [³⁵S]Met-labeled UL25 protein after SDS-PAGE. The sizes of the 143i, 212s, and 560s proteins were consistent with predicted molecular mass for each protein, but the 12s mutant unexpectedly produced a nearly wild-type(wt)-size UL25 protein. (B and C) In vitro capsid-binding assay. In vitro-translated protein was incubated with pooled A and B capsids isolated from Vero cells infected with the UL25-null virus, vΔUL25, and capsids were then purified by sucrose gradient centrifugation. The sucrose gradients were fractionated and analyzed by SDS-PAGE followed by Coomassie staining (top panel, representative gel) and autoradiography (bottom panels). The fractions containing A and B capsids are indicated. The capsid proteins visible in the stained gel are listed at right. Capsid binding was demonstrated by comigration of the UL25 protein with A and B capsids. Aggregation of the truncated 212s protein may explain the presence of this protein in the same dense fractions as in the gradient lacking A and B capsids (bottom of panel C). IVT, in vitro transcription-translation; M, molecular mass markers (from top, 194, 104, 60, 41, 27, 21, 16, and 7 kDa). Lane 1 is the bottom of the gradient and lane 10 (B) or 12 (C) is the top.

FIG. 4.

Capsid binding by UL25 N-terminal mutants. (A) UL25 N-terminal truncation and TAP fusion constructs are shown. The numbers listed on the left indicate the UL25 amino acids included in each construct. The TAP constructs contained the TAP tag fused to the C terminus of UL25. The predicted molecular mass (MM) of each protein is listed in parentheses. wt, wild type. (B) Autoradiograph of in vitro translation (IVT) products after SDS-PAGE. Molecular mass markers in kDa are shown at left. Each protein showed the appropriate molecular mass due to either addition of the TAP tag or deletion of the UL25 N-terminal amino acids. (C) In vitro capsid-binding assay. In vitro-translated protein was incubated with pooled A and B capsids isolated from Vero cells infected with the UL25-null virus, vΔUL25, and then purified by sucrose gradient centrifugation. A representative Coomassie-stained gel of the gradient fractions is shown (top panel). Autoradiographs of dried gels demonstrating the binding of 1-580-TAP or 1-50-TAP but not 51-580-TAP or the 37-580 to the two capsid types (bottom panel). (D) In vitro-translated protein was incubated with pooled wild-type KOS A, B, and C capsids and then purified by sucrose gradient centrifugation. A representative stained gel of the gradient fractions is shown (top panel). Autoradiographs of dried gels demonstrating the binding of 1-50-TAP but not 51-580-TAP to all three capsid types (bottom panel). The fractions containing A, B, and C capsids are indicated. The capsid proteins visible in the stained gel are listed to the right. IVT, in vitro transcription-translation; M, molecular mass markers (from top, 194, 109, 59, 30, 22, 13, and 6 kDa). Lane 1 is the bottom of the gradient, and lane 10 or 11 is the top.

FIG. 5.

Characterization of BAC DNA and HSV-infected cell DNA. (A) Schematic representation of the BamHI region located between nucleotides 48635 and 50929 of the HSV-1 genome that contains the UL25 ORF. The sites where unique PmeI restriction sites are found in the UL25 gene and the resulting BACs and viruses (143i BAC, 155i BAC, 212s BAC, and 560s BAC) are listed on top. Δ1-50 BAC contained a deletion that removes the coding sequences for UL25 amino acids 1 to 50 and inserts a new ATG start codon. UL25-GFP BAC contained an in-frame insertion of the (726-bp) GFP coding sequence after the codon for UL25 amino acid 50. (B) BAC DNA was digested with BamHI and PmeI and run on a 1.2% agarose gel. The wild-type 2.3-kbp BamHI fragment is present in the KOS BAC lane (arrow). This fragment was cleaved to smaller fragments by PmeI in 143i BAC, 155i BAC, 212s BAC, and 560s BAC. The UL25 fragments of Δ1-50 BAC and UL25-GFP BAC are shifted to 2.1 kbp (which comigrates with another viral band) and 3.2 kbp (arrow), respectively. M, DNA markers in kbp. (C) Southern blot of viral DNA. Viral DNA was purified from cells infected with the indicated viruses. Viral DNA (10 μg) was digested with BamHI and PmeI and run on a 1.2% agarose gel. DNA was blotted to a nylon membrane, and the blot was hybridized with the ³²P-labeled UL25-containing BamHI U fragment. The wild-type BamHI fragment containing UL25 is visible at 2.3 kbp in the KOS lane and was cleaved to smaller fragments by PmeI in v143i, v155i, v212s, and v560s. The UL25 fragments of vΔ1-50 and vUL25-GFP are shifted to 2.1 and 3.2 kbp, respectively.

FIG. 6.

Analysis of capsid-bound UL25. Vero cells were infected at an MOI of 5 with KOS or the indicated UL25 mutants. Nuclear lysates were subjected to sucrose gradient sedimentation. (A) Capsids from Vero cells infected (MOI of 5) with KOS or the indicated UL25 mutant virus were harvested at 18 h postinfection, layered onto 20 to 50% sucrose gradients, and centrifuged at 24,000 rpm (SW41 rotor) for 1 h. The positions of A, B, and C capsid bands are indicated. (B) The protein composition for each gradient fraction was determined by SDS-PAGE (stained gel). Lane 1 is the bottom of the gradient, and lane 11 or 12 is the top. Gradient fractions that contained A, B, and C capsids are indicated. The positions of the capsid proteins (major capsid protein, VP5; triplex proteins, VP19C and VP23; scaffold protein, VP22a; smallest capsid protein, VP26) are indicated. (Bottom panels) Gradient fractions were analyzed by SDS-PAGE followed by immunoblotting for UL25 using the 25E10 antibody. The stained gel in the bottom right panel contains capsids isolated from UL25-null virus vΔUL25, which is representative of the mutants (vΔ1-50, v143i, v212s, and v560s) that fail to replicate on Vero cells. The UL25 Western blots (WB) for these mutants are shown below the stained gel with asterisks indicating the fractions that are equivalent to the capsid-containing fractions (lanes 5 to 7) of the vΔUL25-stained gel. Lane a, cell lysate from KOS-infected cells; lane b, cell lysate from UL25 mutant-infected cells. Molecular mass standards are visible in lanes M (from top, 194, 109, 59, 30, 22, 13, and 6 kDa).

FIG. 7.

Processing of virus DNA. (A) Schematic diagram of the HSV genome showing the locations of the HindIII G, I, and N fragments and the BamHI J, K, Q, and S fragments. The different sizes of the BamHI S fragment are due to the presence of one to multiple copies of the a sequence at the U_L terminus. (B to D) Vero cells were infected with the indicated virus at an MOI of 5 PFU per cell. At 18 h postinfection, total infected cell DNA was isolated, digested with BamHI (B and C) or HindIII (D), and subjected to Southern blot analysis. The blots were probed with ³²P-labeled BamHI K (B), BamHI Q (C), or BamHI J (D) fragments of the HSV-1 genome. The ratio of the BamHI joint K fragment to U_S-end Q or U_L-end S fragments and the ratio of the internal HindIII N fragment to the U_S-end G fragment were determined by quantification of the hybridizing bands with phosphorimaging software.

FIG. 8.

Model for the role of UL25 in DNA packaging. Proposed model for the role of UL25 in HSV DNA encapsidation. (Step 1) DNA packaging initiates on concatemers when the terminase complex consisting of UL15, UL28, and UL33 binds (step 2) to packaging sequences and cleaves (step 3) the concatemer to generate a U_L end with the terminase still bound. The terminase-DNA complex then docks at the portal vertex on the procapsid (step 4), and the terminase initiates DNA packaging (step 5). As the procapsid is filled (step 6A) with DNA, it angularizes and in the process, UL25 binding sites are exposed at the capsid vertexes. DNA cleavage is suppressed by a headful mechanism (step 7A) until a full-length genome has entered. Cleavage occurs at a packaging site located at the U_S end of the genome (step 8A). More UL25 binds to the capsid, and the newly packaged genome is sealed within the C capsid (step 9A). In the absence of a functional UL25 protein, the packaging reaction proceeds (step 6B) with the capsid filling and expanding until close to a genome length has entered (step 7B). Without UL25 to stabilize the capsid against the pressure that the packaged genome generates, premature cleavage (step 8B) occurs just prior to the entry of the U_S-end repeat, resulting in genomes with truncated U_S termini. In the absence of UL25, the capsid is not stable. The DNA is released (step 9B), which generates an empty A capsid and a free viral genome that is truncated at the U_S end.