The putative herpes simplex virus 1 chaperone protein UL32 modulates disulfide bond formation during infection

Abstract

During DNA encapsidation, herpes simplex virus 1 (HSV-1) procapsids are converted to DNA-containing capsids by a process involving activation of the viral protease, expulsion of the scaffold proteins, and the uptake of viral DNA. Encapsidation requires six minor capsid proteins (UL6, UL15, UL17, UL25, UL28, and UL33) and one viral protein, UL32, not found to be associated with capsids. Although functions have been assigned to each of the minor capsid proteins, the role of UL32 in encapsidation has remained a mystery. Using an HSV-1 variant containing a functional hemagglutinin-tagged UL32, we demonstrated that UL32 was synthesized with true late kinetics and that it exhibited a previously unrecognized localization pattern. At 6 to 9 h postinfection (hpi), UL32 accumulated in viral replication compartments in the nucleus of the host cell, while at 24 hpi, it was additionally found in the cytoplasm. A newly generated UL32-null mutant was used to confirm that although B capsids containing wild-type levels of capsid proteins were synthesized, these procapsids were unable to initiate the encapsidation process. Furthermore, we showed that UL32 is redox sensitive and identified two highly conserved oxidoreductase-like C-X-X-C motifs that are essential for protein function. In addition, the disulfide bond profiles of the viral proteins UL6, UL25, and VP19C and the viral protease, VP24, were altered in the absence of UL32, suggesting that UL32 may act to modulate disulfide bond formation during procapsid assembly and maturation.

Importance: Although functions have been assigned to six of the seven required packaging proteins of HSV, the role of UL32 in encapsidation has remained a mystery. UL32 is a cysteine-rich viral protein that contains C-X-X-C motifs reminiscent of those in proteins that participate in the regulation of disulfide bond formation. We have previously demonstrated that disulfide bonds are required for the formation and stability of the viral capsids and are also important for the formation and stability of the UL6 portal ring. In this report, we demonstrate that the disulfide bond profiles of the viral proteins UL6, UL25, and VP19C and the viral protease, VP24, are altered in cells infected with a newly isolated UL32-null mutant virus, suggesting that UL32 acts as a chaperone capable of modulating disulfide bond formation. Furthermore, these results suggest that proper regulation of disulfide bonds is essential for initiating encapsidation.

FIG 1

HA32 resembles wild-type virus in protein expression and viral growth properties. (A) Vero cells were infected with KOS or HA32 at an MOI of 3 and collected at the times postinfection (in hours) indicated above the gels. Samples were harvested in reducing SDS sample buffer and resolved by SDS-PAGE. Proteins were detected by immunoblotting with the antibodies indicated on the left. (B) Growth curves of KOS and HA32 propagated on Vero cells at an MOI of 0.1. Mean values from three independent experiments are plotted, and deviations are represented by error bars.

FIG 2

UL32 changes localization during infection. (A) Vero cells were infected with HA32 at an MOI of 0.1. At 6, 9, and 24 h postinfection, cells were fixed in 4% paraformaldehyde and permeabilized in acetone, followed by staining for ICP8 (red, mouse antibody) and HA (green, rat antibody). TOPRO3 staining was performed to visualize the nucleus. Bars = 5 μm. (B) The merged images in panel A were analyzed by ImageJ software. Yellow boxes, the area used to measure fluorescent intensity; blue arrows, the edges of the nucleus indicated by TOPRO3 staining. At least 100 cells were examined at each time point, and the staining patterns shown in all three panels represent those seen in nearly all cells on the coverslip.

FIG 3

UL32 homologs are found in all three subfamilies of herpesviruses. Sequence alignments were performed on UL32 homologs from herpesviruses from the alpha-, beta-, and gammaherpesvirus subfamilies. Residues of interest are highlighted in gray, and the corresponding motifs are indicated in the last row. The consensus sequence (Cons.) is represented by the bold lettering. Lowercase letters represent residues with little to no conservation. The viruses analyzed are indicated in Materials and Methods. MI to MV, motifs I to V, respectively.

FIG 4

Mutagenesis of conserved residues in motifs I, III, IV, and V abolish complementation ability. Alanine substitution mutations were made at every cysteine in motifs I to V, as was a histidine-to-glutamine mutation at amino acid 498. A transient complementation assay was used to test the ability of the mutant proteins to rescue the UL32-null virus hr64FS. Plasmid DNAs containing each mutation were transfected into Vero cells, followed by superinfection with hr64FS, and the resulting titer was determined on 158 cells. Error bars represent the standard deviations from three separate experiments.

FIG 5

UL32 forms slower-migrating species in the presence of H₂O₂. Vero cells were infected with HA32 at an MOI of 3. At 9 hpi, cells were washed with PBS and overlaid with 2 ml of serum-free media either with or without 5 mM H₂O₂. Cells were washed with PBS and collected in RIPA buffer without NEM (lanes 1 and 2) or TCA precipitated, followed by resuspension in NEM buffer (lanes 4 to 8) at the times (in hours) indicated above lanes 5 to 8. Proteins were separated by reducing or nonreducing SDS-PAGE and analyzed by Western blotting using mouse anti-HA antibody. The molecular markers (lane 3) represent, from top to bottom, 250, 150, 100, and 50 kDa.

FIG 6

UL32 mutants, defective in growth, synthesize wild-type levels of viral DNA but are unable to cleave and package it. (A) Growth curves of KOS, hr64FS, or hr64 propagated on Vero or 158 cells. Mean values from three independent experiments are plotted, and deviations are represented by error bars. (B) Vero or 158 cells were infected with KOS, hr64, or hr64FS, and viral DNA was analyzed by Southern blotting, as described in Materials and Methods. The blotted DNA was hybridized with a ³²P-labeled probe containing the BamHI SQ fragment. SQ, viral DNA junctions; S and Q, viral DNA termini.

FIG 7

hr64FS capsids are indistinguishable from KOS capsids. (A) Viral capsids were isolated by sucrose gradient sedimentation from Vero cells infected with KOS, hr64FS, or hr81. Dilutions of B capsids were separated by reducing SDS-PGE and silver stained. Numbers on the left are molecular masses (in kilodaltons). (B) Cryo-EM analysis and reconstruction of B capsids isolated from KOS- or hr64FS-infected cells. Surface representations of the capsid exterior (left) and interior (middle) and a gray-scale central section (right) are shown. Capsid subunits are indicated: hexons (h), pentons (p), triplexes (t), and the capsid vertex specific component (CVSC). Icosahedral 2- and 5-fold symmetry axes are also marked. Bar = 200 Å.

FIG 8

Capsid proteins localize to replication compartments in the absence of UL32. Vero cells were infected with KOS or hr64FS at an MOI of 3. Infected cells were fixed with 4% paraformaldehyde and permeabilized in acetone. Cells were stained with antibodies to ICP8 (red channel) plus the capsid proteins indicated (green channel), as described in Materials and Methods.

FIG 9

UL32 modulates disulfide bond formation in viral proteins. Vero cells were infected with KOS, hr64FS, or hr81 at an MOI of 3 and harvested in 10% TCA at 10 hpi. Free thiols were blocked with NEM, followed by reduction with DTT and then treatment with the thiol alkylating agent AMS. Proteins were resolved by reducing SDS-PAGE and detected by immunoblotting with anti-VP24 or anti-UL6 antibody.