Roles of the Interhexamer Contact Site for Hexagonal Lattice Formation of the Herpes Simplex Virus 1 Nuclear Egress Complex in Viral Primary Envelopment and Replication

Abstract

During the nuclear export of nascent nucleocapsids of herpes simplex virus 1 (HSV-1), the nucleocapsids acquire a primary envelope by budding through the inner nuclear membrane into the perinuclear space between the inner and outer nuclear membranes. This unique budding process, termed primary envelopment, is initiated by the nuclear egress complex (NEC), composed of the HSV-1 UL31 and UL34 proteins. Earlier biochemical approaches have shown that the NEC has an intrinsic ability to vesiculate membranes through the formation of a hexagonal lattice structure. The significance of intrahexamer interactions of the NEC in the primary envelopment of HSV-1-infected cells has been reported. In contrast, the contribution of lattice formation of the NEC hexamer to primary envelopment in HSV-1-infected cells remains to be elucidated. Therefore, we constructed and characterized a recombinant HSV-1 strain carrying an amino acid substitution in a UL31 residue that is an interhexamer contact site for the lattice formation of the NEC hexamer. This mutation was reported to destabilize the interhexamer interactions of the HSV-1 NEC. Here, we demonstrate that the mutation causes the aberrant accumulation of nucleocapsids in the nucleus and reduces viral replication in Vero and HeLa cells. Thus, the ability of HSV-1 to form the hexagonal lattice structure of the NEC was linked to an increase in primary envelopment and viral replication. Our results suggest that the lattice formation of the NEC hexamer has an important role in HSV-1 replication by regulating primary envelopment.IMPORTANCE The scaffolding proteins of several envelope viruses required for virion assembly form high-order lattice structures. However, information on the significance of their lattice formation in infected cells is limited. Herpesviruses acquire envelopes twice during their viral replication. The first envelop acquisition (primary envelopment) is one of the steps in the vesicle-mediated nucleocytoplasmic transport of nascent nucleocapsids, which is unique in biology. HSV-1 NEC, thought to be conserved in all members of the Herpesviridae family, is critical for primary envelopment and was shown to form a hexagonal lattice structure. Here, we investigated the significance of the interhexamer contact site for hexagonal lattice formation of the NEC in HSV-1-infected cells and present evidence suggesting that the lattice formation of the NEC hexamer has an important role in HSV-1 replication by regulating primary envelopment. Our results provide insights into the mechanisms of the envelopment of herpesviruses and other envelope viruses.

Keywords: UL31; UL34; herpes simplex virus; nuclear membrane.

FIG 1

Mutagenesis design in UL31 based on the crystal structure of the HSV-1 NEC. (A) In the HSV-1 NEC structure (16), UL34 is shown in gray and UL31 is shown in green. The locations of amino acids mutated in this study are indicated in blue (L142, E153, and D286) or magenta (V247). Molecular graphics and analyses were performed with the PyMOL molecular graphics system, version 2.0.6 (Schrödinger, LLC). (B) Predicted model for hexagonal lattice formation of NEC from biochemical analysis of purified HSV-1 NEC (16). UL31 and UL34 form a stable NEC heterodimer. Individual NEC heterodimers then assemble into hexameric rings. Finally, the NEC hexamers are linked to each other and form a hexagonal lattice to vesiculate the membrane. The UL34-D25A/E37A and UL31-V247F mutants are expected to be defective for NEC hexamer formation. The UL31-L142E, -E153R, and -D286R mutants are expected to be defective for interhexamer interactions and the lattice formation of the hexamer of the NEC.

FIG 2

Schematic diagrams of the genomic structure of wild-type HSV-1(F) and the relevant domains of the recombinant viruses used in this study. Line 1, the wild-type HSV-1(F) genome (U_L and U_S, unique long and unique short regions, respectively); line 2, domains of the UL30 to UL35 genes; line 3, domains of the UL31 and UL34 genes; lines 4, 5, and 6, recombinant viruses with mutations in the UL31 gene; line 7, recombinant virus with a mutation in the UL34 gene.

FIG 3

Complementation of the replication of the UL31-null mutant virus YK720 (ΔUL31) by expression plasmids for UL31 mutants. (A) Vero cells were transfected with a series of UL31-expressing plasmids or the control plasmid [pcDNA3.1/myc-His(−)A] for 4 h and then superinfected with YK720 (ΔUL31) at an MOI of 3 for 24 h. The progeny virus was collected and titrated on UL31-CV-1 cells. Data are the mean ± standard error from 4 independent experiments. *, P < 0.0001 (Tukey’s test). (B) Vero cells were transfected with the indicated plasmids for 28 h. The cells were analyzed by immunoblotting with anti-UL31 and anti-α-tubulin antibodies. The numbers to the left of the gels are molecular masses (in kilodaltons). Ct, control.

FIG 4

Effects of the D286R mutation in UL31 on the accumulation of viral proteins in HSV-1-infected cells. Vero (A) or HeLa (B) cells were mock infected or infected with wild-type HSV-1(F), YK726 (UL31-D286R), or YK727 (UL31-D286R-repair) at an MOI of 3 for 18 h or at an MOI of 10 for 24 h, respectively. The infected cells were analyzed by immunoblotting with the indicated antibodies. The numbers to the left of the gels are molecular masses (in kilodaltons).

FIG 5

Effects of the D286R mutation in UL31 on its interaction with UL34 in HSV-1-infected cells. Vero cells were mock infected or infected with wild-type HSV-1(F), YK726 (UL31-D286R), or YK722 (ΔUL34) at an MOI of 3 for 18 h, harvested, immunoprecipitated (IP) with anti-UL34 or anti-UL50 antibody, and analyzed by immunoblotting with anti-UL34, anti-UL31, and anti-UL47 antibodies. The numbers to the left of the gels are molecular masses (in kilodaltons).

FIG 6

Effects of the D286R mutation in UL31 on HSV-1 growth in Vero cells. Vero cells were infected with wild-type HSV-1(F), YK726 (UL31-D286R), or YK727 (UL31-D286R-repair) at an MOI of 3 (A) or 0.01 (B), harvested at the indicated times after infection, and assayed on Vero cells. Each data point is the mean ± standard error from 3 independent experiments. *, P < 0.05 (Tukey’s test) for YK726 (UL31-D286R) versus HSV-1(F) and YK726 (UL31-D286R) versus YK727 (UL31-D286R-repair).

FIG 7

Effects of the D286R mutation in UL31 on viral growth in HeLa cells. HeLa cells were infected with wild-type HSV-1(F), YK726 (UL31-D286R), or YK727 (UL31-D286R-repair) at an MOI of 10 (A) or 0.05 (B), harvested at the indicated times postinfection, and assayed on Vero cells. Each data point is the mean ± standard error from 3 independent experiments. *, P < 0.05 (Tukey’s test) for YK726 (UL31-D286R) versus HSV-1(F) and YK726 (UL31-D286R) versus YK727 (UL31-D286R-repair).

FIG 8

Comparison of effects of the D286R and null mutation in UL31 on viral growth in Vero and HeLa cells. (A and B) Vero cells were infected with wild-type HSV-1(F), YK726 (UL31-D286R), YK727 (UL31-D286R-repair), or YK720 (ΔUL31) at an MOI of 3 (A) or 0.01 (B) and harvested at 24 h (A) or 48 h (B) after infection. (C and D) HeLa cells were infected with wild-type HSV-1(F), YK726 (UL31-D286R), YK727 (UL31-D286R-repair), or YK720 (ΔUL31) at an MOI of 10 (C) or 0.05 (D) and harvested at 36 h (C) or 72 h (D) after infection. Cells and supernatants were titrated in UL31-CV-1 cells. Each data point is the mean ± standard error from 3 (A and B) or 4 (C and D) independent experiments. *, P < 0.05 (the unpaired Student's t test).

FIG 9

Effects of the D286R mutation in UL31 on the subcellular localization of UL31 and UL34 in HSV-1-infected Vero cells. (A and B) Vero cells were infected with wild-type HSV-1(F), YK726 (UL31-D286R), or YK727 (UL31-D286R-repair) at an MOI of 3, fixed at 18 h postinfection, permeabilized, stained with anti-UL31 and anti-UL34 antibodies (A) or anti-UL34 and lamin A/C antibodies (B), and examined by confocal microscopy. Bars, 10 μm. (C) Percentage of cells (of 30 to 40 cells in each experiment) showing a nuclear rim localization of UL34 (nuclear rim), needle-like structures of UL34 (needle), and other punctate structures of UL34 (other) in the experiment whose results are presented in panel B. Data are shown as the mean ± SEM from 3 independent experiments.

FIG 10

Effects of the D286R mutation in UL31 on the subcellular localization of UL31 and UL34 in HSV-1-infected HeLa cells. (A and B) HeLa cells were infected with wild-type HSV-1(F), YK726 (UL31-D286R), or YK727 (UL31-D286R-repair) at an MOI of 10, fixed at 24 h postinfection, permeabilized, stained with anti-UL31 and anti-UL34 antibodies (A) or anti-UL34 and lamin A/C antibodies (B), and examined by confocal microscopy. Each image in the far-right columns is the magnified image of the boxed area in the image to its left. Bars, 10 μm (first to third columns) and 2 μm (fourth column). (C) Percentage of cells (of 30 to 40 cells in each experiment) showing a nuclear rim localization of UL34 (nuclear rim), needle-like structures of UL34 (needle), and other punctate structures of UL34 (other) in the experiment whose results are presented in panel B. Data are shown as the mean ± SEM from 3 independent experiments.

FIG 11

Effects of the D286R mutation in UL31 on HSV-1 nuclear egress in Vero cells. Vero cells were infected with wild-type HSV-1(F), YK726 (UL31-D286R), or YK727 (UL31-D286R-repair) at an MOI of 3, fixed at 18 h postinfection, and examined by transmission electron microscopy. N, nucleus; C, cytoplasm; NM, nuclear membrane. Bars, 500 nm.

FIG 12

Effects of the D286R mutation in UL31 on HSV-1 nuclear egress in HeLa cells. HeLa cells were infected with wild-type HSV-1(F), YK726 (UL31-D286R), or YK727 (UL31-D286R-repair) at an MOI of 10, fixed at 24 h postinfection, and examined by transmission electron microscopy. Arrowheads indicate membranous structures in the nucleus. N, nucleus; C, cytoplasm; NM, nuclear membrane. Bars, 500 nm.