The Interaction between Herpes Simplex Virus 1 Tegument Proteins UL51 and UL14 and Its Role in Virion Morphogenesis

Abstract

To investigate the molecular mechanism(s) by which herpes simplex virus 1 (HSV-1) tegument protein UL51 promotes viral replication, we screened for viral proteins that interact with UL51 in infected cells. Affinity purification of tagged UL51 in HSV-1-infected Vero cells was coupled with immunoblotting of the purified UL51 complexes with various antibodies to HSV-1 virion proteins. Subsequent analyses revealed that UL51 interacted with another tegument protein, UL14, in infected cells. Mutational analyses of UL51 showed that UL51 amino acid residues Leu-111, Ile-119, and Tyr-123 were required for interaction with UL14 in HSV-1-infected cells. Alanine substitutions of these UL51 amino acid residues reduced viral replication and produced an accumulation of unenveloped and partially enveloped nucleocapsids in the cytoplasm at levels comparable to those of UL51-null, UL14-null, and UL51/UL14 double-null mutations. In addition, although UL51 and UL14 colocalized at juxtanuclear domains in HSV-1-infected cells, the amino acid substitutions in UL51 produced aberrant localization of UL51 and UL14. The effects of these substitutions on localization of UL51 and UL14 were similar to those of the UL51-null and UL14-null mutations on localization of UL14 and UL51, respectively. These results suggested that the interaction between UL51 and UL14 was required for proper localization of these viral proteins in infected cells and that the UL51-UL14 complex regulated final viral envelopment for efficient viral replication.

Importance: Herpesviruses contain a unique virion structure designated the tegument, which is a protein layer between the nucleocapsid and the envelope. HSV-1 has dozens of viral proteins in the tegument, which are thought to facilitate viral envelopment by interacting with other virion components. However, although numerous interactions among virion proteins have been reported, data on how these interactions facilitate viral envelopment is limited. In this study, we have presented data showing that the interaction of HSV-1 tegument proteins UL51 and UL14 promoted viral final envelopment for efficient viral replication. In particular, prevention of this interaction induced aberrant accumulation of partially enveloped capsids in the cytoplasm, suggesting that the UL51-UL14 complex acted in the envelopment process but not in an upstream event, such as transport of capsids to the site for envelopment. This is the first report showing that an interaction between HSV-1 tegument proteins directly regulated final virion envelopment.

FIG 1

Schematic diagrams of the genome structures of wild-type HSV-1(F) and the relevant domains of the recombinant viruses used in this study. Line 1, wild-type HSV-1(F) genome; line 2, domains of the UL13 to UL15 and UL50 to UL52 genes; line 3, domains of the UL14 and UL51 genes; lines 4 to 12, recombinant viruses with mutations in the UL14 and/or UL51 genes; line 13, domains of the UL10 to UL13 genes; line 14, domains of the UL11 and UL13 genes; line 15, recombinant viruses with mutations in UL11 and UL13. An asterisk denotes a stop codon.

FIG 2

Characterization of the recombinant viruses expressing FEM-tagged UL51 and UL11. (A) Vero cells were mock infected or infected with wild-type HSV-1(F) or YK5019 (UL51-FEM) at an MOI of 5 for 18 h and then analyzed by immunoblotting (IB) with the indicated antibodies. (B) Vero cells were mock infected or infected with wild-type HSV-1(F) or YK5020 (UL11-FEM) at an MOI of 5 for 18 h and then analyzed by immunoblotting with the indicated antibodies. (C and D) Vero cells were infected with wild-type HSV-1(F), YK5019 (UL51-FEM), or YK5020 (UL11-FEM) at an MOI of 5 (C) or 0.01 (D). Total virus from cell culture supernatants and infected cells was harvested at 18 h (C) or 48 h (D) postinfection and assayed on Vero cells. Each data point is the mean ± standard error from the results of triplicate samples. Differences in viral yields between HSV-1(F) and YK5019 (UL51-FEM) and between HSV-1(F) and YK5020 (UL11-FEM) were not statistically significant by analysis of variance (ANOVA) and Dunnett's test. Data are representative of three independent experiments.

FIG 3

Interaction between UL51 and UL14 in HSV-1-infected cells. (A) Vero cells were infected with YK5019 (UL51-FEM) or UK5020 (UL11-FEM) at an MOI of 5 for 18 h, harvested, immunoprecipitated (IP) with anti-Myc antibody (α-Myc), and analyzed by immunoblotting (IB) with the indicated antibodies. WCE, whole-cell extract. (B and C) Vero cells were infected with wild-type HSV-1(F) at an MOI of 5 for 18 h, harvested, immunoprecipitated with anti-UL50 or anti-UL51 antibody (B) or with anti-UL50 or anti-UL14 antibody (C), and analyzed by immunoblotting with the indicated antibodies. (D) Vero cells were mock infected or infected with wild-type HSV-1(F) at an MOI of 5, fixed at 18 h postinfection, permeabilized, stained with anti-UL51 and anti-UL14 antibodies, and examined by confocal microscopy.

FIG 4

Mapping of UL51 domains required for UL51 interaction with UL14. (A) Schematic diagram of the UL51 gene product (top line), with its putative α-helix domains (black boxes), and diagrams of GST-UL51, the GST-UL51 deletion mutants, and the GST-UL51:90-130LIY/AAA mutant. The level of binding of each GST fusion protein to UL14 determined in the pulldown experiments shown in panels B and C is shown at the right of each protein. (B and C) The GST fusion proteins shown in panel A were immobilized on glutathione-Sepharose beads and mock reacted or reacted with lysates of Vero cells that had been infected with wild-type HSV-1(F) at an MOI of 5 for 18 h. The beads were washed extensively and divided into two parts. One part was analyzed by immunoblotting with anti-UL14 antibody (top gels), and the other was electrophoretically analyzed in a denaturing gel and CBB stained (bottom gels). (D) Amount of UL14 pulled down by GST, GST-UL51:90-130, or GST-UL51:90-130LIY/AAA shown in the top of panel C relative to those of the GST fusion protein shown in the bottom of panel C. Each value is the mean ± standard error from three independent experiments and is expressed relative to the mean value of GST, which was normalized to 100. Asterisks indicate significant differences: *, P < 0.05 (by two-tailed Student t test).

FIG 5

Effects of mutation(s) in UL51 and/or UL14 on expression of neighboring genes. (A) Vero cells were mock infected or infected with wild-type HSV-1(F), YK5011 (ΔUL51), YK5012 (ΔUL51-repair), YK5013 (UL51LIY/AAA), or YK5014 (UL51LIY/AAA-repair) at an MOI of 5 for 18 h and then analyzed by immunoblotting with the indicated antibodies. (B) Vero cells were mock infected or infected with wild-type HSV-1(F), YK5015 (ΔUL14), or YK5016 (ΔUL14-repair) at an MOI of 5 for 18 h and then analyzed by immunoblotting with the indicated antibodies. (C) Vero cells were mock infected or infected with wild-type HSV-1(F), YK5017 (ΔUL51/ΔUL14), or YK5018 (ΔUL51/ΔUL14-repair) (C) at an MOI of 5 for 18 h and then analyzed by immunoblotting with the indicated antibodies.

FIG 6

Effects of the UL51LIY/AAA mutations in UL51 on the interaction of UL51 with UL14 in HSV-1-infected cells. Vero cells were infected with wild-type HSV-1(F), YK5013 (UL51LIY/AAA), YK5014 (UL51LIY/AAA-repair), YK5011 (ΔUL51), or YK5015 (ΔUL14) at an MOI of 5 for 18 h, harvested, immunoprecipitated with anti-UL51 (A) or anti-UL14 (B) antibody, and analyzed by immunoblotting with anti-UL51 and anti-UL14 antibodies.

FIG 7

Effects of mutation(s) in UL51 and/or UL14 on localization of UL51 and UL14 in HSV-1-infected cells. Vero cells were infected with wild-type HSV-1(F), YK5015 (ΔUL14), YK5016 (ΔUL14-repair), YK5011 (ΔUL51), YK5012 (ΔUL51-repair), YK5013 (UL51LIY/AAA), YK5014 (UL51LIY/AAA-repair), or R7356 (ΔUL13) at an MOI of 5, fixed at 18 h postinfection, permeabilized, stained with anti-UL51 and anti-UL14 (A) or anti-UL11 (B) antibody, and examined by confocal microscopy.

FIG 8

Effects of mutation(s) in UL51 and/or UL14 on progeny virus yields. (A and B) Vero cells were infected with HSV-1(F), YK5015 (ΔUL14), YK5016 (ΔUL14-repair), YK5011 (ΔUL51), YK5012 (ΔUL51-repair), YK5013 (UL51LIY/AAA), YK5014 (UL51LIY/AAA-repair), YK5017 (ΔUL51/ΔUL14), or YK5018 (ΔUL51/ΔUL14-repair) at an MOI of 5 (A) or 0.01 (B). Total virus from cell culture supernatants and infected cells was harvested at 18 h (A) or 48 h (B) postinfection and assayed on Vero cells. Each data point is the mean ± standard error from the results for triplicate samples. Differences in viral yields between HSV-1(F) and each of the mutant viruses were statistically significant by ANOVA and Dunnett's test (P < 0.05). Differences in viral yields between HSV-1(F) and each of the repaired viruses were not statistically significant by ANOVA and Dunnett's test. Data are representative of three independent experiments.

FIG 9

Effects of mutation(s) in UL51 and/or UL14 on HSV-1 secondary envelopment. Vero cells were infected with wild-type HSV-1(F), YK5015 (ΔUL14), YK5016 (ΔUL14-repair), YK5011 (ΔUL51), YK5012 (ΔUL51-repair), YK5013 (UL51LIY/AAA), YK5014 (UL51LIY/AAA-repair), YK5017 (ΔUL51/ΔUL14), YK5018 (ΔUL51/ΔUL14-repair), or R7356 (ΔUL13) at an MOI of 5, fixed at 18 h postinfection, embedded, sectioned, stained, and examined by transmission electron microscopy. Black arrows indicate unenveloped capsids. White arrows indicate partially enveloped capsids. Scale bar, 200 nm.

FIG 10

Sequence alignment of the UL51 homologs in HSV-1 helix VI in members of the three herpesvirus subfamilies. HSV-2, herpes simplex virus 2; VZV, varicella-zoster virus; MCMV, murine cytomegalovirus; EBV, Epstein-Barr virus; KSHV, Kaposi's sarcoma-associated herpesvirus. HSV-1, HSV-2, VZV, and PRV are in the subfamily Alphaherpesvirinae, HCMV and MCMV are in the Betaherpesvirinae, and EBV and KSHV are in the Gammaherpesvirinae. The residues conserved in at least two herpesviruses are in light, medium, or dark shading. The HSV-1 residues required for interaction with UL14 and the corresponding residues of other herpesviruses are boxed.