A network of protein interactions around the herpes simplex virus tegument protein VP22

Abstract

Assembly of the herpesvirus tegument is poorly understood but is believed to involve interactions between outer tegument proteins and the cytoplasmic domains of envelope glycoproteins. Here, we present the detailed characterization of a multicomponent glycoprotein-tegument complex found in herpes simplex virus 1 (HSV-1)-infected cells. We demonstrate that the tegument protein VP22 bridges a complex between glycoprotein E (gE) and glycoprotein M (gM). Glycoprotein I (gI), the known binding partner of gE, is also recruited into this gE-VP22-gM complex but is not required for its formation. Exclusion of the glycoproteins gB and gD and VP22's major binding partner VP16 demonstrates that recruitment of virion components into this complex is highly selective. The immediate-early protein ICP0, which requires VP22 for packaging into the virion, is also assembled into this gE-VP22-gM-gI complex in a VP22-dependent fashion. Although subcomplexes containing VP22 and ICP0 can be formed when either gE or gM are absent, optimal complex formation requires both glycoproteins. Furthermore, and in line with complex formation, neither of these glycoproteins is individually required for VP22 or ICP0 packaging into the virion, but deletion of gE and gM greatly reduces assembly of both VP22 and ICP0. Double deletion of gE and gM also results in small plaque size, reduced virus yield, and defective secondary envelopment, similar to the phenotype previously shown for pseudorabies virus. Hence, we suggest that optimal gE-VP22-gM-gI-ICP0 complex formation correlates with efficient virus morphogenesis and spread. These data give novel insights into the poorly understood process of tegument acquisition.

Fig 1

Growth characteristics of HSV-1 lacking gE and/or gM. (A) Vero cells in 6-well plates were infected with approximately 100 PFU of WT (sc16), ΔgE, ΔgM, or ΔgEgM viruses and fixed and stained 4 days later. The area of 20 representative plaques for each virus was measured using ImageJ software, and the average relative size was calculated as a percentage of WT plaques. (B) Single-step growth curves of WT, ΔgE, ΔgM, or ΔgEgM viruses were carried out by infecting Vero cells at a multiplicity of 5 and harvested at the indicated times for cell-associated or released virus. All growth curves were carried out in triplicate.

Fig 2

Immunoprecipitation of VP22-specific complexes from cells infected with viruses lacking gE and/or gM. (A to C) Whole-cell lysates harvested 24 h after infection from Vero cells infected with Δ22, sc16 (wt), ΔgE, ΔgM, or ΔgEgM viruses were subjected to immunoprecipitation with an anti-VP22 antibody and analyzed by Western blotting for the presence of gE, gD, or gB (A), gM (B), and VP16 or ICP0 (C). *, immunoglobulin heavy chain; note that the immunoglobulin heavy chain is visible in this gM blot, and no others, because the ordinary rabbit secondary antibody, rather than the TrueBlot secondary antibody, was used here.

Fig 3

VP22 bridges gE and gM to form a multicomponent complex in infected cells. (A to C) Vero cells were infected with WT HSV-1 or the ΔgE or ΔgM viruses, and gE was immunoprecipitated from whole-cell lysates harvested 24 h using antibody ab6510. Samples were subjected to Western blot analysis using antibodies specific for gE, gM, VP22, gD, or VP16. *, immunoglobulin heavy chain. (D) Schematic illustrating our previously described VP22 mutant viruses expressing GFP-tagged full-length VP22 (GFP-22), GFP in place of VP22 (Δ22), GFP-tagged VP22 with a deletion between residues 213 to 226 (ΔgEbind), or GFP tagged to the C-terminal half of VP22 (160 to 301). (E and F) As for panel A, except Vero cells were infected with the viruses described in panel D or the ΔgE mutant and samples analyzed by Western blotting using antibodies specific for gE, gM, VP22, GFP, or ICP0. Molecular weight marker sizes (kDa) are shown on the left.

Fig 4

VP22 interaction with the cytoplasmic tail of gM. (A) Schematic of VP22 variants expressed by recombinant viruses used in panels B and D. (B to D) GST-gM (gM) or GST alone (G) bound to glutathione Sepharose beads was incubated with lysates from Vero cells infected with viruses as indicated and analyzed by SDS-PAGE followed by Western blotting for GFP (B and D) or VP22 (C). Molecular weight marker sizes (kDa) are shown on the left.

Fig 5

The gE-VP22-gM complex incorporates gI. (A to C) Vero cells infected with WT HSV-1, the ΔgE virus, or the ΔgI virus (panel A only) were harvested 24 h after infection and the gE-gI complex was immunoprecipitated using antibody 3063, specific for gE in the context of the gE-gI complex. Samples were analyzed by Western blotting using antibodies as indicated. (D) Vero cells infected with WT HSV-1 or the ΔgE or ΔgI viruses were harvested for 24 h, and gE was immunoprecipitated using antibody 6510, specific for free and gI-complexed gE. Samples were analyzed by Western blotting using antibodies specific for gE, gM, or VP22.

Fig 6

Relative assembly of VP22 into HSV-1 virions isolated from Vero cells infected with glycoprotein mutant viruses. (A and B) Gradient purified extracellular sc16 (WT), ΔgE, ΔgM, or ΔgEgM virions were analyzed by Coomassie blue staining (A) or by Western blotting using antibodies as indicated (B). Molecular weight marker sizes (kDa) are shown on the left. (C and D) Gradient-purified extracellular sc16 (WT) or ΔgI virions were analyzed by SDS-PAGE followed by Coomassie blue staining (C) or Western blotting with antibodies as indicated (D). Molecular weight marker sizes (kDa) are shown on the left. (E and F) Extracellular WT (s17) or Δ22 virions purified from BHK cells were analyzed by Coomassie blue staining (E) or Western blotting using antibodies as indicated (F). Molecular weight marker sizes (kDa) are shown on the left.

Fig 7

HSV-1 lacking glycoproteins E and M is defective in secondary envelopment. HFFF cells infected with the WT or ΔgEgM virus at a multiplicity of 2 were fixed 12 h later and processed for EM. PM, plasma membrane; Ca, capsid; GA, Golgi apparatus; NM, nuclear membrane; WV, wrapped or wrapping virions. Scale bar = 500 nm.

Fig 8

Network of protein-protein interactions around the HSV-1 tegument protein VP22. Solid lines indicate interactions reported in HSV-1 (, , , , , , , , , , –50, 52, 53). Broken lines indicate interactions shown in PRV (19, 27, 28, 55). The glycoprotein-tegument complex characterized here (complex 1), which is required for VP22 and ICP0 assembly into the virion, is shown in the context of previously reported relevant interactions (19, 38, 49). Whether UL11, UL16, or UL13 are incorporated into the VP22-gE-gM-gI-ICP0 complex via their known interactions with gE remains to be determined (22, 42, 53). Others have observed VP22-gD binding in infected cells (5, 17); however, this interaction was not reproducible in our own hands (49), and gD was not detectable in the VP22-gE-gM-gI-ICP0 complex. The previously characterized VP22-VP16 complex, which also incorporates vhs (12, 48, 50) and which was shown here to be separate from the VP22 assembly complex, is also illustrated (complex 2). For completion, the tegument protein VP13/14 has also been included in the figure. VP13/14 has been shown to interact with the cytoplasmic tail of gM (49) and VP16 (8, 52); however, it is not known which if either of these interactions is involved in VP13/14 assembly.