ICP27 phosphorylation site mutants display altered functional interactions with cellular export factors Aly/REF and TAP/NXF1 but are able to bind herpes simplex virus 1 RNA

Abstract

Herpes simplex virus 1 (HSV-1) protein ICP27 is a multifunctional regulatory protein that is phosphorylated. Phosphorylation can affect protein localization, protein interactions, and protein function. The major sites of ICP27 that are phosphorylated are serine residues 16 and 18, within a CK2 site adjacent to a leucine-rich region required for ICP27 export, and serine 114, within a PKA site in the nuclear localization signal. Viral mutants bearing serine-to-alanine or glutamic acid substitutions at these sites are defective in viral replication and gene expression. To determine which interactions of ICP27 are impaired, we analyzed the subcellular localization of ICP27 and its colocalization with cellular RNA export factors Aly/REF and TAP/NXF1. In cells infected with phosphorylation site mutants, ICP27 was confined to the nucleus even at very late times after infection. ICP27 did not colocalize with Aly/REF or TAP/NXF1, and overexpression of TAP/NXF1 did not promote the export of ICP27 to the cytoplasm. However, in vitro binding experiments showed that mutant ICP27 was able to bind to the same RNA substrates as the wild type. Nuclear magnetic resonance (NMR) analysis of the N terminus of ICP27 from amino acids 1 to 160, compared to mutants with triple substitutions to alanine or glutamic acid, showed that the mutations affected the overall conformation of the N terminus, such that mutant ICP27 was more flexible and unfolded. These results indicate that these changes in the structure of ICP27 altered in vivo protein interactions that occur in the N terminus but did not prevent RNA binding.

FIG. 1.

ICP27 is confined to the nucleus in infection with phosphorylation site mutants. HeLa cells were infected with HSV-1 KOS, S16A, S18A, S114A, S16,18A, S16,18,114A, S16,18,114E, dLeu, and d3-4 at an MOI of 5. At 4, 8, 12, and 16 h postinfection, cells were fixed with 3.7% formaldehyde and immunostained with anti-ICP27 monoclonal antibody. Cells were viewed with a Zeiss Axiovert S100 microscope. Magnification, ×100.

FIG. 2.

ICP27 remains associated with splicing speckles throughout infection with phosphorylation site mutants. HeLa cells were infected with HSV-1 KOS, S16A, S18A, S114A, and S16,18,114A at an MOI of 5. At 4 and 8 h after infection, cells were fixed and stained with antibodies to SC35 and ICP27. Images were captured with the LSM 510 confocal microscope. Magnification, ×63.

FIG. 3.

ICP27 does not colocalize with Aly/REF in infections with glutamic acid substitution mutants. HeLa cells were transfected with pEGFP-Aly/REF and 24 h later were infected with HSV-1 KOS, S16E, S16,18,114E, or d2-3. At 4 and 8 h after infection, cells were fixed, and immunostaining was performed with anti-ICP27 antibody. Green fluorescent protein (GFP) fluorescence was visualized directly. The white arrows point to Aly/REF sites that do not colocalize with ICP27. Images were viewed with Zeiss Axiovert S100 microscope. Magnification, ×100.

FIG. 4.

Aly/REF coimmunoprecipates with mutant ICP27. HeLa cells were mock infected or were infected with HSV-1 KOS or S16,18,114A at an MOI of 5 for 8 h. Immunoprecipitation was performed with anti-ICP27 antibody (left). Western blot analysis was performed with anti-ICP27 antibody and anti-Aly/REF antibody (left). Portions of the whole-cell extracts were fractionated directly on an SDS-polyacrylamide gel, and Western blot analysis was also performed (right). The sizes of the molecular mass markers (in kilodaltons) are indicated on the left.

FIG. 5.

ICP27 and TAP/NXF1 do not colocalize in infections with phosphorylation site mutants. HeLa cells were transfected with pEGFP-TAP and were subsequently infected with HSV-1 KOS, S16E, or S16,18,114E. Cells were fixed at 4 and 8 h after infection and immunostained with anti-ICP27 antibody. GFP fluorescence was visualized directly. (A) Images were captured with a Zeiss Axiovert S100 microscope. Magnification, ×100. (B) Images were viewed with an LSM 510 confocal microscope. Magnification, ×63. White arrows point to GFP-TAP sites, and yellow arrows mark ICP27 sites in the merged images.

FIG. 6.

Alanine and glutamic acid substitution mutants do not complement. (A) Schematic representation of ICP27 illustrating the CK2 and PKA phosphorylation sites denoted by arrows. (B) HeLa cells were infected with the viruses indicated in the figure. Single-virus infections were performed at an MOI of 5. Coinfections were performed at an MOI of 2.5 for each virus. Virus yields were determined by plaque assay on the ICP27 complementing cell line 2-2. Each virus is depicted by a different color, and coinfections are a mix of the two colors for the viruses used in the coinfections.

FIG. 7.

Expression of ¹⁵N-labeled ICP27 N terminus and S16,18,114A and S16,18,114E phosphorylation point mutants. Rosetta E. coli cells were transformed with either the pET21b wild-type ICP27 N terminus or phosphorylation point mutant S16,18,114A or S16,18,114E expression plasmids. Cells were grown in Neidhart's minimal media supplemented with ¹⁵NH₄Cl. Protein expression was induced with IPTG, and 6× His-tagged proteins were purified using Ni-NTA agarose under native protein purification conditions. A portion of the elution fractions from the Ni-NTA column was separated on a 10 to 20% Tris gradient gel, and the gel was stained with Coomassie blue.

FIG. 8.

Alanine and glutamic acid substitution mutations in the ICP27 N terminus show structural changes in two-dimensional (2D) NMR HSQC spectra. (A) The ¹H-¹⁵N HSQC spectrum for the wild-type ICP27 N terminus. Panels include all peaks detected except for tryptophan. (B) ¹H-¹⁵N HSQC spectrum for the S16,18,114A mutant. (C) ¹H-¹⁵N HSQC spectrum for the S16,18,114E mutant. (D) A portion of the full ¹H-¹⁵N HSQC spectra (boxes in panels A and B) with an overlay of the wild-type ICP27 N terminus (blue) and S16,18,114A mutant (red). Arrows highlight some peaks specifically appearing in either wild-type ¹H-¹⁵N spectrum (blue arrow) or mutant spectrum (red arrow). (E) A portion of the full ¹H-¹⁵N HSQC spectra with an overlay of wild-type ICP27 N terminus (blue) and S16,18,114E mutant (orange). Arrows highlight some peaks specifically appearing in either wild-type spectrum (blue arrow) or mutant spectrum (orange arrow).

FIG. 9.

Alanine and glutamic acid substitution mutants bind HSV-1 gC sequences. (A) Twenty fmol of radiolabeled gC 11-40, gC 1-30, or gC 31-60 30-mer oligonucleotides (see Materials and Methods) were either incubated with no protein (−) or with 2.5 to 62.5 μM wild-type ICP27 N terminus or S16,18,114A N terminus. Samples were fractionated on a prerun acrylamide gel, and dried gels were exposed to film. Arrows indicate the migration of free probe and the shift due to protein binding. (B) Twenty fmol of radiolabeled gC 11-40, gC 1-30, and gC 31-60 30-mer oligonucleotides were either incubated with no protein or 2.5 to 62.5 μM wild-type ICP27 N-terminal protein or S16,18,114E N terminus and were analyzed as described in the legend for panel A.