The interaction of the cellular export adaptor protein Aly/REF with ICP27 contributes to the efficiency of herpes simplex virus 1 mRNA export

Abstract

Herpes simplex virus 1 (HSV-1) protein ICP27 enables viral mRNA export by accessing the cellular mRNA export receptor TAP/NXF, which guides mRNA through the nuclear pore complex. ICP27 binds viral mRNAs and interacts with TAP/NXF, providing a link to the cellular mRNA export pathway. ICP27 also interacts with the mRNA export adaptor protein Aly/REF, which binds cellular mRNAs and also interacts with TAP/NXF. Studies using small interfering RNA (siRNA) knockdown indicated that Aly/REF is not required for cellular mRNA export, and similar knockdown studies during HSV-1 infection led us to conclude that Aly/REF may be dispensable for viral RNA export. Recently, the structural basis of the interaction of ICP27 with Aly/REF was elucidated at atomic resolution, and it was shown that three ICP27 residues, W105, R107, and L108, interface with the RNA recognition motif (RRM) domain of Aly/REF. Here, to determine the role the interaction of ICP27 and Aly/REF plays during infection, these residues were mutated to alanine, and a recombinant virus, WRL-A, was constructed. Virus production was reduced about 10-fold during WRL-A infection, and export of ICP27 protein and most viral mRNAs was less efficient. We conclude that interaction of ICP27 with Aly/REF contributes to efficient viral mRNA export.

Fig 1

ICP27 mutant WRL-A does not interact with Aly/REF. (A) HeLa cells were cotransfected with pEGFP-Aly/REF and wild-type (WT) ICP27 or plasmids expressing ICP27 mutant WRL-A, D6-21-A, or D6-21-A+WRL-A as indicated. Transfected cells were infected with ICP27 null mutant virus 27Lac-Z 24 h after transfection. Four hours after infection, cells were fixed, and immunofluorescence staining was performed with anti-ICP27 antibody. Green fluorescent protein (GFP) fluorescence was visualized directly. The white arrows show the colocalization of Aly/REF and ICP27. The yellow arrows point to Aly/REF sites that do not colocalize with ICP27 harboring the WRL mutations. (B) HeLa cells were transfected with WT ICP27 or mutant WRL-A or with pUC18 as a negative control. Twenty-four hours after transfection, cells were infected with 27-LacZ. Eight hours after infection, cells were harvested, and immunoprecipitation (IP) was performed on cell lysates with anti-ICP27 antibody (α-ICP27). Western blots (WB) were probed with anti-Aly/REF and anti-ICP27 antibodies.

Fig 2

Virus production is reduced in ICP27 WRL-A mutant virus infection. (A) Schematic of the ICP27 coding sequence illustrating the leucine-rich region (LRR), acidic region (D/E), nuclear localization signal (NLS), RGG box RNA binding domain, predicted KH domains, and a zinc finger-like region (CCHC). The Aly/REF binding site is shown. The sites of the mutations in D6-21-A are also shown. HSV-1 mutant d2-3, which has a deletion of amino acids (aa) 64 to 108, is shown by the dotted line. (B) A one-step virus growth curve was performed by infecting Vero cells with wild-type (WT) HSV-1 KOS or ICP27 mutant WRL-A or d2-3 at an MOI of 1. Infected cells were harvested at 0, 4, 8, 12, and 16 h after infection. Virus titers were determined by plaque assays. The experiments were performed three times, and in each experiment, each time point was performed in triplicate. The data shown are from a representative experiment.

Fig 3

ICP27 mutant WRL-A does not interact with Aly/REF during infection. (A) HeLa cells were infected with WT HSV-1 KOS, WRL-A, or d2-3. Eight hours after infection, cells were harvested, and immunoprecipitation was performed with anti-ICP27 antibody. Western blots were probed with anti-Aly/REF and anti-ICP27 antibodies. Antibody to YY1 was used as the loading control. The positions of molecular mass markers (MM) (in kilodaltons) are indicated to the right of the gels. (B) HeLa cells were transfected with pEGFP-Aly/REF, and 24 h later, the cells were infected with WT KOS, WRL-A, or d2-3. Four hours after infection, cells were fixed, and immunofluorescence staining was performed with anti-ICP27 antibody. GFP fluorescence was visualized directly. The arrows point to Aly/REF sites that do not colocalize with WRL-A and d2-3.

Fig 4

ICP27 is exported to the cytoplasm less efficiently in WRL-A-infected cells. HeLa cells were infected with WT KOS or ICP27 mutant WRL-A. Eight hours after infection, cells were fixed, and immunofluorescence staining was performed with anti-ICP27 antibody. (Top) Distribution of cells in which ICP27 was nuclear, cytoplasmic, or both cytoplasmic and nuclear (C/N). For WRL-A, 141 cells were scored, and for WT KOS, 139 cells were scored. (Bottom) Representative images of ICP27 cellular location. The two white arrows in WRL-infected cells show the nuclear localization, and the yellow arrow in KOS-infected cells shows the cytoplasmic localization.

Fig 5

Poly(A)⁺ RNA accumulates in the nucleus in cells infected with WRL-A. HeLa cells were either mock infected or infected with WT KOS, WRL-A, or d2-3 as indicated. Eight hours after infection, cells were fixed, and in situ hybridization was performed by using a biotinylated oligo(dT) probe to detect poly(A)⁺ RNA. ICP27 was detected by staining with anti-ICP27 antibody. In the bottom row, mock-infected cells were treated with RNase.

Fig 6

Viral mRNA export is less efficient for many HSV-1 transcripts in WRL-A infection. HeLa cells were infected with WT KOS or with WRL-A, d2-3, or ΔRGG. Eight hours after infection, cells were harvested, and nuclear and cytoplasmic viral poly(A)⁺ RNA was isolated and reverse transcribed. HSV-1 transcripts from each fraction were hybridized in triplicate to HSV-1-specific microarray chips. After quantification using Array Vision software, the average cytoplasmic/nuclear (cyto/nuc) ratio was calculated and plotted, with the y axis representing the ratio for each transcript and the x axis representing individual HSV-1 transcripts. The experiments were performed in triplicate, and the results of a representative experiment are shown. The red line shows a 1:1 ratio of cytoplasmic/nuclear poly(A)⁺ RNA. Note that the scale for the y axis for KOS is from 0 to 3.0, whereas the scale for the WRL-A, d2-3, and ΔRGG mutants is from 0 to 2.0. HSV-1 transcripts are shown as immediate early (IE), early (E), and late (L).