Regulation of the subcellular distribution of key cellular RNA-processing factors during permissive human cytomegalovirus infection

Abstract

Alternative splicing and polyadenylation of human cytomegalovirus (HCMV) immediate-early (IE) pre-mRNAs are temporally regulated and rely on cellular RNA-processing factors. This study examined the location and abundance of essential RNA-processing factors, which affect alternative processing of UL37 IE pre-mRNAs, during HCMV infection. Serine/threonine protein kinase 1 (SRPK1) phosphorylates serine/arginine-rich proteins, necessary for pre-spliceosome commitment. It was found that HCMV infection progressively increased the abundance of cytoplasmic SRPK1, which is regulated by subcellular partitioning. The essential polyadenylation factor CstF-64 was similarly increased in abundance, albeit in the nucleus, proximal to and within viral replication compartments (VRCs). In contrast, the location of polypyrimidine tract-binding protein (PTB), known to adversely affect splicing of HCMV major IE RNAs, was temporally regulated during infection. PTB co-localized with CstF-64 in the nucleus at IE times. By early times, PTB was detected in punctate cytoplasmic sites of some infected cells. At late times, PTB relocalized to the nucleus, where it was notably excluded from HCMV VRCs. Moreover, HCMV infection induced the formation of nucleolar stress structures, fibrillarin-containing caps, in close proximity to its VRCs. PTB exclusion from HCMV VRCs required HCMV DNA synthesis and/or late gene expression, whereas the regulation of SRPK1 subcellular distribution did not. Taken together, these results indicated that HCMV increasingly regulates the subcellular distribution and abundance of essential RNA-processing factors, thereby altering their ability to affect the processing of viral pre-mRNAs. These results further suggest that HCMV infection selectively induces sorting of nucleolar and nucleoplasmic components.

Fig. 1.

HCMV infection progressively increases the abundance of SRPK1 in the cytoplasm of infected G₀-HFFs. G₀-HFFs were HCMV infected (m.o.i.=3) or mock infected. At 12 (a), 24 (b), 48 (c), 72 (d) and 96 (e) h p.i., cells were harvested and sequentially incubated with anti-SRPK1 (red, 1 : 1000) and mAb 810 (specific for MIE, green, 1 : 500), followed by the corresponding secondary antibodies. Probed cells were imaged by confocal microscopy. Panels on the left and in the middle show greyscale imaging, whereas panels on the right show the merged colour images.

Fig. 2.

Subcellular distribution of SRPK1 in HCMV-infected G₀-HFFs. HFFs (6.0×10⁷ cells) were growth arrested and mock infected or HCMV infected (m.o.i.=3), harvested at the indicated times, and fractionated into nuclear and cytoplasmic fractions. Fractionated proteins (20 μg) were resolved by electrophoresis, transferred to nitrocellulose and probed with anti-SRPK1 (1 : 1000), anti-lamin B (1 : 1000) and anti-LDH (1 : 1000). Probed blots were stripped of bound antibodies after exposure and reprobed with other antibodies. The fold inductions in SRPK1 abundance were determined by comparison of the densities of the bands from HCMV-infected cells divided by the densities of the corresponding mock-infected cells.

Fig. 3.

HCMV induction of cytoplasmic SRPK1 during PFA treatment. G₀-HFFs were HCMV infected (m.o.i.=10) and untreated or treated with PFA (400 μg ml⁻¹), following absorption, for 96 h p.i. Cells were harvested, stained for MIE (green) and SRPK1 (red) and visualized as in Fig. 1.

Fig. 4.

Temporal relocalization of PTB and CstF-64 during HCMV infection. G₀-HFFs were treated as in Fig. 1. HCMV-infected and mock-infected cells were methanol fixed at 12 (a), 24 (b), 48 (c), 72 (d) and 96 (e) h p.i., and sequentially incubated with anti-PTB (green, 1 : 250), anti-CstF-64 (red, 1 : 250) and mAb 810 (blue, 1 : 500; MIE) and then with the corresponding secondary antibodies. Cells were imaged by confocal microscopy. The left and middle panels are greyscale, whilst the right panel shows the merged images. (f) PTB is selectively excluded from HCMV VRCs at late times of infection. G₀-HFFs were HCMV infected (m.o.i.=3) and fixed at 72 h p.i. as in Fig. 1 and sequentially incubated with anti-PTB (green, 1 : 250) and anti-ppUL57 (blue, 1 : 250). Cells were then probed with secondary antibodies and imaged using a Zeiss Axiovert inverted UV microscope with an ApoTome attachment. The left and middle panels show greyscale images whilst the right panels show an overlay of both channels.

Fig. 5.

PFA treatment blocks HCMV-induced redistribution of PTB at late times of infection. G₀-HFFs were HCMV infected (m.o.i.=10) and untreated or PFA treated as in Fig. 3. Cells were harvested at 96 h p.i. and stained for PTB (green), CstF-64 (red) and ppUL57 (blue) and imaged by confocal microscopy as in Fig. 4.

Fig. 6.

Subcellular distribution of PTB and CstF-64 in HCMV-infected G₀-HFFs. G₀-HFFs were HCMV infected (m.o.i.=3) or mock infected, fractionated and blotted as in Fig. 2. The same membranes were stripped and reprobed with anti-PTB (1 : 250), rabbit anti-CstF-64 (1 : 250), anti-lamin B (1 : 1000) and anti-LDH (1 : 1000). Probed blots were repeatedly stripped of bound primary and secondary antibodies after exposure and reprobed as required. The increases in PTB and CstF-64 abundance were determined using the density of the bands from HCMV-infected cells divided by the density of the corresponding mock-infected band, as in Fig. 2. Lighter exposures of the nuclear CstF-64 blot were used for scanning and quantification.

Fig. 7.

(a) HCMV infection induces the formation of fibrillarin-containing caps. G₀-HFFs were mock infected or HCMV infected and harvested at 96 h p.i. as in Fig. 3. Cells were stained with anti-fibrillarin (green, 1 : 250), anti-PSF (red, 1 : 250) and anti-ppUL57 (blue, 1 : 250) antibodies and corresponding secondary antibodies. The left and middle panels are greyscale and the rightmost panels show all three channels merged. The bottom panel shows an enlarged HCMV-infected cell. (b) Co-sedimentation of cellular RNA-processing factors and nucleolar components with HCMV ppUL57, a VRC component. G₀-HFFs were mock infected (M) or HCMV infected (H, m.o.i.=1) as in Fig. 2 and harvested at 96 h p.i. Cells were fractionated into cytoplasmic fraction, subnuclear pellet and supernatant (Sup) by modification of the nucleolar isolation procedure of Andersen et al. (2002). The cytoplasmic (Cyto) fraction was concentrated 5-fold and proteins (15 μg per well) were resolved by SDS-PAGE and blotted as described in Fig. 2. The blots were reacted with antibodies against CstF-64 (1 : 2000), SRPK1 (1 : 1000), fibrillarin (1 : 1000), PSF (1 : 2000), ppUL57 (1 : 500) or LDH (1 : 1000). Probed blots were stripped of bound primary and secondary antibodies after exposure and reprobed as required. The fold inductions in CstF-64, SRPK1, fibrillarin and PSF abundance were determined by comparison of the densities of the bands from HCMV-infected cells divided by the densities of the corresponding mock-infected cells as in Fig. 2 except where the mock- or HCMV-infected band was not detectable (nd).