HCMV infection temporally alters the abundance and subcellular distribution of key post-transcriptional processing factors during infection. At IE times, SRPK1, PTB and CstF-64 co-localized in nuclear punctate sites with the MIE proteins in HCMV-infected cells. IE1 and IE2, visualized as single punctate foci, are enriched in sites called nuclear domain 10 (ND10) at IE times (Ahn & Hayward, 1997
). Because IE1 becomes diffused in the nucleus (Korioth et al., 1996
) and IE2 remains at punctate sites, the cellular RNA-processing factors appear to contain IE2 at former ND10 sites. ND10s are active sites of HCMV IE gene transcription (Ishov et al., 1997
). The combined presence of SRPK1, PTB and CstF-64 will predictably affect alternative splicing and polyadenylation of HCMV IE transcripts. Phosphorylation of SR proteins by SRPK1 targets them to sites of active transcription. PTB is known to negatively regulate MIE pre-mRNA splicing (Cosme et al., 2009
), whereas CstF-64 selectively increases HCMV UL37x1 poly(A) site usage (Adair et al., 2004
; Su et al., 2003a
During the cell cycle, SRPK1 is translocated to the nucleus during M phase (Ding et al., 2006
). In contrast, during HCMV infection, which causes pseudo-mitosis of the infected cell (Hertel et al., 2007
), SRPK1 is increased mainly in the cytoplasm. Regulation of SRPK1 is achieved by partitioning through an anchoring mechanism (Ding et al., 2006
) and by its interactions with molecular co-chaperones (Zhong et al., 2009
). Heat-shock protein 70, part of the macromolecular complex used to anchor SRPK1 in the cytosol, is induced from IE to late times of HCMV infection (Santomenna & Colberg-Poley, 1990
) with kinetics similar to those we observed for the progressively increasing abundance of cytoplasmic SRPK1. HCMV infection is well documented to block cellular stress pathways that would result in apoptosis (Goldmacher et al., 1999
; Reeves et al., 2007
; Skaletskaya et al., 2001
; Terhune et al., 2007)
. Thus, HCMV infection may effectively block the cellular stress responses that could result in release of SRPK1 from the co-chaperone complex and nuclear translocation. Of particular note, HCMV infection redundantly blocks mitochondrial stress responses (Goldmacher et al., 1999
; Reeves et al., 2007
), possibly modulating oxidative stress, its consequent release of SRPK1 from its co-chaperone complex and its translocation from the cytoplasm to the nucleus (Zhong et al., 2009
The SRPK1 cytoplasmic location may allow continued RNA splicing during HCMV infection but inhibit its nuclear functions. HCMV virion egress requires the destabilization of the nuclear lamina through recruitment of the HCMV pUL97 by p32 and the structural modifications of HCMV proteins ppUL50 and ppUL53 through their interaction with protein kinase C (Camozzi et al., 2008
; Marschall et al., 2005
; Milbradt et al., 2007
). Phosphorylation of lamin B receptor (LBR) by SRPK1 induces its association with lamin B at the inner nuclear membrane (Papoutsopoulou et al., 1999
). SRPK1 phosphorylation of LBR also stimulates the binding of the protein to chromatin and the stabilization of the nuclear membrane (Takano et al., 2004
). It is therefore possible that continued anchoring of SRPK1 to the cytoplasm sequesters its activity from the nucleus allowing efficient egress of the HCMV nucleocapsids while allowing continued RNA splicing to occur through late times of infection.
As PTB inhibits splicing of the MIE transcripts and has negative effects on HCMV growth (Cosme et al., 2009
), HCMV tightly regulates its subcellular distribution during infection. During IE times of infection, PTB, CstF-64 and MIE proteins are enriched in punctate structures, potentially perinucleolar compartments (PNCs). PNCs are subcellular structures, located at the periphery of nucleoli with high concentrations of PTB (Ghetti et al., 1992
; Matera et al., 1995
). PNCs are thought to be active sites of RNA polymerase II and III transcription. At early times of HCMV infection, PTB was partially translocated to discrete cytoplasmic sites of some infected cells. As PTB was relocalized, the nuclear PNCs in HCMV-infected cells appeared to disintegrate. This dissolution of PNCs following cytoplasmic PTB relocalization is consistent with the requirement for localization of PTB at the periphery of the nucleoli to maintain their integrity (Wang et al., 2003
) and may partially underlie the sorting of fibrillarin to nucleolar caps.
By late times of infection, we observed exclusion of PTB from increasingly prominent subnuclear structures and the inclusion of CstF-64 in these. These proved to be VRCs by co-localization with the HCMV DNA-binding protein ppUL57. When HCMV DNA replication was blocked, the formation of the VRCs and the redistribution PTB was reduced, suggesting a role for HCMV DNA replication and/or late gene expression in PTB exclusion.
The presence of CstF-64 and exclusion of PTB suggest that HCMV VRCs are active sites of transcription and post-transcriptional processing, in addition to sites of DNA replication. RNA polymerase II, phosphorylated at Ser-2, was found in HCMV VRCs at 48 h p.i. in HCMV-infected G0
-HFFs (Tamrakar et al., 2005
). By subnuclear fractionation, we intriguingly found that HCMV ppUL57 mostly co-sedimented with induced nuclear CstF-64 and SRPK1, suggesting that these factors may be functionally associated with HCMV VRCs during infection.
The abundance of CstF-64 is the rate-limiting factor for the assembly of the core polyadenylation machineries, and increases in CstF-64 abundance positively influence 3′-end processing and the use of weak polyadenylation sites. Analysis of the HCMV UL37x1 polyadenylation signal, contained within an intron, showed that this signal is increasingly used during infection and that its usage is enhanced by a downstream element (Adair et al., 2004
; Su et al., 2003a
). The presence of CstF-64 at sites of HCMV post-transcriptional processing could serve to favour alternative polyadenylation of its transcripts, despite increased levels of viral transcripts at late times of infection.
The HCMV-induced sorting of nuclear components included the redistribution of nucleolar components. Sorting of nucleolar components in response to inhibition of transcription results in the formation of dynamic structure bodies (Shav-Tal et al., 2005
). Fibrillarin is normally localized in the dense fibrillar component domain within nucleoli and segregates to LNCs following actinomycin D treatment. We observed similar segregation of fibrillarin to nucleolar caps at late times of HCMV infection and in close proximity to VRCs. During transcription inhibition, DNCs enriched in CstF-64 and PSF are formed (Shav-Tal et al., 2005
). Similar to stress-responsive DNCs, HCMV VRCs include CstF-64 and do not include PTB. However, in contrast to DNCs, HCMV VRCs partially exclude PSF.
The question of what promotes nucleolar cap formation in HCMV-infected cells remains unanswered. Several HCMV proteins are known to localize to nucleoli (Salsman et al., 2008
). Sorting of nucleolar components into nucleolar caps can result in the disintegration of PNCs (Shav-Tal et al., 2005
). PNCs disintegrate at early times of HCMV infection when PTB shuttles to the cytoplasm and nuclear VRCs begin to form.
Nucleolar cap formation has been attributed to transcriptional arrest (Shav-Tal et al., 2005
). Transcription of cellular and viral genes continues to occur throughout the HCMV life cycle. However, HCMV infection might differentially inhibit transcription of sensor genes, resulting in the formation of nucleolar caps. Alternatively, the presence of HCMV proteins in nucleoli and Cajal bodies (Salsman et al., 2008
) may segregate nucleolar components and cause formation of nucleolar caps. Finally, the addition of pyruvate, whose concentration is markedly increased in HCMV-infected cells (Munger et al., 2008
), can lead to the formation of nucleolar caps, as it does in other cell types (Shav-Tal et al., 2005
). Taken together, our studies show that HCMV-induced restructuring of subnuclear domains includes relocalization of cellular RNA-processing machineries and nucleolar and nucleoplasmic components during permissive infection.