cells Article The Sub-Nuclear Localization of RNA-Binding Proteins in KSHV-Infected Cells Ella Alkalay, Chen Gam Ze Letova Refael, Irit Shoval, Noa Kinor, Ronit Sarid and Yaron Shav-Tal * The Mina & Everard Goodman Faculty of Life Sciences and The Institute of Nanotechnology and Advanced Materials, Bar-Ilan University, Ramat Gan 5290002, Israel; [email protected] (E.A.); [email protected] (C.G.Z.L.R.); [email protected] (I.S.); [email protected] (N.K.); [email protected] (R.S.) * Correspondence: [email protected] Received: 14 August 2020; Accepted: 21 August 2020; Published: 25 August 2020 Abstract: RNA-binding proteins, particularly splicing factors, localize to sub-nuclear domains termed nuclear speckles. During certain viral infections, as the nucleus fills up with replicating virus compartments, host cell chromatin distribution changes, ending up condensed at the nuclear periphery. In this study we wished to determine the fate of nucleoplasmic RNA-binding proteins and nuclear speckles during the lytic cycle of the Kaposi’s sarcoma associated herpesvirus (KSHV). We found that nuclear speckles became fewer and dramatically larger, localizing at the nuclear periphery, adjacent to the marginalized chromatin. Enlarged nuclear speckles contained splicing factors, whereas other proteins were nucleoplasmically dispersed. Polyadenylated RNA, typically found in nuclear speckles under regular conditions, was also found in foci separated from nuclear speckles in infected cells. Poly(A) foci did not contain lncRNAs known to colocalize with nuclear speckles but contained the poly(A)-binding protein PABPN1. Examination of the localization of spliced viral RNAs revealed that some spliced transcripts could be detected within the nuclear speckles. Since splicing is required for the maturation of certain KSHV transcripts, we suggest that the infected cell does not dismantle nuclear speckles but rearranges their components at the nuclear periphery to possibly serve in splicing and transport of viral RNAs into the cytoplasm. Keywords: KHSV; nuclear speckles; SRSF2; nuclear structure; RNA 1. Introduction Kaposi’s sarcoma-associated herpesvirus (KSHV), also known as human herpesvirus 8 (HHV-8), is a member of the lymphotropic gammaherpesvirus subfamily and one of seven known oncogenic human viruses [1]. This obligatory parasite is the etiologic agent of Kaposi’s sarcoma (KS), an angioproliferative multifocal neoplasm of the skin and other organs, that predominantly occurs in immunodeficient individuals, such as AIDS patients, but also among apparently healthy individuals, in particular the elderly. KSHV is also causally associated with two lymphoproliferative diseases, namely primary effusion lymphoma (PEL) and multicentric Castleman’s disease (MCD), as well as with a condition that mimics acute severe sepsis termed KSHV-inflammatory cytokine syndrome (KICS) [2–6]. KSHV can potentially infect a wide variety of human cell types such as keratinocytes, epithelial, endothelial and lymphoid cells, as well as several types of animal cells [7]. Like all herpesviruses, replication and transcription of the KSHV double-stranded DNA genome take place in the nucleus of the infected cells [8,9]. Once the viral genome reaches the host nucleus, one of two alternative infection cycles may occur: latent or lytic. During latent infection the viral DNA exists in the cell nucleus in the form of a circular chromatinized episome, no new virions are produced and only a small proportion of Cells 2020, 9, 1958; doi:10.3390/cells9091958 www.mdpi.com/journal/cells Cells 2020, 9, 1958 2 of 22 the KSHV gene repertoire is expressed. Latent gene products assist in maintaining the viral genome by tethering it to the host chromosomes, supporting its replication and directing its segregation into daughter cells during mitosis, thus establishing a long term infection [10–12]. In contrast, the lytic infection cycle involves extensive viral DNA replication which ends by the assembly and release of new virions. This cycle is characterized by temporal expression of most KSHV genes, which are classified as immediate-early (IE), early (E) and late (L) genes based on their transcription kinetics. Lytic infection results in dramatic reorganization of the nuclear architecture involving marginalization of host chromatin [13,14], which enables the formation of nuclear viral replication compartments where viral DNA replication and capsid assembly take place. In addition, as the virus hijacks the host machineries to preferentially produce viral transcripts, transcription and splicing factors are found adjacent to these replication compartments [15]. The nucleus of mammalian cells contains a variety of nuclear bodies that harbor various RNA-binding proteins [16,17]. Immunostaining with antibodies to splicing factors in cells under regular conditions has shown that splicing factors are diffusely dispersed in the nucleoplasm and are enriched in sub-nuclear bodies termed nuclear speckles [18–24]. These dynamic structures are located throughout the nucleus of mammalian cells and their number may be over 20, serving not only as a hub for splicing factors but also for kinases and a variety of other nuclear factors involved in mRNA biogenesis. The exact function of these nuclear bodies is not known, but spatially they can be closely associated with active genes [25–27]. RNA fluorescence in situ hybridization (FISH) performed with probes that hybridize to the polyA tail of RNAs has shown that nuclear speckles are enriched in polyadenylated RNAs [28,29], of which some are long non-coding RNAs (lncRNAs) such as MALAT1 in nuclear speckles and Neat1 in paraspeckles [30]. Nuclear speckles have been suggested to function as storage and recycling centers for splicing factors returning from splicing to be re-phosphorylated by kinases [31,32]. Recently, it was suggested that nuclear speckles may regulate the levels of splicing factors in the nucleoplasm and thereby control gene activity by modulating the availability of splicing factors in the nucleoplasm [24,33]. Pre-mRNA splicing is a vital process occurring in all higher eukaryotes that increases transcriptome diversity and complexity of the single organism. The core splicing factors are the U1, U2, U4, U5 and U6 snRNPs that interact directly with the pre-mRNA [34]. Many other auxiliary splicing factors impact spliceosome function, and some of these belong to the family of serine/arginine (SR)-rich proteins [35,36]. The SR family is comprised of 12 factors termed Serine Arginine Splicing Factors (SRSFs) that share two main features: an ‘RNA recognition motif’ (RRM) at the N-terminal of the proteins, which binds RNA, and a ‘Serine Arginine (SR) domain’ at the C-terminus, which can be phosphorylated, and promote interactions of SR proteins with RNA segments at active genes or with other proteins [37]. SR proteins facilitate the binding of the U1 and U2 snRNP spliceosome core complexes to the 30 and 50 splice sites in the pre-mRNA molecule [38,39] and contribute to the stabilization of the early spliceosomal complex. Furthermore, they take part in escorting the U4/U6 U5 snRNPs to the spliceosomal complex during the later stages of splicing [40]. SR proteins can also promote alternative splicing by binding to alternative exons [41]. Finally, since most SR proteins can bind multiple exons, they compete with each other such that their binding to the pre-mRNA can amplify or repress the inclusion of a certain exon, thus broadly affecting splicing [42]. An important SR protein is Serine Arginine Splicing Factor 2 (SRSF2) also known as SC35, which is used as a prominent marker of nuclear speckles. This splicing factor enables either constitutive splicing or alternative splicing for weak exon upon binding to regulatory sequences at the pre-mRNA, thereby determining transcripts fate [39]. Since the lytic cycle of KSHV infection is associated with modification of the global distribution of chromatin in the host nucleus, we were interested in examining the localization of RNA-binding proteins that function in gene expression pathway, in particular, during splicing and mRNA export. Furthermore, we aimed to determine the fate of sub-nuclear structures and their components during the lytic cycle of KSHV infection. Cells 2020, 9, 1958 3 of 22 2. Materials and Methods 2.1. Cell Culture and Viruses Human SLK or iSLK-PURO cells kindly provided by Prof. Don Ganem, (University of California) and Prof. Rolf Renne (University of Florida) [43] were maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (HyClone Laboratories, Logan, UT, USA), 50 IU/mL penicillin and 50 µg/mL streptomycin (Biological Industries, Kibbutz Beit Haemek, Israel). To maintain the Tet-On transactivator and K-Rta expression cassette in iSLK cells, 250 µg/mL G418 and 1 µg/mL puromycin (A.G. Scientific Incorporation, San Diego, CA, USA), respectively, were added to iSLK-PURO growth medium. Recombinant viruses BAC16 GFP (kindly provided by Prof. Jae J. Jung, University of Southern California) [44] and BAC16 GFP-mCherry-ORF45 were previously described [45]. Infected iSLK-PURO were selected with 600 µg/mL Hygromycin (Sigma-Aldrich, St. Louis, MI, USA). Lytic reactivation was induced with 50 ng/mL Doxycycline (Dox) (Sigma-Aldrich) and 1 mM n-Butyrate (Sigma-Aldrich) for 48 or 72 h. Inhibition of viral DNA replication was achieved by adding 0.5 µM Phosphonoacetic acid (PAA) (Sigma-Aldrich) to the culture 2 h before lytic reactivation and during reactivation (48 h). 2.2. Immunofluorescence 5 105 cells were grown on 18 mm coverslips (Bar Naor Ltd. Ramat-Gan, Israel) in 12-well × plates. Cells were washed with 1xPBS (Invitrogen, Waltham, MA, USA) and fixed for 15 min in 4% paraformaldehyde (PFA) (Electron Microscopy Science, Hatfield, PA, USA). Alternatively, cells were fixed with ice cold methanol (5 min) (Daejung, Shieung-si, South Korea) and acetone (2 min) (Carlo Erba Reagents, Val de Reuil, France), for U1A, SF3B1 and Prp8 staining. After fixation, cells were permeabilized in 0.5% Triton X-100 ((Sigma-Aldrich) for 2 min and blocked with 0.5% bovine serum albumin (MP Biomedical, Solon, OH, USA).