
==== Front
Virus Res
Virus Res
Virus Research
0168-1702
1872-7492
Elsevier

S0168-1702(24)00149-7
10.1016/j.virusres.2024.199456
199456
Article
The cellular paraspeckle component SFPQ associates with the viral processivity factor ORF59 during lytic replication of Kaposi's Sarcoma-associated herpesvirus (KSHV)
Payen Shannon Harger a
Andrada Kayla a
Tara Evelyn a
Petereit Juli b
Verma Subhash C. a
Rossetto Cyprian C. ccrossetto@med.unr.edu
a⁎
a University of Nevada, Reno School of Medicine, Department of Microbiology & Immunology, Reno, NV 89557, USA
b University of Nevada, Reno, Nevada Bioinformatics Center (RRID: SCR_017802), Reno, NV 89557, USA
⁎ Corresponding author. ccrossetto@med.unr.edu
07 9 2024
11 2024
07 9 2024
349 1994563 6 2024
19 8 2024
20 8 2024
© 2024 The Author(s)
2024
https://creativecommons.org/licenses/by-nc-nd/4.0/ This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Highlights

• KSHV viral proteins interact with cellular paraspeckle components SFPQ and NONO.

• The KSHV DNA polymerase processivity factor, ORF59, interacts with SFPQ and NONO within the region of amino acids 101–150.

• A small polypeptide of ORF59 spanning 101–150 aa acts as a dominant negative inhibitor between SFPQ and ORF59 during lytic reactivation resulting in a decrease of infectious virus production.

• Herpesvirus DNA polymerase processivity factors from HSV-1, EBV, and HCMV interact with SFPQ in the absence of other viral factors.

Kaposi's sarcoma-associated herpesvirus (KSHV) relies on many cellular proteins to complete replication and generate new virions. Paraspeckle nuclear bodies consisting of core ribonucleoproteins splicing factor proline/glutamine-rich (SFPQ), Non-POU domain-containing octamer-binding protein (NONO), and paraspeckle protein component 1 (PSPC1) along with the long non-coding RNA NEAT1, form a complex that has been speculated to play an important role in viral replication. Paraspeckle bodies are multifunctional and involved in various processes including gene expression, mRNA splicing, and anti-viral defenses. To better understand the role of SFPQ during KSHV replication, we performed SFPQ immunoprecipitation followed by mass spectrometry from KSHV-infected cells. Proteomic analysis showed that during lytic reactivation, SFPQ associates with viral proteins, including ORF10, ORF59, and ORF61. These results are consistent with a previously reported ORF59 proteomics assay identifying SFPQ. To test if the association between ORF59 and SFPQ is important for replication, we first identified the region of ORF59 that associates with SFPQ using a series of 50 amino acid deletion mutants of ORF59 in the KSHV BACmid system. By performing co-immunoprecipitations, we identified the region spanning amino acids 101–150 of ORF59 as the association domain with SFPQ. Using this information, we generated a dominant negative polypeptide of ORF59 encompassing amino acids 101–150, that disrupted the association between SFPQ and full-length ORF59, and decreased virus production. Interestingly, when we tested other human herpesvirus processivity factors (EBV BMRF1, HSV-1 UL42, and HCMV UL44) by transfection of each expression plasmid followed by co-immunoprecipitation, we found a conserved association with SFPQ. These are limited studies that remain to be done in the context of infection but suggest a potential association of SFPQ with processivity factors across multiple herpesviruses.

Keywords

Kaposi's sarcoma-associated herpesvirus (KSHV)
Splicing factor proline and glutamine rich (SFPQ)
Non-POU domain-containing octamer-binding protein (NONO)
Paraspeckles, DNA polymerase processivity factor
Abbreviations

KSHV Kaposi's sarcoma-associated herpesvirus

SFPQ splicing factor proline and glutamine-rich

NONO Non-POU domain-containing octamer-binding protein

EBV Epstein–Barr virus

HCMV human cytomegalovirus

HSV-1 herpes simplex virus one

KS Kaposi's sarcoma

MCD multicentric Castleman's disease

PEL primary effusion lymphoma

DBHS drosophila behavior/ human splicing

lncRNA long non-coding rna

PSPC1 paraspeckle component 1

EMCV encephalomyocarditis virus

HDV hepatitis delta virus

HIV human immunodeficiency virus

EBER2 EBV-encoded RNA 2

Dox doxycycline

TPA phorbol 12-tetradecanoate 13-acetate

NaB sodium butyrate

HA hemagglutinin

TRAM1 translocation-associating membrane protein 1

EIF4E eukaryotic translation initiation factor 4E

CYFIP1 cytoplasmic FMR1 interacting protein 1

ANTXR1 anthrax toxin receptor 1

ATRX ATP-dependent helicase X-linked helicase II

SSBP1 single-stranded DNA binding protein 1

APOL2 apolipoprotein L2

PARP1 poly [ADP-ribose] polymerase 1

Rae1 ribonucleic acid export 1

dNTP deoxynucleoside triphosphate

BAC bacterial artificial chromosome

GFP Green fluorescent protein

RFP red fluorescent protein
==== Body
pmc Summary

During lytic replication, KSHV relies on both viral and host-encoded proteins to successfully produce new virions. The requirement and function of many cellular proteins used during KSHV replication remain unclear. In this study, we focus on the cellular paraspeckle component SFPQ, which has been speculated to play a role in virus replication. Using SFPQ immunoprecipitations and proteomics analysis, we identified ORF10, ORF59, and ORF61 as having increased protein abundance associating with SFPQ during KSHV lytic reactivation.. We further tested ORF59 and found the amino acids 101–150 of ORF59 interact with SFPQ. With this information, we generated a dominant negative polypeptide of ORF59 encompassing amino acids 101–150 that effectively decreased infectious virus production when introduced into cells before KSHV reactivation. Interestingly, we found SFPQ was able to be immunoprecipitated with other human herpesvirus processivity factors, including EBV BMRF1, HSV-1 UL42, and HCMV UL44, in transfection experiments.

Alt-text: Unlabelled box

1 Introduction

Human herpesviruses in the family Herpesviridae contain large double-stranded DNA genomes and cause lifelong infections in the host. There are eight recognized human herpesviruses classified into three groups; α herpesviruses include herpes simplex virus types 1 and 2 (HSV-1, HSV-2), and varicella-zoster virus (VZV); β herpesviruses include human cytomegalovirus (HCMV), and human herpesviruses 6 and 7 (HHV-7, HHV-7); and γ herpesviruses include Epstein-Barr virus (EBV) and Kaposi's sarcoma-associated herpesvirus (KSHV). KSHV is an oncogenic virus and the causative agent for Kaposi's sarcoma (KS), Multicentric Castleman's disease (MCD), and Primary Effusion Lymphoma (PEL) (Verma and Robertson, 2003; Ganem, 2006). After initial infection, KSHV remains latent as a circular episome within the host cell nucleus. Reactivation is initiated by the activation of the ORF50 promoter, leading to the expression of the replication and transcription activator (K-RTA), the primary regulator of lytic replication (Rossetto et al., 2011; Sun et al., 1998). Early lytic genes encode viral proteins essential for DNA replication or viral gene expression. In contrast, late lytic genes encode viral structural proteins, including envelope and capsid proteins, crucial for the assembly of virions (Mesri et al., 2010; Chen et al., 2001; Lukac et al., 1998).

Paraspeckles are small, irregular nuclear bodies found within the interchromatin space. Paraspeckle components from the Drosophila behavior/ human splicing (DBHS) protein family include Non-POU domain-containing octamer-binding protein (NONO), splicing factor proline/glutamine-rich (SFPQ) and paraspeckle protein component 1 (PSPC1). DBHS proteins are a family of predominantly nuclear proteins involved in gene regulation, carcinogenesis, and paraspeckle formation (Passon et al., 2012; Knott et al., 2016). Members of DBHS proteins are known to interact with double- and single-stranded DNA and RNA (Fox and Lamond, 2010). NONO acts as a pre-mRNA splicing factor as a heterodimer with SFPQ (Knott et al., 2016). Both NONO and SFPQ are ubiquitous in all tissues, excluding lymphocytes and retina cells in which unique forms of NONO are expressed (Yang et al., 1993). Only DBHS dimers containing SFPQ exhibit DNA binding activity, which is attributed to the unique sequences in the N-terminal intrinsically disordered regions (Ha et al., 2011; Song et al., 2005). Additionally, DBHS proteins interact with long non-coding RNA NEAT1 during paraspeckle formation in the nucleus (Passon et al., 2012; Sasaki et al., 2009; Clemson et al., 2009).

The role of paraspeckles includes regulating RNA through the retention of mRNAs, recruitment of RNA-binding proteins, and microRNA formation regulation (Fox et al., 2023; Yasuhara et al., 2022). Additionally, previous research has shown that the interaction between SFPQ and NEAT1 lncRNA is essential for regulating the host's innate immune response to viruses (Ahmed and Liu, 2018). SFPQ has been found to perform various roles in the facilitating replication of RNA viruses such as encephalomyocarditis virus (EMCV), hepatitis delta virus (HDV), influenza A, human immunodeficiency virus (HIV), Sindbis virus, and SARS-CoV-2 viruses (Zhou et al., 2019; Greco-Stewart et al., 2006; Landeras-Bueno et al., 2011; Zolotukhin et al., 2003; Labeau et al., 2022; Girardi et al., 2023). Studies have suggested that SFPQ and other paraspeckle components may be involved in viral replication for DNA viruses. One study using HSV-1 found paraspeckles containing NONO and PSPC1 localized to the HSV-1 genome and were involved in regulating viral gene expression (Wang et al., 2017). For EBV, oriP transcripts were enriched in NONO-containing paraspeckle complexes during reactivation and suggests that this interaction facilitates lytic gene expression (Cao et al., 2015). Additionally, EBV-encoded RNA 2 (EBER2) was demonstrated to interact with SFPQ in an RNA-protein crosslinking experiment (Lee et al., 2016). A recent study using a CRISPR/Cas9 knockout of SFPQ demonstrated that SFPQ is involved in regulating EBV latency (Murray-Nerger et al., 2024). The underlying mechanism and full extent of the reliance on paraspeckle components to facilitate replication for HSV-1 and EBV is still unknown. Interestingly, in a previously published immunoprecipitation proteomics study of the KSHV protein, ORF59 performed 48 h after viral reactivation, SFFQ and NONO were identified as binding partners of ORF59 (Strahan et al., 2017). ORF59 is the virally encoded DNA polymerase processivity factor, an early lytic protein, and interacts with the viral DNA polymerase, ORF9 (Pol-8), during DNA replication (Ruby Chan and Chandran, 2000; Chan and Chandran, 2000; Chen et al., 2005). Because of its diverse cellular and viral binding partners, ORF59 is speculated to have a role in viral replication beyond the classical processivity factor function (Ruby Chan and Chandran, 2000). The requirement and function of the association between ORF59 and SFPQ/NONO during KSHV replication have not previously been explored.

To better understand the role of SFPQ in KSHV replication, we performed SFPQ immunoprecipitation followed by mass spectrometry to identify proteins interacting with SFPQ during lytic replication. We identified several viral proteins with significantly higher protein abundance in SFPQ-IP-induced cell proteomics compared to latent cells. The KSHV viral proteins ORF10, ORF61 and ORF59 were detected with high confidence and further analyzed. We confirmed the associations through co-immunoprecipitation and visualized the localization of SFPQ with ORF10, ORF61, and ORF59 with immunofluorescent assays (IFA). We further identified the specific domains within ORF59 that interact with SFPQ. The 101–150 amino acid domain of ORF59 was found to associate with SFPQ and NONO, and immunofluorescent analysis of ORF59 Δ101–150 showed aberrant size and aggregates of SFPQ and NONO-containing paraspeckles. Using this information, we generated a plasmid that expressed the 101–150 polypeptide of ORF59 to test as a dominant negative inhibitor. The addition of the 101–150 polypeptide before lytic reactivation of WT KSHV in iSLK cells resulted in a decrease in infectious virion production. Additionally, we tested if SFPQ associates with processivity factors of other human herpesviruses including EBV BMRF1, HSV-1 UL42, and HCMV UL44. Interestingly, in co-immunoprecipitation and immunofluorescent localization experiments, we found that other herpesvirus processivity factors associate, with SFPQ and have areas of overlap within the nucleus. Although we have not explored this association in the context of infection, the limited results with exogenous expression suggest there may be a conserved function and requirement for SFPQ, and paraspeckle bodies, during lytic replication of herpesviruses.

2 Materials and methods

2.1 Cells and viruses

Human HEK293L (RRID: CVCL_M775) and HEK293FT (RRID: CVCL_6911) were cultured and maintained in antibiotic-free Dulbecco's modified Eagle medium (DMEM) (Corning, cat. # 45,000–304) supplemented with 10 % fetal bovine serum (Corning, Ref. # 35–010-CV). Human 293 L cells were obtained from the NIH AIDS reagent program. iSLK cells were maintained in DMEM with 10 % fetal bovine serum, 1 μg/ml of puromycin, and 250 μg/ml of G418 (InvivoGen). iSLK cells containing the KSHV bacterial artificial chromosome 16 (BAC16) were maintained in identical conditions with the addition of 1.2 ug/mL hygromycin B (US Biologicals). KSHV BAC16 used in these studies include WT with an in-frame HA tag ORF59, ORF59Δ51–100, ORF59Δ101–150, ORF59Δ251–300, and ORF59Δ351–396 have previously been described (Gutierrez et al., 2021a). To induce lytic reactivation in the described cell lines, 0.25 mM sodium butyrate (NaB) (Sigma-Aldrich), 1 μg/ml doxycycline (U.S. Biologicals), and 10 ng/ml of phorbol 12-myristate 13-acetate (TPA) (Sigma-Aldrich) were administered at 24 h post-plating. Cell lines were grown and maintained at 37 °C in a humidified incubator supplemented with 5 % CO2.

2.2 Plasmids

The pCMV-ORF10 Flag expression plasmid was previously described and kindly provided by Dr. Ting-Ting Wu (University of California, Los Angeles) (Gong et al., 2016). To generate the ORF61-HA expression plasmid, the phCMV-XI plasmid (pXI) vector was linearized to insert the ORF61-HA gBlock DNA (IDT). The insert was cloned into the pXI vector using GeneArt Seamless Cloning and Assembly (Thermo Fisher Scientific) and transformed into One Shot TOP10 chemically competent E. coli (cat. C404010) following the manufacturer's protocol. Plasmids were subjected to restriction digest and sanger sequencing to verify the correct insert. The ORF61-HA sequence is as follows with the HA tag in bold and the underline region homologous to the vector for insertion:

cgagctcaagcttcgaattcATGTCTGTCCGGACATTTTGTCAGGTTCACTTGGGAGCTGTCCCACCAGAGCGACGCGGGTCTCAGGTGTCTGCGGGACAATTGTCGGATTTTGATATGTGTGCCCAATCTCTCATAGACTTTCTAAAGGTTAGGGTCGGGTGGGACGTGCGGGCCAATGCCATGGCGGGGCGGCTGTGGCACCAAATTATGGAGGCTAGGTGCCCAGCGACTCTCAAGCAGTATCTTGGGATTTTTCGAGGCGTGTTGGGACATCGGGTGGAATCGTTTATTCAAAAGAACATTGATGCCTTGGAAGATATGTTGTGCGCCTACAGACGGTCCAAAGCTTACGAGGACACACTTAACTGTGGCTACCTATCGGCCGTGAGGTTGTATGATACTTACGTCCTAAGGACCATGGGGACAGAGCCTGTCTATGAAAGCGTGGCGCAAATGTTTATGAGGGTTTCCGTGTTTGTTGCCTGTCAGTGCTTAGAGCATGAATGCCTGTATTGGTTGGCGCGGGACCTTATTGAGGACGCTAAGTCTGTATCTGAGATGGCTATAGTGGAGTACGTTTTTGGCTACCTCGCTGCGCAGCACGTGTGCTGCGCCACGCCAATACTGCGCTCAGCTGGAGTCGAAGGCGGACAACTCGCCAGCTGTTTTATTCTGCAGCCCTCCATGATGAATGAGTCGGGTACTTTGGACGCCCTGTACCACGATATGAGTCCGTTGCTGGCCAGTAAGTCAGGCGTGGGTCTGGATGTTACGTCATTCTCCCACCAAAAGAACATAGCCAGTTGCCTCAAACTGGTGGATGCCCAGGTTCATTATTTCAATGACAATAACATCAGGCCCGTTGGCGCGAGCGCATACATGGAACTCTGGCACAGTCAAATCTGTGATTTTTTGAACGCCAAACTACCGGAGAATCCCGACAGATGTCACAGTCTCTTTCAGGGCGTGTGCATCCCAACATTGTTTTTCAGGATGTATGAAAAGGACCCATCCAAGTTATGGTATTTGTTCGATCCAGCCACCGCCCCCAATTTGATTAAGCTATACGGTGCTGCGTTCGATAACGAATACGAGCGCCTGGTGCGGGCTGGAAAGTACGTTAGTTGCATGCCGTTGAAGTCTATGATGTTCACTCTTATCCACACCATCATAAAGACAGGCTCACCGTACGTGCTTTTAAAAGAAGCGCTCAATGAACACCACTGGACCGACACTCAGGGCATGGCCATTAACTGCTCAAACTTGTGTGCAGAGATAGTCCAACTGCCAGGAAGAAACACTTCGGTGTGCAACCTGGCAAATATCTGCCTTCCAAAGTGTCTGAGAACTGTTGAAAGCGCGAGGGTTGGCACCACGGACGCCAACAGGCCGTTTTTTTGTTTTGAGGCCTTGGGCGACGCAGTAAGGGTGGCAGTCCTCGTCATCAACGCCTGCATTCTGGGTGGTTCCCATCCAACACCGGGGGTCGAGCGAGGTCAAAAGGAGAGGTCTATGGGGATTGGTGTTCAGGGGTTGGCGGATGTGTTTGCGGAATTGGGGTACGGGTACCTCGATGCTGAGAGCGCTGAGTTAGACAAGAATATATTCCAGTCTATGTACTACACCGCGGTGGAGACTAGTCACAACCTTGTTTTGGAGGGACAGGGTGTCCCATTTCATGGCTGGGAGGTCAGTAACTTCGCCAAGGGCAGGTTTCATTGGCAAACATGGGAGGGGGAGGATGCTTCGTTTGTACCACGCCATCGCTGGGACGCGTTGGGAAAGAGCATTGCCGAGCATGGCATATTCAATTCTCAGTTTCTGGCGGTGATGCCGACGGCTGGGACCTCGCAAGTAACTGGCTATGCCGAGTCTGTGTACCCCTTTTTTGCTAATATATCTTCCAAGGTCACCAATAAAGAAGAGGTGTTGAGGCCCAATGTGACTTTTTTTAAGAAGGTTCTCCCCGACGACCTTCGGGTGGTTAGACAGTATGGTGGGGACGTTTCCACGTTCCCCAAGCATCACAGGGAAAGATATAGGGTATTCCTAACAGCCTTTGATTACTGTCCATTTAAGCTACTGGACAGGGCTCGTGCCAGGGCGCCGTTTGTAGACCAGAGCCAGTCCATGAGCTTCTTCCTCAAGGAGGACAGAGTCAGGAATGCGAGCTATCTTAGGGACCTGCTGCTACATGGATACAGGCTTGGTCTAAAAACCCTCATGTACTACTGTAGGGTTCAGAAGCAATCTAGTCTGACGGCTCTTCAGTGCCTGGCAGATCCAGGGTCACCGCCTCACTCTGGGATGAAACAAGATGGGGCGTGGTTACCCGGACCTAAAAACCCGGAGGAGGAGTCCTGCGCGGCAGACCCCGAGTGCCTGGTCTGTCAGTACCCATACGATGTTCCAGATTACGCTTAGggctaagaattctgcagtcgacggta

The ORF59 101–150DsRed expression plasmid was cloned as described above. The pXI vector was linearized using EcoRI restriction enzyme (New England Biolabs #R0101M), and the ORF59 101–150 sequence was obtained as gBlock (IDT) containing an in-frame DsRed tag. The 101–150DsRed sequence is as follows with the DsRed tag in bold and the underline region homologous to the vector for insertion:

gcttcgaattccaccATGAAAACCGGAGTAGTGCAAGTGCACGGATCGGCTTGCACGCCAACCCTCAGTGTGCTGTCCAGCGTGGGGACAGCTGGCGTTCTGGGGTTAAGAATAAAGAATGCCCTTACGCCCCTGGTGGGACACACGGAAGGCAGTGGAGACGTTAGCTTCAGCTTCAGGAATACGTCCGTCGGTAGCGGCTTCACGCACACGCGTGAGCTATTCGGTATGGTGCGCTCCTCCAAGAACGTCATCAAGGAGTTCATGCGCTTCAAGGTGCGCATGGAGGGCACCGTGAACGGCCACGAGTTCGAGATCGAGGGCGAGGGCGAGGGCCGCCCCTACGAGGGCCACAACACCGTGAAGCTGAAGGTGACCAAGGGCGGCCCCCTGCCCTTCGCCTGGGACATCCTGTCCCCCCAGTTCCAGTACGGCTCCAAGGTGTACGTGAAGCACCCCGCCGACATCCCCGACTACAAGAAGCTGTCCTTCCCCGAGGGCTTCAAGTGGGAGCGCGTGATGAACTTCGAGGACGGCGGCGTGGTGACCGTGACCCAAGACTCCTCCCTGCAGGACGGCTGCTTCATCTACAAGGTGAAGTTCATCGGCGTGAACTTCCCCTCCGACGGCCCCGTAATGCAGAAGAAGACCATGGGCTGGGAGGCCTCCACCGAGCGCCTGTACCCCCGCGACGGCGTGCTGAAGGGCGAGATCCACAAGGCCCTGAAGCTGAAGGACGGCGGCCACTACCTGGTGGAGTTCAAGTCCATCTACATGGCCAAGAAGCCCGTGCAGCTGCCCGGCTACTACTACGTGGACTCCAAGCTGGACATCACCTCCCACAACGAGGACTACACCATCGTGGAGCAGTACGAGCGCACCGAGGGCCGCCACCACCTGTTCCTGtagggctaagaattctgcagtcgacggta. The pDsRed-N1 (Clontech) was used as a control plasmid for DsRed expression alone.

Expression plasmids encoding HA-tagged ORF59 and UL44 have previously described, pXI ORF59-HA and pTarget UL44-HA (Kagele et al., 2012; McDowell et al., 2013). In this study, we generated pSI BMRF1-HA and pSI UL42-HA using the GeneArt Seamless Cloning and Assembly as described above. The BMRF1-HA insert was purchased as a DNA gBlock (IDT), lowercase indicates sequence homologous to the pSI vector and the HA tag is in bold:

tacttaatacgactcactataggctagcctcgagaattcagccaccATGGAAACCACTCAGACTCTCCGCTTTAAGACCAAGGCCCTAGCCGTCCTGTCCAAGTGCTATGACCATGCCCAGACTCATCTCAAGGGAGGAGTGCTGCAGGTAAACCTTCTGTCTGTAAACTATGGAGGCCCCCGGCTGGCCGCCGTGGCCAACGCAGGCACGGCCGGGCTAATCAGCTTCGAGGTCTCCCCTGACGCTGTGGCCGAGTGGCAGAATCACCAGAGCCCAGAGGAGGCCCCGGCCGCCGTGTCATTTAGAAACCTTGCCTACGGGCGCACCTGTGTCCTGGGCAAGGAGCTGTTTGGCTCGGCTGTGGAGCAGGCTTCCCTGCAATTTTACAAGCGGCCACAAGGGGGTTCCCGGCCTGAATTTGTTAAGCTCACTATGGAATATGATGATAAGGTGTCCAAGAGCCACCACACCTGCGCCCTGATGCCCTATATGCCCCCGGCCAGCGACAGGCTGAGGAACGAGCAGATGATTGGGCAGGTGCTGTTGATGCCCAAGACGGCTTCCTCGTTGCAGAAGTGGGCACGCCAGCAAGGCTCAGGCGGCGTTAAGGTGACACTCAATCCGGATCTCTACGTCACCACGTATACTTCTGGGGAGGCCTGCCTCACCCTAGACTACAAGCCTCTGAGTGTGGGGCCATACGAGGCCTTCACTGGCCCTGTGGCCAAGGCTCAGGACGTGGGGGCCGTTGAGGCCCACGTTGTCTGCTCGGTAGCAGCGGACTCGCTGGCGGCGGCGCTTAGCCTCTGCCGCATTCCGGCCGTTAGCGTGCCAATCTTGAGGTTTTACAGGTCTGGCATCATAGCTGTGGTGGCCGGCCTGCTGACGTCAGCGGGGGACCTGCCGTTGGATCTTAGTGTTATTTTATTTAACCACGCCTCCGAAGAGGCGGCCGCCAGTACGGCCTCTGAGCCAGAAGATAAAAGTCCCCGGGTGCAACCACTGGGCACAGGACTCCAACAACGCCCCAGACATACGGTCAGTCCATCTCCTTCACCTCCGCCACCTCCTAGGACCCCTACTTGGGAGAGTCCGGCAAGGCCAGAGACACCCTCGCCTGCCATTCCCAGCCACTCCAGCAACACCGCACTGGAGAGGCCTCTGGCTGTTCAGCTCGCGAGGAAAAGGACATCGTCGGAGGCCAGGCAGAAGCAGAAGCACCCCAAGAAAGTGAAGCAGGCCTTTAACCCCCTCATTTACCCATACGATGTTCCAGATTACGCTTAGTAaagtcgacccgggcggccgcttcgagcagacatgataagat.

For the UL42-HA insert, primers were used to amplify the UL42 DNA sequence from HSV-1 DNA (KOS). The primers used are the following, lowercase indicates sequence homologous to pSI vector and HA tag is in bold. FWD: 5′-tacttaatacgactcactataggctagcctcgagaattcagccaccATGACGGATTCCCCTGGCGGT-3′

REV: 5′-TATGGTTTTGGATTCCCCTACCCATACGATGTTCCAGATTACGCTTAGTGaagtcgacccgggcggccgcttcgagcagacatgataagat-3′. Plasmids were subjected to restriction digest and sanger sequencing to confirm the correct insert.

2.3 Western blotting and co-immunoprecipitation (Co-IP)

iSLK KSHV cells were plated in 10 cm dishes at 6  ×  105 seed density. Cells were induced with 0.25 mM NaB, 0.5 ug/ml DOX, and 10 ng/ml TPA, for 48 h. Total protein lysate was prepared following three washes with ice-cold PBS. A total of 1 mL IP lysis buffer (Pierce, Thermo Fisher) with protease inhibitors (1:100) (catalog number P8340; Sigma-Aldrich) were added to each dish and lysed for 10 min on ice, rocking. Lysates were then sonicated using four 10-s pulses with a probe disruptor (Misonix 200; Thermo Fisher) and centrifuged (13,000xg for 10 min at 4 °C). For input controls, 100 µl was combined with 100 µl of 2X Laemmli buffer (Bio-Rad cat # 161–0737) with β-mercaptoethanol and boiled for 5 min at 95 °C. Inputs were stored at −20 °C until it was run on an SDS-PAGE gel with the immunoprecipitation samples.

For the co-IP samples, lysates were rotated with either NONO, SFPQ, or HA primary antibody overnight at 4 °C. Additionally, samples containing either mouse or rabbit IgG antibody isotypes were included as controls. Following incubation, Dynabeads™ Protein G for Immunoprecipitation (Catalog number: 10003D) was added (1:10) and rotated at room temperature for 2 h. Immunoprecipitants were collected by centrifuging at 6000 x g for 1 min at 4 °C. The supernatant was aspirated and discarded without disturbing the beads. The beads were washed three times for 5 min with IP lysis buffer. After the final wash, the supernatant was aspirated, discarded, and beads resuspended with 100 µl of 2X Laemmli buffer with β-mercaptoethanol and boiled for 5 min at 95 °C.

Western blots were performed as previously described (Gutierrez et al., 2021b). Briefly, a total of 35 µl of protein was resolved by SDS-PAGE gel and transferred to a polyvinylidene difluoride (PVDF) membrane (Bio-Rad). Blots were blocked for 15 min with 5 % skim milk in Tris-buffered saline with 0.1 % Tween 20 (TBST) (10 mM Tris–HCl [pH 7.5], 150 mM NaCl, 0.05 % Tween 20) and then incubated with the following specific antibodies: rabbit anti-HA at 1:2000 (H6908; Sigma-Aldrich), mouse anti-NONO at 1:1000 (SC-135746; Santa Cruz Biotechnology), rabbit anti-SFPQ at 1:1000 (ab177149; abcam ), and mouse or rabbit IgG isotype control at 1:2000. All primary antibodies were incubated at 4 °C overnight, followed by washing with TBST and incubation with either IR-dye 680 and 800 anti-mouse or anti-rabbit (Li-Cor) secondary antibody for 30 minutes, and proteins were detected using a ChemiDoc MP imaging system (Bio-Rad).

2.4 Immunofluorescence assay (IFA)

In a 12-well dish with glass coverslips, cells of iSLK BAC16–59HA WT, and Δ101–150 ORF59 deletion mutant were seeded at a density of 60,000 cells per well. Cells were either left uninduced or induced for 6, 24, or 48 h. A similar setup was used for the 293FT cells transfected with the processivity factors (ORF59-HA, BMRF1-HA, UL42-HA, and UL44-HA) Following induction or transfected, samples were fixed using 4 % paraformaldehyde in PBS for 30 min and subjected to three washes before permeabilization with 0.5 % Triton for 10 min. Samples were blocked with 3 % goat serum for 30 min at room temperature, rocking. The primary antibodies (2.5 µg/ml HA-Rabbit, 200 µL/well,), (2.5 µg/ml SFPQ-Rabbit, 200 µl/well), and/or (2.5 µg/ml NONO-Mouse, 200 µl/well) was incubated for 2 h, rocking. The primary antibody was removed, and samples were washed three times with 1 % BSA. The secondary antibody (1:500, Alexa Fluor Rabbit 405, Alexa Fluor Rabbit/Mouse 594, and/or Alexa Fluor Rabbit/Mouse 488 200 uL/well) was incubated for 30 min at room temperature, rocking. The secondary antibody was removed, and samples were washed three times with 1 % BSA. Coverslips were mounted onto glass slides with Prolong Gold Antifade with DAPI or without DAPI (Thermo Fisher) as indicated. Samples were imaged using Zeiss AxioVision fluorescent microscope. The fluorescence filters used to capture the images were 405 nm (Blue), 488 nm (Green), and/or 594 nm (Red).

2.5 Viral DNA quantification using qPCR

Viral DNA was quantified as previously described (Gutierrez et al., 2021a,b). Briefly, iSLK BAC16–59HA WT cells were seeded at a density of 2.5  ×  105 cells per well. Samples comprising WT, WT transfected with 101–150DsRed (5ug), and WT transfected with DsRed (5ug) were induced with 0.25 mM NaB, 0.5 ug/ml DOX, and 10 ng/ml TPA for 72 h. A non-induced set was included as a control. DNA extraction was carried out by phenol-chloroform extraction. Subsequently, DNA was subjected to qPCR analysis using SsoAdvanced Universal Probes Supermix (Bio-Rad) and primer/probes from IDT according to the manufacturer's instructions. KSHV-specific primers were used to quantify viral DNA synthesis and normalized to cellular DNA and each qPCR assay was conducted in biological triplicates (Gutierrez et al., 2021a,b).

2.6 Reverse transcription (RT)-qPCR

For quantitating NEAT1 transcripts, iSLK BAC16 ORF59-HA WT, and 59Δ101–150 cells were induced with 0.25 mM NaB, 1 μg/ml doxycycline and 10 ng/ml TPA. Total RNA was and harvested at 0, 24, 48, and 72 hpi using a PureLink RNA minikit (Thermo Fisher, Invitrogen). DNA was removed from the samples using a Turbo DNA-free kit (Thermo Fisher, Invitrogen). cDNA was generated using iScript cDNA synthesis kit (Bio-Rad) and amplified on a T100 thermal cycler (Bio-Rad) and qPCR was performed using SsoAdvanced universal probe supermix (Bio-Rad) according to the manufacturer's instructions. Specific primers used for cellular NEAT1 and GAPDH were purchased from IDT predesigned assays NEAT1 (Hs.PT.58.26695822.g) and GAPDH (Hs.PT.39a.22214836). qPCR was conducted from three biological replicates. The ΔΔCt method was used to calculate the fold change in NEAT1, samples were normalized using GAPDH and compared to 0 hpi.

2.7 Infectious virus assay

The viral inoculum was obtained as previously described (Gutierrez et al., 2021a,b). Briefly, iSLK BAC16 ORF59-HA WT cells were seeded at a density of 5  ×  106 cells. The WT cells and those transfected with either 101–150DsRed or DsRed-N1 alone were induced using 0.25 mM NaB, 0.5 ug/ml DOX, and 10 ng/ml of TPA. After 5 days of induction, cell supernatant was harvested, frozen at −80 °C, and subjected to three thawing cycles at 37 °C. The clarified media was obtained by three consecutive spins of 10 min at 4000 x g. Virus was pelleted by centrifugation at 28,000 x g for 90 min in an SW28 rotor. The virus pellet was reconstituted in 0.5 ml of antibiotic-free medium and serially diluted. Subsequently, 293 L cells plated at 2.5  ×  105 were infected, and GFP foci were counted at 72 h post-infection.

2.8 SFPQ and ORF59 three-dimensional modeling

ChimeraX using ColabFold v1.5.5 along with AlphaFold was used for prediction modeling of the protein docking between ORF59 dimer (PDB ID 3HSL) with SFPQ dimer (PDB ID 4WIJ). The disordered portion of the proteins were not used in the modeling because lack of known structure from crystallography. Protein docking and folding predictions were performed as previously described (Jumper et al., 2021; Mirdita et al., 2022).

2.9 Proteomics

For protein digestion, cut gel bands were washed twice with 25 mM ammonium bicarbonate and acetonitrile, reduced with 10 mM dithiothreitol, alkylated with 100 mM iodoacetamide, and digested with a trypsin/Lys-C protease mixture (Promega #V5071) following the provided protocol. Samples were cleaned up for analysis using C18 tips (Glygen #TT2C18). Samples were reconstituted in 0.1 % formic acid in water for analysis. For liquid Chromatography - Samples were analyzed using an UltiMate 3000 RSLCnano system (Thermo Scientific, San Jose, CA). The peptides were trapped before separation on a 300 um i.d. x 5 mm C18 PepMap 100 trap (Thermo Scientific, San Jose, CA) for 5 min at 10 ul/min. Separation was performed on a 50 cm uPAC C18 nano-LC column (PharmaFluidics, Ghent, Belgium) on an EasySpray source (Thermo Scientific, San Jose, CA) fitted with a 30 um ID stainless steel emitter (PepSep, Marslev, Denmark). Separation was performed at 350 nl/min using a gradient from 1 % - 45 % for 60 min (Solvent A 0.1 % Formic Acid, Solvent B Acetonitrile, 0.1 % Formic Acid). Mass spectrometry – We performed Data Independent Analysis (DIA) using an Eclipse Tribrid Orbitrap mass spectrometer (Thermo Scientific, San Jose, CA) (1,2). A chromatographic library was generated using the Spectronaut (v 16.0, Biognosys, Schlieren, Switzerland) integrated database search engine Pulsar to generate a hybrid using DIA and DDA spectra using an aliquot from each sample to form a pool of peptides. The library was generated using a combination of six gas phase fractions (GPF) and full scan data-dependent analysis of the biological sample pool. The GPF acquisition used 4 m/z precursor isolation windows in a staggered pattern (GPF1 398.4–502.5 m/z, GPF2 498.5–602.5 m/z, GPF3 598.5–702.6 m/z, GPF4 698.6–802.6 m/z, GPF5 798.6–902.7 m/z, GPF6 898.7–1002.7 m/z) at a resolution of 60,000, AGC target was set to custom with a normalized target of 1000 %, maximum injection time was set to dynamic with a minimum of nine points across the peak, and an NCE of 33 using higher-energy collision dissociation (HCD). Three DDA full scan runs were performed on the biological pool. The MS1 precursor selection range is from 375 to 1500 m/z at a resolution of 120 K with a normalized automatic gain control (AGC) target of 250 % and an automatic maximum injection time. Quadrupole isolation of 0.7 Th for MS2 isolation and CID fragmentation in the linear ion trap with a collision energy of 35 % and a 10 ms activation time. The MS2 AGC was in standard mode with a 35 ms maximum injection time. The instrument was operated in a data-dependent mode with a 3-second cycle time and the most intense precursor priority, and the dynamic exclusion was set to an exclusion duration of 60 with a 10 ppm tolerance. Biological samples were run on an identical gradient as the GPFs using a staggered window scheme of 8 m/z over a mass range of 385–1015 m/z. Precursor isolation was performed in the Orbitrap at 60,000 resolutions with a dynamic maximum injection allowing for a minimum of nine points across the peak, a custom AGC normalized to 1000 %, and an NCE of 33 using HCD.

A custom library containing species-specific Uniprot protein FASTA databases for Homo sapiens and Kaposi's sarcoma-associated herpesvirus, as well as known contaminants (Crap_uniprot_with_human_MRSonbeadV2) was used in the Spectronaut analysis. Variable modifications considered were: Carbamidomethylation C. Identification cutoffs for precursor and protein Qvalue cutoffs were set to 0.01 and quantity was based on the area of MS2 ions. Initial data investigation was performed using Spectronaut. Unscaled, not-imputed intensity values by protein group were exported from Spectronaut and data were further analyzed within the statistical software R v4.3.1. To retain only quality-controlled protein groups for the analysis, we employed three filters: 1) proteins that did not map to Homo sapiens or Human herpesvirus were removed; 2) keratin proteins (e.g., KRT ) and VIM and ACTB were removed as they were considered possible contaminates; 3) proteins not well-quantified within at least one of the SFPQ experimental groups were also removed. The resulting dataset was again assessed for quality used for downstream analysis. SFPQ samples were then scaled and transformed using multiple standard protocols, and equal median normalization from the Bioconductor package DEqMS v1.18.0 outperformed the other transformation methods (e.g., variance stabilization, cyclic loess, or log2 quantile normalization). Equal median normalized data were further investigated for quality, e.g., Principal Component Analysis (PCA). Proteins with significant abundances differences in the lytic reactivated samples relative to the non-induced control were identified by performing empirical Bayes moderated t-test using Bioconductor package limma v3.56.2. Limma uses moderated t-statistic and empirical Bayes methods for assessing differential expression and has been described as “hierarchical” method since it runs a traditional F-test followed by t-tests approach (F-then-t). DEqMS takes into account the dependence of variance on the number of peptides used for protein quantification Proteins were deemed to be statistically significantly differentially abundant if the p-value was <0.01 and the absolute value of log2 Fold Change was greater than 1. Differentially abundant proteins were visualized with heatmaps, volcano plots, and box plots, with an emphasis on the viral proteins.

2.10 Statistical analysis

Unless indicated, statistical analyses were completed using one-way ANOVA with Tukey's multiple comparison tests or two-way ANOVA with Bonferroni's multiple comparison test. All non-proteomic statistical analyses were completed using GraphPad Prism software.

3 Results

3.1 SFPQ proteomics from KSHV-infected iSLK cells

SFPQ is a versatile nuclear protein with various functions including paraspeckle formation, DNA damage repair, and transcriptional regulation (Lim et al., 2020). Prior proteomics of ORF59 immunoprecipitation (IP) from KSHV-infected cells identified SFPQ and NONO interacting with ORF5929. To better understand the role of SFPQ during KSHV replication, we first sought to identify viral and cellular proteins interacting with SFPQ. We performed IPs using anti-SFPQ antibodies incubated with iSLK cell lysate from latent and reactivated cells, followed by mass spectrometry of the SFPQ-enriched sample (Fig. 1A). iSLK cells containing WT KSHV were induced to undergo lytic reactivation by treatment with doxycycline (Dox), Phorbol 12-tetradecanoate 13-acetate (TPA), and sodium butyrate (NaB) for 48 h, and non-induced latent cells were used as a control. In addition to samples incubated with the anti-SFPQ antibody, a set incubated with an isotype anti-IgG antibody was included as a control. Samples were split and used for western blots (Fig. 1B) to confirm the presence of SFPQ and NONO in the SFPQ-IP and processed for mass spectrometry. Given the well-documented interaction between SFPQ and NONO in various cell types, we used the presence of NONO in the SFPQ-IP as a positive control. The four biological replicates of each SFPQ-IP and control were submitted for LC/MS and protein identification at the Mitch Hitchcock Nevada Proteomics Center, University of Nevada, Reno according to their standard protocol. Samples were processed for data-independent acquisition (DIA) analysis and underwent multiple quality assessments at the Nevada Bioinformatics Center. A total of 2864 unique proteins were identified in at least one experimental sample, after quality control filters the resulting dataset was composed of 1316 proteins (Supplemental Table 1).Fig. 1 SFPQ proteomics from KSHV infected cells.

(A) Schematic of experimental design for mass spectrometry analysis. iSLK WT cells containing latent WT KSHV BAC16 were plated for control (non-induced) and induced (Dox, TPA, NaB). At 48 h post-induction, cell lysate was harvested, and proteins were immunoprecipitated using anti-SFPQ or anti-IgG (control) antibodies and protein G magnetic beads. The input and IP samples were mixed with 2X Laemmli buffer with β-mercaptoethanol, boiled, and divided for western blots and LC-MS/MS proteomics. (B) Input and IP samples were resolved by SDS-PAGE and western blots were incubated with anti-SFPQ and anti-NONO antibodies. The western blot is representative of the four biological replicates sent for proteomics analysis.

Fig. 1

3.2 Proteomic analysis of SFPQ-IP in KSHV infected cells

The enrichment of cellular and viral proteins interacting with SFPQ was determined by comparing the proteomics data from the latent with induced lytic iSLK cells. Proteomics and bioinformatics for this project were conducted using three sample groups: SFPQ-IP from the non-induced lysate, SFPQ-IP from the induced lysate, and isotype control IgG-IP from the induced lysate. Each sample group contained four biological replicates. Fig. 2A and B highlight the reproducibility of experimental samples; although one sample (SFPQ-IP non-induced 3, labeled as SFPQ_NI.3) demonstrates higher variability, overall, the data cluster within their experimental groups. The IgG sample was used as a background control during the quality assessment. Replicates of each sample group were clustered based on total protein quantification (Fig. 2A). Initial analysis with clustered heatmaps shows that the replicates for each of the sample groups cluster together, except for latent replicate 3 (SFPQ_NI.3), which clustered more closely with the induced samples. Considering expression (Euclidean distance), the induced samples present greater similarity, whereas based on behavior across measurements, the SFPQ non-induced and induced samples display higher similarity than the IgG controls (Fig. 2B). Differential analysis resulted in 77 proteins demonstrating statistically significant differences (p < 0.01) with most proteins showing a higher abundance in induced compared to non-induced cells. The heatmap shows the fold enrichment of the protein abundance in the SFPQ-induced samples compared to the non-induced (Fig. 2C). Overall, more proteins had an increased protein abundance with SFPQ in the induced sample, with a few notable decreased abundances. Among the few decreased proteins was TRAM1, a stimulatory protein required for the translocation of secretory proteins across the ER membrane (Klein et al., 2020). The eukaryotic translation initiation factor 4E or EIF4E which acts as a mediator for translation repression when complexed with CYFIP1 had a decreased protein abundance with SFPQ in the induced samples versus non-induced (DeRubeis et al., 2013).Fig. 2 SFPQ-IP proteomics from KSHV infected cells.

Four biological replicates from each group; SFPQ-IP from non-induced cells “SFPQ-Non-Ind” Rep.1–4, SFPQ-IP from induced cells “SFPQ-Ind” Rep.1–4, and IgG-IP from induced cells “IgG-Ind” Rep1–4, were subjected to liquid chromatography with mass spectrometry (LC-MS) and protein identification. (A) Clustered heatmap of log2 raw protein intensities to assess sample quality; white indicates missing values. (B) Clustered heatmap of pairwise Spearman's rank correlation coefficients of raw protein intensities. (C) Clustered heatmap of row scaled equal median normalized protein abundances of differentially abundant proteins; black indicates missing values. (D) Heatmap of equal median normalized viral proteins; white indicates missing values. (E) Volcano plot: purple indicates differential proteins with higher abundance in SFPQ-IP from induced cells compared to non-induced cells, green highlights differential proteins with higher abundance in non-induced cells, black shows all viral proteins independent of differential abundance, gray all other proteins in the data.

Fig. 2

Cellular proteins with an increased protein abundance in the SFPQ-Induced samples during lytic reactivation included ANTXR1, ATRX, SSBP1, APOL2, and PARP1. Anthrax toxin receptor 1 (ANTXR1), is a cell-surface protein that exhibits high conservation across species and is frequently overexpressed on the vasculature within tumor infiltrates (Chaudhary et al., 2012). Interestingly, ANTXR1 is used as a prognostic biomarker in certain cancers (Huang et al., 2020). ATRX (alpha-thalassemia mental retardation X-linked) is a tumor suppressor gene commonly mutated in human cancers (Aguilera and López-Contreras, 2023). ATRX has been linked to involvement in molecular pathways, including the regulation of chromatin state, gene expression, and DNA damage repair (Watson et al., 2013; Truch et al., 2022; Voon et al., 2015). Apolipoprotein L2 (ApoL2) is transcriptionally induced by interferon and is translocated to the mitochondria in H3N2 swine influenza virus infection where it acts as an anti-apoptotic regulator (Galindo-Moreno et al., 2014; Wu et al., 2013). Poly (ADP-ribose) polymerase 1 (PARP1) is a multifunctional enzyme, that has roles in mediating DNA damage repair pathways, stabilization of DNA replication forks, and remodeling chromatin (Chaudhuri and Nussenzweig, 2017). A list of KSHV proteins enriched in the lytic SFPQ-IP sample is shown in Fig. 2D. Amongst the high abundance proteins included ORF69, ORF59, ORF61, and ORF10. Although there were several cellular and viral proteins of interest, we focused on confirming immunoprecipitations with ORF10, and ORF61, and characterizing the association with ORF59.

3.3 Viral proteins ORF10 and ORF61 interact with SFPQ

During KSHV infection, there is a restriction of host factors to reduce competition for finite cellular resources, ultimately aiding in a successful replication cycle. One mechanism KSHV uses to accomplish this is through inhibition of host nuclear mRNA export (Gong et al., 2016). ORF10, a KSHV-encoded protein selectively interferes with host nuclear mRNA export by binding with the Rae1 and Nup98 complex (Gong et al., 2016). Mutations in ORF10 that abrogated interactions with Rae1/Nup98 fail to retain poly(A)+ RNA in the nucleus, indicating the interaction is necessary for nuclear mRNA accumulation (Gong et al., 2016). From our SFPQ proteomics, we identified an increased abundance of ORF10 in the lytic SFPQ-IP samples compared to controls.

To test the association between ORF10 and SFPQ we performed co-immunoprecipitations (co-IPs) in 293FT cells transfected with an ORF10 Flag-tagged expression plasmid to examine the protein-protein immunoprecipitation. Forty-eight hours post-transfection (48 hpt) cell lysate from transfected and non-transfected cells was harvested and co-IPs were performed using an anti-SFPQ antibody along with protein G magnetic beads and an anti-IgG isotype control. Western blots were probed with anti-Flag antibody and re-probed with anti-SFPQ antibody. Western blot analysis ORF10 was able to immunoprecipitated with SFPQ, while the non-transfected and IgG controls did not (Fig. 3A). We next wanted to assess the localization of ORF10 with SFPQ. 293FT cells were transfected for 48 h and an IFA was performed using anti-SFPQ and anti-Flag antibodies. We observed nuclear and cytoplasmic localization for ORF10-Flag and there was some area within the nucleus that formed speckle-like aggregates overlapping with SFPQ (Fig. 3B).Fig. 3 KSHV proteins ORF10 and ORF61 interact with SFPQ.(A) 293FT cells transfected with ORF10-Flag were used in co-immunoprecipitation (co-IP) assays to test the association between ORF10 and SFPQ. Forty-eight hours post transfection cell lysate was harvested and incubated with anti-SFPQ antibody, protein G magnetic beads and rotated overnight. Input lysate and co-IPs were resolved by SDS-PAGE and western blots were incubated with anti-SFPQ and anti-Flag. Non-transfected cells and IgG isotype antibodies were used as controls. (B) 293FT cells were transfected with ORF10-Flag or non-transfected cells for 48 h. Cells were fixed and permeabilized, and IFA was performed with anti-SFPQ antibody followed by anti-Flag antibody before being incubated with secondary antibodies labeled with Alexa Fluor 594 (Red) and Alexa Fluor 488 (Green). Nuclei were stained with DAPI (Blue). Images were obtained by fluorescent microscopy at 63x magnification. To the right of the main panels is a zoomed image of a representative cell. (C) 293FT cells transfected with ORF61-HA were used for co-IP assays to test the association between SFPQ with ORF61. The assay was performed as described above, western blots were visualized using SFPQ and HA-specific antibodies. (D) IFA from 293FT cells transfected with ORF61-HA or non-transfected cells was performed as described above. Cells were incubated with anti-SFPQ antibody followed by anti-HA antibody before being incubated with secondary antibodies labeled with Alexa Fluor 594 (Red) and Alexa Fluor 488 (Green). Nuclei were stained with DAPI (Blue). Images were obtained by fluorescent microscopy at 63x magnification.

Fig. 3

Another KSHV protein that showed a significant abundance in the SFPQ-IP from induced samples was the ribonucleotide reductase large subunit, ORF61. The transcript of ORF61 is a 6.0 kbp polycistronic mRNA containing open reading frames 61, 60, 59, and 58 (Wang et al., 2010). The ribonucleotide reductase (RNR) is an enzyme that catalyzes the synthesis of deoxyribonucleotides by reducing the 2′‑hydroxyl from ribonucleotides and producing deoxynucleoside triphosphate (dNTP) necessary for DNA synthesis and DNA damage repair (Torrents, 2014). One of the functions of SFPQ is mediating homology directed DNA repair and DNA damage responses (Rajesh et al., 2011).

We tested the association of SFPQ and ORF61 by performing co-IPs from 293FT cells transfected with an ORF61 HA-tagged expression plasmid in a similar method as described above. Lysate from the transfected and non-transfected cells was harvested at 48 hpt and incubated with anti-SFPQ antibody and magnetic protein G beads. Western blots were probed with anti-SFPQ and anti-HA antibodies and showed enrichment of ORF61 in the SFPQ-IPs compared to controls (Fig. 3C). We examined the localization of ORF61 and SFPQ by transfecting 293FT cells with ORF61-HA plasmid and performing IFA at 48 hpt using anti-HA and anti-SFPQ antibodies. Interestingly, although the co-IPs showed an association between ORF61 and SFPQ, in the IFA ORF61 localized adjacent to the nucleus, sometimes within highly aggregated pockets, making it difficult to assess the localization with SFPQ and determine if there were areas of overlap. (Fig. 3D). Additionally, the localization patterns could be attributed to the lack of other viral factors which are absent in the transfected cells.

3.4 The 101–150 amino acid region of ORF59 is required for the association with SFPQ

To investigate the association between ORF59 and SFPQ during reactivation, we performed co-IPs of both SFPQ and NONO in iSLK cells containing either WT or recombinant KSHV BACmids with 50 amino acid deletion of ORF59. The WT and ORF59 deletion mutants contain an in-frame hemagglutinin (HA) tag and have previously been described and characterized (I.V. Gutierrez et al., 2021). iSLK cells containing either WT, Δ51–100, Δ101–150, Δ251–300, and Δ351–396 ORF59 BACmid were treated with Dox, TPA, and NaB, and cell lysates were harvested at 48 h postinduction (hpi). Protein-protein complexes were immunoprecipitated with either anti-NONO or anti-SFPQ antibodies. Control immunoprecipitations were done with non-induced cells as well as IgG isotype antibody. Western blots were probed with anti-NONO, anti-SFPQ, and anti-HA antibodies (Fig. 4A). The non-induced control samples show no ORF59 expression, and upon reactivation with TPA, NaB and Doxycycline there is expression of ORF59. In our previous paper characterizing the ORF59 deletion mutants, there were no apparent defects in immediate early reactivation of the mutants and show relatively similar amounts of ORF59 expressed by the mutants compared to WT (I.V. Gutierrez et al., 2021). The co-IP's in Fig. 4A from the ORF59 mutant viruses are not meant to be quantitative and do not contain load controls for the blots. The association between ORF59 and SFPQ, and ORF59 and NONO, remained in all deletion mutants, as observed in the WT, except for the iSLK BACmid cell lysate which contained an internal Δ101–150 ORF59 deletion. Also, we note that the association between SFPQ and NONO is not disrupted during reactivation of WT or any of the ORF59 internal deletion mutant viruses.Fig. 4 The 101–150 amino acid region of ORF59 is required for association with SFPQ.

(A) iSLK BAC16 59HA-WT along with ORF59Δ51–100, ORF59Δ101–150, ORF59Δ251–300 and ORF59Δ351–396 deletion mutants were either non-induced (Control) or treated with NaB, TPA, and Dox for 48 h. Lysate was harvest and divided for co-IPs using either anti-SFPQ or anti-NONO antibodies along with protein G magnetic beads and incubated rotating overnight. Immunoprecipitations were resolved by SDS-PAGE and western blot were incubated with anti-SFPQ, anti-NONO, and anti-HA antibodies. (B) iSLK BAC16 59HA-WT cells were induced with NaB, TPA, and Dox or non-induced (Control) for 24 h. IFA was performed by incubating fixed and permeabilized cells with anti-SFPQ antibody followed by anti-HA antibody before being incubated with secondary antibodies labeled with Alexa Fluor 594 (Red) and Alexa Fluor 405 (Blue). KSHV BAC16 constitutively expressed GFP. Images were obtained by fluorescent microscopy at 63x magnification. To the right is a zoomed image of a representative cell.

Fig. 4

The non-induced and induced iSLK cells containing WT KSHV were used for immunofluorescence assay to visualize ORF59 and SFPQ during latency and lytic reactivation. Cells were incubated with anti-HA antibodies to detect ORF59 and anti-SFPQ antibodies followed by secondary antibodies Alexa Fluor-594 (red) anti-rabbit for SFPQ and Alexa Fluor-405 (blue) anti-mouse for HA and mounted using ProLong Gold Antifade. The KSHV BAC16 used for the WT and ORF59 deletion mutants contains a constitutively expressed green fluorescent protein (GFP) which is visible in both the non-induced and induced cells, and for simplicity, the GFP signal is not shown. Consequently, because we used a secondary antibody that contains a fluorophore within the blue wavelength (405) we could not use DAPI to stain the nucleus; the images shown are the relative localization and overlap between ORF59 and SFPQ, and not necessarily the nuclear localization. Cells were examined by fluorescent microscopy at 63X to visualize localization between ORF59 and SFPQ and a zoomed panel to the right of the main image shows a representative cell (Fig. 4B). We note that there is no substantial change in the localization pattern of SFPQ comparing the latent and reactivated samples. This is interesting because SFPQ aggregates have been observed and reported following infection of other viruses.

3.5 Localization of SFPQ during reactivation of ORF59Δ101–150 virus reveals increased size and abundance of paraspeckles compared to WT

The localization and formation of SFPQ and NONO-containing paraspeckles are often associated with the function of these proteins. Because there was an absence in the association between ORF59 and SFPQ/NONO in the ORF59Δ101–150 containing iSLK cell line, we investigated the localization of SFPQ and NONO in WT and ORF59Δ101–150 at 0-, 6-, 24-, and 48 hpi (Fig. 5A). IFA were performed as previously described using antibodies to either SFPQ or NONO followed by secondary Alexa Fluor-594 (red) antibodies. Coverslips were mounted with ProLong Gold Antifade with DAPI and cells were examined by fluorescent microscopy at 63X. In WT, SFPQ remained relatively diffuse within the nucleus with some more defined small aggregates accumulating within 48 h post-induction. However, at 24 h post-induction for the ORF59Δ101–150 cell line, both NONO and SFPQ began to form large aggregates that were more visually apparent. These larger paraspeckles were consistently observed through 48 h post-induction. Examining the zoomed ORF59 Δ101–150 revealed large aggregates of SFPQ and NONO compared to WT (Fig. 5B). These data suggest ORF59 may play a role in the dispersion of SFPQ and NONO during reactivation, and loss of the association results in the accumulation and formation of paraspeckles. Because the formation of paraspeckles is often correlated with an increase in the association between NEAT1, SFPQ, and NONO we measured the amount of total NEAT1 following reactivation of WT and ORF59Δ101–150 (Fig. 5C). Interestingly, the amount of NEAT1 substantially decreased in WT iSLK cells by 24 hpi and remained low, while the ORF59Δ101–150 iSLK cells had only a small decrease in the amount of NEAT1 at 24 hpi and the levels rebounded to non-induced levels by 48 hpi. The relative decrease of NEAT1 RNA in KSHV+ cells following reactivation is similar to what has previously been reported in BCBL-1 cells (Borah et al., 2011). The relative amount of NEAT1 in the ORF59Δ101–150 compared to WT may contribute to the observed paraspeckle formation in the ORF59Δ101–150. Note that the ΔΔCT method used to calculate the relative amount of NEAT1 may not accurately reflect the numbers, as the primer amplification efficiency between the target (NEAT1) and reference (GAPDH) were not assessed during reactivation.Fig. 5 Localization of SFPQ and NONO in ORF59 Δ101–150 deletion mutant.

(A) IFA was performed to visualize SFPQ and NONO localization in iSLK BAC16 59HA-WT and ORF59 Δ101–150 cell lines induced with NaB, TPA, and Dox for 6-, 24-, and 48-hours. Non-induced cells were used as a control. Cells used in IFA were fixed, permeabilized and incubated with an anti-SFPQ antibody or an anti-NONO antibody before being incubated with secondary antibodies labeled with Alexa Fluor 594 (Red). Nuclei were stained with DAPI (Blue). Cells were visualized at 63x magnification. (B) Zoomed images of NONO and SFPQ at 0- and 24-hours post-induction in the iSLK BAC16 59HA-WT and ORF59 Δ101–150 cell lines. (C) NEAT1 RNA was measured by RT-qPCR at 0-, 24-, 48-, and 72-hours post-induction in iSLK BAC16 59HA-WT and ORF59Δ101–150 cell lines. Fold change of NEAT1 RNA during reactivating was calculated using ΔΔCT, normalized with cellular GAPDH and compared to the non-induced samples at 0 hpi. Two-way ANOVA with Bonferroni's multiple comparison test was performed, ** p = 0.0016.

Fig. 5

3.6 The addition of 101–150 aa ORF59 polypeptide acts as a dominant negative inhibitor to block SFPQ association with full-length ORF59, leading to a decrease in viral replication

To gain a better understanding of the three-dimensional structure of ORF59 and SFPQ, we performed prediction modeling of the protein docking between the ORF59 dimer (Blue) with the SFPQ dimer (Gold) with ChimeraX using ColabFold v1.5.5 along with AlphaFold (Fig. 6A). Protein docking and folding predictions were performed following the previous published instructions (Jumper et al., 2021; Mirdita et al., 2022). The 101–150 amino acid sequence of ORF59 is highlighted in pink, and with the predicted modeling this region had the highest probability of interacting with SFPQ. The predicted model along with the co-IPs of SFPQ from the ORF59 deletion BACmid cell lines (Fig. 4A), supports the hypothesis that the 101–150 amino acid region is where SFPQ associates with ORF59.Fig. 6 Dominant negative 101–150 aa of ORF59 disrupts viral replication.

(A) Crystal structures of ORF59 dimer (Blue, PDB ID 3HSL) predicted association site with SFPQ dimer (Gold, PDB ID 4WIJ), highlighting the 101–150 amino acid region of ORF59 (Pink). (B) 293FT cells were transfected with 101–150DsRed and DsRed-N1 (Control) plasmids and 48 hpt DsRed expression was visualized with a live cell fluorescent microscope. (C) Western blots from 293FT cells transfected with increasing concentrations (0.5, 1, and 3 µg) of the 101–150DsRed expression plasmid. Protein expression of the non-transfected (NT) and 48-hour 101–150DsRed plasmid transfected lysate was analyzed by SDS-PAGE and visualized using an anti-DsRed antibody. (D) To determine if the 101–150DsRed polypeptide could disrupt the association between full-length ORF59 and SFPQ, pXI-ORF59HA expression plasmid was transfected into 293FT cells alone or along with either 101–150DsRed or DsRed-N1 expression plasmid. A set of 293FT non-transfected cells was used as a control. 48 hpt protein complexes were isolated using anti-HA antibody and protein G magnetic beads. Input lysate and IPs were resolved by SDS-PAGE and western blots were incubated with anti-SFPQ and anti-HA antibodies. (E) Viral DNA was harvested and purified at 0-, 48-, and 72-hours post-induction from non-transfected iSLK BAC16 59HA-WT cells, and iSLK BAC16 59HA-WT cells transfected with either 101–150DsRed or DsRed-N1. Purified DNA was measured by qPCR using primers and probes specific to KSHV (ORF26). Relative viral DNA was normalized with cellular DNA and fold change was calculated using untreated samples. Results represent three biological replicates. Bar graphs represent means ± the standard deviation (SD). One-way ANOVA with Tukey's multiple comparison test was performed, ** p = 0.0045 and *** p = 0.0008. (F) Quantification of infectious viruses from iSLK BAC16 59HA-WT transfected with either 101–150DsRed or DsRed-N1 expression plasmids. iSLK BAC16 59HA-WT were transfected 24 h before induction with Dox, TPA, and NaB. KSHV was harvested from cells and supernatant at 5 dpi. A 10-fold serial dilution of the inoculum was used to infect 293 L cells and GFP+ foci were counted at 3 dpi. iSLK BAC16 59HA-WT cells transfected with 101–150DsRed expression plasmid produce infectious virus (GFP+/ml) at lower amounts than control iSLK BAC16 59HA-WT and the group transfected with DsRed-N1. Results represent three biological replicates. Bar graphs represent means ± the standard deviation (SD). One-way ANOVA with Tukey's multiple comparison tests was performed, ** p = 0.0014 and *** p = 0.0003.

Fig. 6

We next wanted to determine if the association between ORF59 and SFPQ is required for viral replication. We created an expression plasmid containing the 101–150 amino acids from ORF59, along with an in-frame DsRed tag to visualize the polypeptide within transfected cells. The 101–150DsRed plasmid was transfected into 293FT cells and red fluorescent protein (RFP) was visualized from the 101–150DsRed plasmid along with a set transfected with the DsRed-N1 vector alone as a control (Fig. 6B). The same amount of plasmid DNA and transfection reagent was used for the DsRed vector alone and 101–150DsRed plasmid. As shown in Fig. 6 panel B, the fluorescent signal from the 101–150DsRed and DsRed vector are similar, but we did not further quantify the amounts and there may be slight variability in the expression levels. To examine protein expression, 293FT cells were transfected with increasing concentrations of the 101–150DsRed plasmid, and protein was assessed by western blot incubated with DsRed antibody (Fig. 6C). A non-transfected sample was used as a control.

To test if the 101–150DsRed could act as a dominant negative polypeptide and prevent association between ORF59 and SFPQ, co-transfection experiments were performed in 293FT cells. ORF59-HA expression plasmid was transfected into 293FT cells along with either 101–150DsRed or DsRed-N1 expression plasmid. A set of 293FT non-transfected cells was used as a control. Co-IPs were performed at 48 hpt with anti-HA antibody. Input lysate and co-IP samples were resolved by SDS-PAGE and western blots were incubated with anti-HA to detect ORF59 and anti-SFPQ antibodies. In the samples transfected with 101–150DsRed and ORF59-HA, we did not detect SFPQ in the HA-IP. In cells transfected solely with ORF59-HA or with ORF59-HA with DsRed-N1, we detected SFPQ in the HA-IP, suggesting that the 101–150DsRed effectively hindered the association between full-length ORF59 and SFPQ. The direct association between 101 and 150DsRed and SFPQ was not tested, as such we cannot draw any conclusion regarding the association of 101–150DsRed to SFPQ.

After confirming the 101–150DsRed polypeptide was able to block the association between SFPQ and full-length ORF59, we next wanted to examine the effects on viral DNA replication. To accomplish this, we transfected the 101–150DsRed plasmid into iSLK-BAC16 59HA-WT cells and induced with Dox, TPA, and NaB for 48- and 72- hours. We measured the fold change in viral DNA accumulation by qPCR using Taqman primers and probes specific for viral DNA (ORF26) compared to non-induced controls and normalized to cellular DNA. A DsRed-N1 vector alone transfected set was used as a control. We found no significant difference at 48 h between WT, 101–150DsRed, and the DsRed-N1 control. At 72 h the 101–150DsRed had significantly less viral DNA compared to WT and DsRed-N1 control which had increased to similar levels, indicating the addition of the 101–150DsRed peptide acts as an inhibitor (Fig. 6E).

To assess the functional association between SFPQ and ORF59 during the lytic reactivation, we measured the amount of infectious virus produced in the presence of the dominant negative peptide. iSLK cells were either transfected with 101–150DsRed or DsRed-N1 plasmid alone for 24 h before being induced with Dox, TPA, and NaB for 5 days. Cells and supernatant were harvested, and the purified virus was isolated. The 5-dpi purified virus was added to 293 L cells and incubated for 3 days. At 3 dpi, the GFP-positive foci were counted and calculated as GFP+/mL. We found a small amount of virus still being produced in the 101–150DsRed transfected WT iSLK cells, but a significantly lower amount compared to the non-transfected WT iSLK cells and WT iSLK cells transfected with DsRed-N1 control (Fig. 6F).

3.7 SFPQ associates with KSHV, EBV, HSV-1, and HCMV-encoded processivity factors

To determine if the association of SFPQ extends to other human herpesvirus processivity factors, we performed co-immunoprecipitations following transfections of plasmids encoding ORF59, EBV BMRF1, HSV-1 UL42, and HCMV UL-44 (Fig. 7A). Each processivity factor was cloned into an expression plasmid containing an in-frame HA tag and transfections of the plasmids were done in 293FT cells. Following a 48-hour incubation, cell lysates were harvested and subjected to immunoprecipitation using anti-HA and control anti-IgG antibodies. Input lysate and co-IP samples were resolved by SDS-PAGE. Western blots were incubated with anti-HA and anti-SFPQ antibodies. Interestingly, our findings indicate that all the examined viral processivity factors interact with SFPQ.Fig. 7 SFPQ associates with KSHV, EBV, HSV, and HCMV encoded processivity factors. (A) Co-IPs were performed from 293FT cells transfected with KSHV ORF59-HA, EBV BMRF1-HA, HSV UL42-HA, or HCMV UL44-HA expression plasmids. Lysate was harvested from the transfected and non-transfected (NT) cells 48 hpt and incubated with anti-HA antibody along with protein G magnetic beads and rotated overnight. Input lysate and co-IP samples were resolved by SDS-PAGE and western blot were incubated with anti-SFPQ and anti-HA antibodies. (B) IFA was performed on 293FT cells transfected with KSHV ORF59-HA, EBV BMRF1-HA, HSV UL42-HA, or HCMV UL44-HA expression plasmids. 48 hpt, cells fixed and permeabilized before incubating with an anti-SFPQ antibody followed by a secondary antibody labeled with Alexa Fluor 594 (Red) and an anti-HA antibody followed by a secondary antibody labeled with Alexa Fluor 488 (Green). Cells were visualized at 63x magnification. The bottom panel is a zoom image of a single cell.

Fig. 7

To further characterize the associations between the herpesvirus processivity factors and SFPQ, we conducted immunofluorescent assays using 293FT cells transfected with ORF59-HA, EBV BMRF1-HA, HSV-1 UL42-HA, and HCMV UL44-HA plasmids. IFA was performed at 48 hpt, and SFPQ was visualized with an anti-SFPQ antibody and a secondary Alexa Fluor-594 (red) antibody. Each of the HA-tagged viral processivity factors was visualized with an anti-HA primary antibody and secondary Alexa Fluor-488 (green) antibody. Coverslips were mounted onto glass slides using ProLong Gold Antifade with DAPI. Cells were examined by fluorescent microscopy at 63X (Fig. 7B). Interestingly, the images and localization patterns between SFPQ and the viral processivity factors vary, the zoomed images highlight some of these differences. There are some cells in which the processivity factor (for example BMRF1) forms aggregates devoid of SFPQ among diffuse areas that show some overlap. While in the cells transfected with UL42 there are larger aggregates of SFPQ among areas that show potential overlap between UL42 and SFPQ. These differences may be from additional unique cellular binding partners for each of the processivity factors. Our findings with the co-IPs and IFA suggest an association between SFPQ and herpesvirus processivity factors in the absence of other viral factors.

4 Discussion

Successful completion of KSHV lytic replication requires utilization of viral and host cell factors. A cellular protein that had not previously been explored during KSHV replication is the paraspeckle component SFPQ. We became interested in SFPQ in part because of a prior report that identified the cellular paraspeckle components SFPQ and NONO in a proteomics screen of ORF59. SFPQ is multifunctional protein that has additional roles outside of paraspeckles, including modulating gene expression, antiviral response and DNA damage repair response. The literature regarding the role of SFPQ in various RNA and DNA virus infections is quite diverse, so we sought to explore SFPQ in KSHV lytic replication, In this study, we examined the protein interactome of SFPQ in KSHV-infected cells and subsequently characterize the associations between SFPQ and viral proteins ORF10, ORF61 and ORF59. Our data suggests that SFPQ may have a role in KSHV replication, but there remain questions about the underlying mechanism of how SFPQ supports KSHV replication.

SFPQ and NONO are commonly associated with paraspeckle complexes, which are a class of subnuclear bodies in mammalian cells (Fox and Lamond, 2010; Fox et al., 2002). These RNA-protein structures are formed through interactions between a long nonprotein-coding RNA, NEAT1, and members of the DBHS family of proteins including SFPQ, NONO, and PSPC110. These proteins have been implicated in a wide variety of functions including regulating gene expression, mRNA processing, innate immune response to viral infection, and cellular stress responses (Alkalay et al., 2020). They can also be directly involved in viral infection. For example, in Influenza A infected cells, silencing of SFPQ leads to reduced accumulation of viral messenger and genomic RNAs (Landeras-Bueno et al., 2011). A recent study revealed that SFPQ interacts with SARS-CoV-2 RNA, enhancing viral replication (Labeau et al., 2022).

In addition to its roles in RNA splicing and 3′-end processing, the SFPQ/NONO complex plays a crucial role in facilitating the nuclear retention of specific mRNAs within paraspeckles (Malnar and Rogelj, 2021). The retention signal is established by ADAR (Adenosine De-Aminase RNA specific) enzymes, which catalyze the conversion of adenosines (A) to inosines (I) within target mRNAs by hydrolytic deamination (Poison et al., 1991; DeCerbo and Carmichael, 2005). The SFPQ/NONO complex exhibits a strong affinity for hyper-edited RNAs, effectively sequestering them within paraspeckles and thereby preventing their export to the cytoplasm. Previous research found oriP transcripts of EBV to be hyper-edited, allowing interaction with NONO to support viral transcription (Cao et al., 2015). Additionally, studies have shown EBV-encoded RNA 2 (EBER2) interacts with SFPQ in an RNA-protein crosslinking experiment and the interaction was suggested to regulate viral gene expression (Lee et al., 2016). It has been reported that HSV-1 infection enhances paraspeckle formation in HeLa cells within 4 h post-infection. The increase in paraspeckles coincides with the localization of NONO, PSPC1, and NEAT1 to the HSV-1 genome, implying their involvement in viral gene expression and replication (Wang et al., 2017). Interestingly, a recent study on EBV using a CRISPR/Cas9 knockout of SFPQ demonstrated that SFPQ is involved in the regulation of latency, as there was an increase in lytic gene expression following SFPQ knockdown and subsequent decrease in H1 linker histone occupancy of the EBV genome (Murray-Nerger et al., 2024). There is the potential that SFPQ has multiple regulatory roles for herpesviruses, acting as a repressor for cellular factors which control reactivation, and associating with viral proteins during lytic replication. Perhaps the association between SFPQ and early lytic proteins like ORF59, titrates SFPQ away from the cellular genome to further augment lytic reactivation.

Another potential role of SFPQ and paraspeckle components is related to the immune response to KSHV infection. One study using HSV-1 and influenza virus demonstrated that SFPQ binding sites within the IL-8 promoter are de-repressed upon infection with these viruses but not with the measles virus (Imamura et al., 2014). The study also showed that there was excessive formation of paraspeckles and an induction in the expression of the cellular NEAT1 lncRNA, which interacts with SFPQ and NONO in the paraspeckle formation. In the previously referenced EBV study, they noted in Akata cells there was an increase in NEAT1 following reactivation (Cao et al., 2015). They also scanned the EBV genome for SFPQ binding sites, similar to the IL-8 promoter sites, and found 17 sites in the viral genome and speculated these may be de-repressed or act as transcription activators during the reorganization of SFPQ but did not determine if these were functional binding sites for SFPQ. In assessing the KSHV genome we did not detect any of these potential SFPQ sites, but we cannot rule out cryptic SFPQ binding sites. For NEAT1 expression in KSHV infection, a study in BCBL-1 showed during reactivation there is a significant and sustained decrease in NEAT1, but the cause for the decrease is unknown (Borah et al., 2011). Interestingly, KSHV PAN RNA and NEAT1 both contain a unique triple helix structure located at the 3′-end of the RNAs (Mitton-Fry et al., 1979; Wilusz et al., 2012; Brown et al., 2012). Further investigation will need to be carried out to determine if there is a connection between the decrease in NEAT1 during KSHV infection and the expression of PAN RNA, which has also been shown to regulate some innate immune responses upon KSHV reactivation (Rossetto and Pari, 2011).

The SFPQ proteomics from KSHV-infected cells identified specific viral proteins interacting with SFPQ during lytic replication. Two viral proteins tested, ORF61 and ORF10, have roles in regulating RNA. ORF61 is the ribonucleotide reductase large subunit (RNR, RIR1) and is known to relocate APOBEC3A (A3A) and APOBEC3B (A3B) to perinuclear bodies and inhibit DNA deaminase activity (Cheng et al., 2019). ORF10 selectively inhibits cellular mRNA nuclear export, induces nuclear accumulation Poly(A)+ RNA, and is required for late viral gene expression (Gong et al., 2016). ORF10 has also been reported to block type 1 interferon signaling by forming inhibitor complexes with the Janus kinases, Jak1 and Tyk2, and the STAT2 transcription factor (Bisson et al., 2009). The association of ORF10 with SFPQ could be involved in immune response suppression and mRNA regulation. We note that the association between SFPQ and the viral proteins may be mediated by nucleic acid, and our detection of these proteins may be an indirect association as SFPQ, ORF61 and ORF10 are all known to associate with RNA.

Because of our prior work with ORF59, we focused the remainder of the SFPQ study on the association between ORF59 and SFPQ. We used internal deletion mutants of ORF59 to identify the region between 101 and 150 amino acids that associates with SFPQ. In our previous publication, we discovered that several internal deletions of ORF59, including ORF59Δ101–150, exhibited a defect in DNA synthesis due to the impaired ability of ORF59 to function as a processivity factor. Therefore, when investigating the association between SFPQ and ORF59 using the ORF59Δ101–150 mutant virus, we limited our observations to no later than 48 h post-infection (hpi) (I.V. Gutierrez et al., 2021). For later time points we used a dominant negative peptide to disrupt the association of SFPQ and ORF59 in the context of WT infection. We would like to note that because we did not perform experiments, such as nuclease treatment of the co-IPs, to assess the direct interaction between SFPQ and ORF59 we have characterized the relationship between SFPQ and ORF59 as an association and not a direct interaction. Further investigation will be needed to determine if there is a direct interaction between SFPQ and ORF59 or if there are other co-factor, such as DNA or RNA, mediating the interaction.

We created a dominant negative polypeptide containing amino acids 101–150 of ORF59 which was able to block the association between full-length ORF59 and SFPQ. After the transfection of the 101–150DsRed plasmid and lytic reactivation by TPA, NaB and doxycycline, we observed a decrease in viral replication, measured by a reduction in viral DNA and infectious virus production. Further experiments will be needed to determine if there is a direct interaction between the 101–150DsRed ORF59 peptide and SFPQ, or if the 101–150DsRed association with SFPQ is transient. Additionally, it will be important to determine if the association between SFPQ and ORF59 directly effects DNA synthesis or if the reduction in virus production with the 101–150DsRed peptide disrupts auxiliary functions of ORF59.

There are limitations to the interpretation of these results, the first being that the observed decrease in viral replication may be from indirect effects rather than disruption of the SFPQ-ORF59 association. For example, the 101–150DsRed peptide may disrupt the ability of ORF59 to dimerize or form higher order structures necessary for DNA synthesis. The previous identified ORF59 dimerization domains are within amino acids 1–21 and 277–307, even though the 101–150DsRed peptide is outside these regions there remains the possibility that ORF59 dimerization was compromised by the 101–150DsRed peptide. Further tests will need to be performed to determine the tertiary and quaternary structure of ORF59 in the presence of the 101–150DsRed peptide. If ORF59 is unable to form higher order structures, its ability to properly function in DNA synthesis would be affected. We did not explore the potential of ORF59, directly or indirectly, to regulate the expression of SFPQ and NONO, or the stability and retention of SFPQ and NONO protein. The amounts of SFPQ and NONO may be an important consideration, given the increase in paraspeckles observed in the ORF59Δ101–150 virus compared to WT. Additionally, the internal deletion of 101–150 was previously found to encompass the binding domain for histones H1 and H2A (I.V. Gutierrez et al., 2021). Although there were multiple regions outside of 101–150 ORF59 that interacted with H1 and H2A, there is the potential that the associations were also disrupted by the dominant negative polypeptide.

One of the more exciting aspects of our study was testing if other human herpesvirus processivity factors interact with SFPQ. We tested EBV BMRF1, HSV-1 UL42, and HCMV UL44 along with ORF59 in co-IP experiments and found that all the viral processivity factors were able to interact with SFPQ. This became even more interesting when we looked at the differences in the localization pattern of the processivity factors and SFPQ. We acknowledge the limitations in these studies and temper our conclusions until further studies can be conducted. One limitation of these experiments is that they were only performed using expression plasmids, but in future experiments it will be important to test these association in the context of infected cells. Also, we did not rule out the possibility that nucleic acid may be mediating the association between the viral processivity factors and SFPQ. Together, our results indicate that paraspeckle components, such as SFPQ, may be an important factor for successful viral replication.

Author statement

We declare that we have no financial or personal relationships with others or organizations that could inappropriately influence our work. While preparing this work, the authors did not use any generative AI or AI-assisted technologies in the writing process. All content was written, reviewed, and edited manually by the authors, who take full responsibility for the content of the publication. We confirm that the work described in this manuscript has not been published previously and is not under consideration for publication elsewhere. All authors have approved the submission of this manuscript, and if accepted, it will not be published elsewhere in the same form, in English, or any other language, including electronically, without the written consent of the copyright holder.

Funding

This work was supported by the National Institute of Health (NIH) NIAID (R01 AI123011 to CCR, and grants from the 10.13039/100000057 National Institute of General Medical Sciences (GM103440 and 5 U54 GM104944 ).

CRediT authorship contribution statement

Shannon Harger Payen: Conceptualization, Validation, Formal analysis, Visualization, Investigation, Writing – original draft, Writing – review & editing. Kayla Andrada: Investigation, Formal analysis, Visualization. Evelyn Tara: Visualization, Formal analysis, Investigation. Juli Petereit: Writing – review & editing, Visualization, Formal analysis. Subhash C. Verma: Writing – review & editing, Formal analysis, Conceptualization. Cyprian C. Rossetto: Writing – review & editing, Writing – original draft, Visualization, Supervision, Project administration, Funding acquisition, Formal analysis, Conceptualization.

Declaration of competing interest

The authors declare that they have no conflicts of interest with the contents of this article.

Appendix Supplementary materials

Image, application 1

Data availability

Data will be made available on request.

Acknowledgments

We thank the Mitch Hitchcock, Ph.D. Nevada Proteomics Center (RRID: SCR_017761), and Nevada Bioinformatics Center (RRID: SCR_017802), University of Nevada, Reno for preparing and analyzing proteomic samples.

The ORF10-Flag plasmid was a kind gift from Dr. Ting-Ting Wu (University of California, Los Angeles).

Molecular graphics and analyses performed with UCSF ChimeraX, developed by the Resource for Biocomputing, Visualization, and Informatics at the University of California, San Francisco, with support from National Institutes of Health R01-GM129325 and the Office of Cyber Infrastructure and Computational Biology, National Institute of Allergy and Infectious Diseases.

Supplementary material associated with this article can be found, in the online version, at doi:10.1016/j.virusres.2024.199456.
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