Journal: Virology

Article Title: Long non-coding RNA SUN2-AS1 acts as a negative regulator of ISGs transcription to promote flavivirus infection.

doi: 10.1016/j.virol.2024.110245

Figure Lengend Snippet: Fig. 1. Characterization of SUN2-AS1. (A) A schematic representation of human SUN2-AS1 transcribed from the antisense strand of the protein-coding gene SUN2 on chromosome 22. (B) 5′- and 3′ RACE was conducted to determine the full length of SUN2-AS1. Full-length SUN2-AS1 was chemically synthesized and cloned into the pcDNA3.1(+) vector. Total RNA from ZIKV-infected A549 cells was extracted at 24 h.p.i. The RNA was then reverse transcribed and amplified using the SMARTer RACE cDNA amplification kit. (C) The CPAT (Coding Potential Assessment Tool) was used to predict the coding probability of SUN2-AS1. (D) 293T cells were transfected with pcDNA3.1-GFP-HA (vector control), SUN2-HA (positive control), pcDNA3.1-HA-SUN2-AS1, or pcDNA3.1-SUN2-AS1-HA plasmid. At 36 h post- transfection, cells were harvested for a Western blot analysis to detect anti-HA bands. Blots were representative of at three independent experiments. (E) The relative expression of human β-actin (cytoplasmic control), U6 (nuclear control), and the expression of SUN2-AS1 were analyzed by using qRT-PCR in the nuclear and cytoplasmic fractions.

Article Snippet: The primary antibodies used in this study were as follows: Rabbit polyclonal antibodies against ZIKV E (GeneTex, GTX133314), rabbit polyclonal antibodies against SUN2 (Proteintech, 27556-1-AP), anti-phospho-STAT1 (HUABIO, ET1611-20), anti-STAT1 (HUABIO, ET1612-22), anti-MX1 (Proteintech, 13750-1-AP), anti-PKR (Proteintech, 18244-1-AP), anti-HA-tag mAb (MBL, M180-3) and rabbit polyclonal antibodies against GAPDH (Proteintech, I0494-I-AP).

Techniques: Synthesized, Clone Assay, Plasmid Preparation, Infection, Reverse Transcription, Amplification, Transfection, Control, Positive Control, Western Blot, Expressing, Quantitative RT-PCR

Journal: Virology

Article Title: Long non-coding RNA SUN2-AS1 acts as a negative regulator of ISGs transcription to promote flavivirus infection.

doi: 10.1016/j.virol.2024.110245

Figure Lengend Snippet: Fig. 2. Differential expression of SUN2-AS1 induced by various viruses. (A) A549 cells were infected with ZIKV at MOI 3, and total RNAs were harvested at indicated time points (0, 6, 12, 18, and 24 h.p.i.) to detect the SUN2-AS1 level by qRT-PCR (n = 5). (B) A549 cells were infected with ZIKV at indicated MOI (0, 0.05, 0.5, 5, and 10), and total RNAs were harvested at 24 h.p.i. to detect the SUN2-AS1 level by qRT-PCR (n = 5). (C) A549 cells were infected with ZIKV, DENV2 NGC, JEV, VSV, or HSV-1 (MOI 3), and total RNAs were harvested at 24 h.p.i. to detect the SUN2-AS1 level by qRT-PCR (n = 5). (D) A549, Huh7, LN229, hMDM (monocyte-differentiated macrophages), or 293T cells were infected with ZIKV at MOI 3 and harvested for total RNA extraction at 24 h.p.i. qRT-PCR was performed to detect the level of SUN2-AS1. Human U6 level was measured as an internal control and normalized to uninfected cells (MOI 0, 0 h, or wt A549) (n = 3). Bio logically independent experiments were conducted. Data were shown as means ± S.D. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. NS, not significant.

Article Snippet: The primary antibodies used in this study were as follows: Rabbit polyclonal antibodies against ZIKV E (GeneTex, GTX133314), rabbit polyclonal antibodies against SUN2 (Proteintech, 27556-1-AP), anti-phospho-STAT1 (HUABIO, ET1611-20), anti-STAT1 (HUABIO, ET1612-22), anti-MX1 (Proteintech, 13750-1-AP), anti-PKR (Proteintech, 18244-1-AP), anti-HA-tag mAb (MBL, M180-3) and rabbit polyclonal antibodies against GAPDH (Proteintech, I0494-I-AP).

Techniques: Quantitative Proteomics, Infection, Quantitative RT-PCR, RNA Extraction, Control

Journal: Virology

Article Title: Long non-coding RNA SUN2-AS1 acts as a negative regulator of ISGs transcription to promote flavivirus infection.

doi: 10.1016/j.virol.2024.110245

Figure Lengend Snippet: Fig. 3. SUN2-AS1 is an inducible host lncRNA through the type I IFN pathway. (A) 293T cells were transfected with varying amounts of cellular RNA (without ZIKV infection) or viral RNA cocktail (from 0 to 1 μg), which was extracted by the QIAamp Viral RNA Mini Kit. The levels of SUN2-AS1 were determined by qRT-PCR at 24 h post-transfection (n = 5). (B) 293T cells were transfected with various plasmids encoding Zika virus nonstructural proteins (1.0 μg). The levels of SUN2-AS1 were determined by qRT-PCR at 24 h post-transfection. (C) A549 cells were treated with 400 ng/ml poly(I:C), and the total cells were collected at indicated time points for RNA extraction to detect the level of SUN2-AS1 using qRT-PCR. (D) A549 cells were treated with 500 units/mL of IFN-β for 24 h and total RNAs were harvested to determine the level of SUN2-AS1 by qRT-PCR. (E) Putative transcription factor binding sites on the promoter region of SUN2-AS1. (F-G) RNA Immunoprecipitation (RIP) was performed on A549 cells using NF-κB and STAT1 antibodies. qRT-PCR analysis for SUN2-AS1, NKILA (binding to NF-κB p65) (F), and LUCAT1 (binding to STAT1) (G) expression in A549 (n = 3). (H–L) Control cells, IFNAR1KO or STAT1KO cells were treated with 500 units/mL of IFN-β for 24 h. Cell lysates were collected for Western blot to detect the phospho-STAT1, STAT1 and MX1 protein level (H for IFNAR1KO cells, and I for STAT1KO cells). Total RNAs were harvested to determine the level of IFNB1, ISG15 and MX1 by qRT-PCR (J-L). (M) A549 control cells, IFNAR1KO cells, or STAT1KO cells were infected with mock or ZIKV (MOI 3). Total RNAs were harvested at 24 h.p.i. to detect the level of SUN2-AS1 by qRT-PCR. Human U6 level was measured as an internal control and normalized to uninfected cells. Biologically independent experiments were conducted. Data were shown as means ± S.D. *P < 0.05, **P < 0.01, ***P < 0.001. NS, not significant.

Article Snippet: The primary antibodies used in this study were as follows: Rabbit polyclonal antibodies against ZIKV E (GeneTex, GTX133314), rabbit polyclonal antibodies against SUN2 (Proteintech, 27556-1-AP), anti-phospho-STAT1 (HUABIO, ET1611-20), anti-STAT1 (HUABIO, ET1612-22), anti-MX1 (Proteintech, 13750-1-AP), anti-PKR (Proteintech, 18244-1-AP), anti-HA-tag mAb (MBL, M180-3) and rabbit polyclonal antibodies against GAPDH (Proteintech, I0494-I-AP).

Techniques: Transfection, Infection, Quantitative RT-PCR, Virus, RNA Extraction, Binding Assay, RNA Immunoprecipitation, Expressing, Control, Western Blot

Journal: Virology

Article Title: Long non-coding RNA SUN2-AS1 acts as a negative regulator of ISGs transcription to promote flavivirus infection.

doi: 10.1016/j.virol.2024.110245

Figure Lengend Snippet: Fig. 4. SUN2-AS1 promotes ZIKV replication. (A) Two SUN2-AS1 knockout A549 cells were generated by CRISPR/Cas9 technology. The knockout effects of SUN2- AS1 were determined by qRT-PCR under both mock and ZIKV infection. (B) Cell viability of control, SUN2-AS1KO-1, and SUN2-AS1KO-2 cells was determined by CCK8 with or without ZIKV infection at 24 h. (C-E) Control and SUN2-AS1KO cells were infected with ZIKV at an MOI of 3 for 24 h. The viral RNA was extracted by QIAamp Viral RNA Mini Kit to detect the ZIKV RNA level by qRT-PCR (C). The cells and supernatants were harvested for Western blot (D) and plaque assay (E). (F) The pcDNA3.1 plasmid (empty vector) or pcDNA3.1-HA-SUN2-AS1 plasmid (1.0 μg) (SUN2-AS1OE) was transfected into A549 cells followed by infection with mock or ZIKV (MOI 3) for 24 h. Cells were harvested at 24 h.p.i., and the expression levels of SUN2-AS1 were determined by qRT-PCR. (G) Cell viability of pcDNA3.1 and SUN2-AS1OE cells was determined by CCK8 with or without ZIKV infection at 24 h. (H-J) pcDNA3.1 and SUN2-AS1OE cells were infected with ZIKV at MOI 3 for 24 h. The viral RNA level (H), E protein level (I), and titers of ZIKV particles (J) were measured by qRT-PCR, Western blot, and plaque assay, respectively. Human β-actin level was measured as an internal control and normalized to uninfected cells. All experiments were independently repeated three times. Data were shown as means ± S.D. *P < 0.1; ***P < 0.001; NS, not significant.

Article Snippet: The primary antibodies used in this study were as follows: Rabbit polyclonal antibodies against ZIKV E (GeneTex, GTX133314), rabbit polyclonal antibodies against SUN2 (Proteintech, 27556-1-AP), anti-phospho-STAT1 (HUABIO, ET1611-20), anti-STAT1 (HUABIO, ET1612-22), anti-MX1 (Proteintech, 13750-1-AP), anti-PKR (Proteintech, 18244-1-AP), anti-HA-tag mAb (MBL, M180-3) and rabbit polyclonal antibodies against GAPDH (Proteintech, I0494-I-AP).

Techniques: Knock-Out, Generated, CRISPR, Quantitative RT-PCR, Infection, Control, Western Blot, Plaque Assay, Plasmid Preparation, Transfection, Expressing

Journal: Virology

Article Title: Long non-coding RNA SUN2-AS1 acts as a negative regulator of ISGs transcription to promote flavivirus infection.

doi: 10.1016/j.virol.2024.110245

Figure Lengend Snippet: Fig. 5. SUN2-AS1 is indispensable for flavivirus infection. (A-B) Control and SUN2-AS1KO cells were infected with DENV2 NGC (A) or JEV (B) (MOI = 5). The supernatant was collected at 24 h.p.i. The viral titers were determined using the focus-forming assay (FFA). (C-D) Vector control (pcDNA3.1) and SUN2-AS1OE cells were infected with DENV2 NGC (C) or JEV (D) (MOI = 5). The supernatant was collected at 24 h.p.i. and the viral titers were determined by focus forming assay (FFA). (E-F) Control and SUN2-AS1KO cells were infected with VSV (MOI = 1) or HSV-1 (MOI = 1). The supernatant of VSV (E) and HSV-1 (F) was collected at 24 h.p. i. and the viral titers were determined by the plaque-forming assay. All experiments were independently repeated three times. The data were shown as means ± S.D. *p ≤0.1; **p ≤0.01; NS, not significant.

Article Snippet: The primary antibodies used in this study were as follows: Rabbit polyclonal antibodies against ZIKV E (GeneTex, GTX133314), rabbit polyclonal antibodies against SUN2 (Proteintech, 27556-1-AP), anti-phospho-STAT1 (HUABIO, ET1611-20), anti-STAT1 (HUABIO, ET1612-22), anti-MX1 (Proteintech, 13750-1-AP), anti-PKR (Proteintech, 18244-1-AP), anti-HA-tag mAb (MBL, M180-3) and rabbit polyclonal antibodies against GAPDH (Proteintech, I0494-I-AP).

Techniques: Infection, Control, Focus Forming Assay, Plasmid Preparation

Journal: Virology

Article Title: Long non-coding RNA SUN2-AS1 acts as a negative regulator of ISGs transcription to promote flavivirus infection.

doi: 10.1016/j.virol.2024.110245

Figure Lengend Snippet: Fig. 6. SUN2-AS1 inhibits the expression of ISGs via regulating the transcription of ISGs mRNA. (A) Control and SUN2-AS1KO cells were infected with ZIKV at an MOI of 3 for 24 h. Total cellular RNA was extracted by TRIzol to detect the mRNA level of IFNβ (A), ISG15 (B), MX1 (C), PKR (D), or OASL (E) by qRT-PCR. Human β-actin level was measured as an internal control and normalized to uninfected cells. (F-J) mRNA stability assay. Control and SUN2-AS1KO cells were treated with actinomycin D for 0, 3, 6, and 9 h. Cellular RNAs were extracted for qRT-PCR to measure the mRNA levels of GAPDH (F), ISG15 (G), MX1 (H), PKR (I), or OASL (J). Human β-actin level was measured as an internal control and normalized to untreated cells. Biologically independent experiments (n = 3) were conducted, and all data are shown as means ± S.D. P-values were calculated by one-way ANOVA. **P < 0.01, ***P < 0.001; ns, not significant.

Article Snippet: The primary antibodies used in this study were as follows: Rabbit polyclonal antibodies against ZIKV E (GeneTex, GTX133314), rabbit polyclonal antibodies against SUN2 (Proteintech, 27556-1-AP), anti-phospho-STAT1 (HUABIO, ET1611-20), anti-STAT1 (HUABIO, ET1612-22), anti-MX1 (Proteintech, 13750-1-AP), anti-PKR (Proteintech, 18244-1-AP), anti-HA-tag mAb (MBL, M180-3) and rabbit polyclonal antibodies against GAPDH (Proteintech, I0494-I-AP).

Techniques: Expressing, Control, Infection, Quantitative RT-PCR, Stability Assay

Journal: Virology

Article Title: Long non-coding RNA SUN2-AS1 acts as a negative regulator of ISGs transcription to promote flavivirus infection.

doi: 10.1016/j.virol.2024.110245

Figure Lengend Snippet: Fig. 7. The proviral role of SUN2-AS1 is mediated by suppressing ISGs. (A) Cytotoxic effect of DMSO and Ruxolitinib in control or SUN2-AS1KO cells was determined by CCK8. (B-E) Control and SUN2-AS1KO cells were infected with ZIKV at an MOI of 3 for 1 h. Then treated with 1 μM ruxolitinib. Cellular RNAs were extracted for qRT-PCR to measure the mRNA levels of ISG15 (B), MX1 (C), PKR (D), or OASL (E). Human β-actin level was measured as an internal control and normalized to uninfected cells. (F-H) Control and SUN2-AS1KO cells were infected with ZIKV (MOI = 3). Then treated with 1 μM ruxolitinib for 24 h. ZIKV RNA was extracted by the QIAamp Viral RNA Mini Kit and determine by qRT-PCR (F). Cell lysates were collected for Western blot to detect the MX1, PKR and ZIKV E protein level (G). And the supernatants were collected at 24 h.p.i for plaque assay (H). Biologically independent experiments (n = 3) were conducted, and all data are shown as means ± S.D. P-values were calculated by one-way ANOVA. *P < 0.1, **P < 0.01, ***P < 0.001; ns, not significant.

Article Snippet: The primary antibodies used in this study were as follows: Rabbit polyclonal antibodies against ZIKV E (GeneTex, GTX133314), rabbit polyclonal antibodies against SUN2 (Proteintech, 27556-1-AP), anti-phospho-STAT1 (HUABIO, ET1611-20), anti-STAT1 (HUABIO, ET1612-22), anti-MX1 (Proteintech, 13750-1-AP), anti-PKR (Proteintech, 18244-1-AP), anti-HA-tag mAb (MBL, M180-3) and rabbit polyclonal antibodies against GAPDH (Proteintech, I0494-I-AP).

Techniques: Control, Infection, Quantitative RT-PCR, Western Blot, Plaque Assay

Journal: bioRxiv

Article Title: SV40 exploits the Nesprin-2-SUN1-KPNA4 axis for stepwise targeting and entry into the host nucleus to promote infection

doi: 10.64898/2026.03.15.711898

Figure Lengend Snippet: (A) CV-1 cells transfected with the indicated siRNA were infected with SV40 and the resulting whole cell extracts were subjected to SDS-PAGE followed by immunoblotting. The extent of depletion by the KPNA1, KPNA2, and KPNA4 siRNA is shown in the Western blots below. Data were normalized to the Scr control. (B) The T-Ag band intensity in A was quantified by the FIJI software. Data were normalized to the Scr control. (C) CV-1 cells transfected with the indicated siRNA were infected with SV40 and the resulting whole cell extracts were subjected to SDS-PAGE and immunoblotting. (D) The T-Ag band intensity in C was quantified by the FIJI software. Data were normalized to the Scr control. (E) CV-1 cells were fixed and stained for Nesprin-2 (red) and either one of the KPNA1, KPNA2, or KPNA4 protein (green) and counterstained with DAPI (blue). Scale bar: 10 µm. (F) Pearson’s coefficient was used to quantify colocalization between KPNA1, KPNA2, or KPNA4 with Nesprin-2. Each data point represents one field of view with at least 10 cells. * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001

Article Snippet: Antibodies, along with the companies that were purchase from and the corresponding catalog numbers, are indicated: SUN1 (Invitrogen, MA5-47231), SUN2 (Proteintech, 27556-1-AP), β actin (Cell Signaling, 4967S), SV40 large T-antigen (Western blot: Santa Cruz Biotechnology, SC-53448; Immunofluorescence: Santa Cruz Biotechnology, SC-147), Nesprin-2 (IP and Immunofluorescence: Invitrogen, MA5-18075; Western blot: Bethyl, A305-393A), SV40 VP1 (Abcam, ab53977), SV40 VP2/3 (Abcam, ab53983), Bap31 (Invitrogen, MA 3002), Hsp90 (Santa Cruz Biotechnology, sc-13119), M2 FLAG (Millipore, F3165-1MG), FLAG (Millipore, F7425), BicD2 (Abcam, ab117818), KPNA1 (Proteintech, 18137-1-AP), KPNA2 (Invitrogen, 108191AP150UL), KPNA4 (Invitrogen, PA518239), Mab414 (Abcam, ab24609).

Techniques: Transfection, Infection, SDS Page, Western Blot, Control, Software, Staining

Journal: bioRxiv

Article Title: SV40 exploits the Nesprin-2-SUN1-KPNA4 axis for stepwise targeting and entry into the host nucleus to promote infection

doi: 10.64898/2026.03.15.711898

Figure Lengend Snippet: (A) CV-1 cells transfected with a scrambled (Scr) control siRNA or siRNA against SUN1 or SUN2 were harvested and the resulting whole cell extracts subjected to SDS-PAGE and immunoblotting with the indicated antibodies. β actin was used as a loading control. (B) CV-1 cells transfected with the indicated siRNA were infected with SV40, fixed, and stained for large T antigen (T-Ag). Data were normalized to the Scr control. (C) CV-1 cells transfected with the indicated siRNA were infected with SV40 and the resulting whole cell extracts were subjected to SDS-PAGE and immunoblotting. (D) The T-Ag band intensity in C was quantified by the FIJI software. Data were normalized to the Scr control. (E) Schematic of full-length (FL) SUN1 containing an N-terminal GFP tag (GFP-SUN1 FL), and a truncated SUN1 lacking the coiled-coil and SUN domain with also an N-terminal GFP tag (GFP-SUN1 ΔLU). (F) CV-1 cells transfected with the indicated constructs were fixed and stained for GFP (green) and counterstained with DAPI (blue). Scale bar: 10 µm. (G) CV-1 cells transfected with the Scr control siRNA or siRNA against SUN1 were also transfected with either the GFP-FLAG control construct or the indicated GFP-SUN1 construct. Cells were infected with SV40, fixed, and stained to assess T-Ag expression, as in 1B. Only GFP-expressing cells were analyzed and data were normalized to the Scr control with GFP-FLAG. (H) CV-1 cells were transfected with either Scr or siRNA against SUN1 or SUN2. The resulting whole cell extracts were subjected to SDS-PAGE and immunoblotted with the indicated antibodies. (I) The Nesprin-2 band intensity in H was quantified by the FIJI software and data were normalized to the Scr control. (J) CV-1 cells transfected with either Scr or siRNA against SUN1 or SUN2 were fixed and stained for Nesprin-2 (red) and counterstained with DAPI (blue). * p ≤ 0.05; ** p ≤ 0.01; ns= not significant. Scale bar: 10 µm

Article Snippet: Antibodies, along with the companies that were purchase from and the corresponding catalog numbers, are indicated: SUN1 (Invitrogen, MA5-47231), SUN2 (Proteintech, 27556-1-AP), β actin (Cell Signaling, 4967S), SV40 large T-antigen (Western blot: Santa Cruz Biotechnology, SC-53448; Immunofluorescence: Santa Cruz Biotechnology, SC-147), Nesprin-2 (IP and Immunofluorescence: Invitrogen, MA5-18075; Western blot: Bethyl, A305-393A), SV40 VP1 (Abcam, ab53977), SV40 VP2/3 (Abcam, ab53983), Bap31 (Invitrogen, MA 3002), Hsp90 (Santa Cruz Biotechnology, sc-13119), M2 FLAG (Millipore, F3165-1MG), FLAG (Millipore, F7425), BicD2 (Abcam, ab117818), KPNA1 (Proteintech, 18137-1-AP), KPNA2 (Invitrogen, 108191AP150UL), KPNA4 (Invitrogen, PA518239), Mab414 (Abcam, ab24609).

Techniques: Transfection, Control, SDS Page, Western Blot, Infection, Staining, Software, Construct, Expressing

Journal: bioRxiv

Article Title: SV40 exploits the Nesprin-2-SUN1-KPNA4 axis for stepwise targeting and entry into the host nucleus to promote infection

doi: 10.64898/2026.03.15.711898

Figure Lengend Snippet: (A) CV-1 cells transfected with a scrambled (Scr) control siRNA or siRNA against SUN1 or SUN2 were harvested and the resulting whole cell extracts subjected to SDS-PAGE and immunoblotting with the indicated antibodies. β actin was used as a loading control. (B) CV-1 cells transfected with the indicated siRNA were infected with SV40, fixed, and stained for large T antigen (T-Ag). Data were normalized to the Scr control. (C) CV-1 cells transfected with the indicated siRNA were infected with SV40 and the resulting whole cell extracts were subjected to SDS-PAGE and immunoblotting. (D) The T-Ag band intensity in C was quantified by the FIJI software. Data were normalized to the Scr control. (E) Schematic of full-length (FL) SUN1 containing an N-terminal GFP tag (GFP-SUN1 FL), and a truncated SUN1 lacking the coiled-coil and SUN domain with also an N-terminal GFP tag (GFP-SUN1 ΔLU). (F) CV-1 cells transfected with the indicated constructs were fixed and stained for GFP (green) and counterstained with DAPI (blue). Scale bar: 10 µm. (G) CV-1 cells transfected with the Scr control siRNA or siRNA against SUN1 were also transfected with either the GFP-FLAG control construct or the indicated GFP-SUN1 construct. Cells were infected with SV40, fixed, and stained to assess T-Ag expression, as in 1B. Only GFP-expressing cells were analyzed and data were normalized to the Scr control with GFP-FLAG. (H) CV-1 cells were transfected with either Scr or siRNA against SUN1 or SUN2. The resulting whole cell extracts were subjected to SDS-PAGE and immunoblotted with the indicated antibodies. (I) The Nesprin-2 band intensity in H was quantified by the FIJI software and data were normalized to the Scr control. (J) CV-1 cells transfected with either Scr or siRNA against SUN1 or SUN2 were fixed and stained for Nesprin-2 (red) and counterstained with DAPI (blue). * p ≤ 0.05; ** p ≤ 0.01; ns= not significant. Scale bar: 10 µm

Article Snippet: Antibodies, along with the companies that were purchase from and the corresponding catalog numbers, are indicated: SUN1 (Invitrogen, MA5-47231), SUN2 (Proteintech, 27556-1-AP), β actin (Cell Signaling, 4967S), SV40 large T-antigen (Western blot: Santa Cruz Biotechnology, SC-53448; Immunofluorescence: Santa Cruz Biotechnology, SC-147), Nesprin-2 (IP and Immunofluorescence: Invitrogen, MA5-18075; Western blot: Bethyl, A305-393A), SV40 VP1 (Abcam, ab53977), SV40 VP2/3 (Abcam, ab53983), Bap31 (Invitrogen, MA 3002), Hsp90 (Santa Cruz Biotechnology, sc-13119), M2 FLAG (Millipore, F3165-1MG), FLAG (Millipore, F7425), BicD2 (Abcam, ab117818), KPNA1 (Proteintech, 18137-1-AP), KPNA2 (Invitrogen, 108191AP150UL), KPNA4 (Invitrogen, PA518239), Mab414 (Abcam, ab24609).

Techniques: Transfection, Control, SDS Page, Western Blot, Infection, Staining, Software, Construct, Expressing

Journal: bioRxiv

Article Title: SV40 exploits the Nesprin-2-SUN1-KPNA4 axis for stepwise targeting and entry into the host nucleus to promote infection

doi: 10.64898/2026.03.15.711898

Figure Lengend Snippet: (A) CV-1 cells transfected with either a scrambled (Scr) control siRNA or siRNA against SUN1 or SUN2 were infected with SV40 in the presence of actinomycin D (ActD). Cells were stained with anti-VP2/3 (green), mAb414 (red), and counterstained with DAPI (blue). Scale bar: 10 µm. (B) The percent of cells with a discrete VP2/3+ signal on or proximal to (i.e. within 0.3 µm) the nuclear membrane were quantified and normalized to the Scr control. (C) As in A, except stained with Bap31 (red). Scale bar: 10 µm. (D) CV-1 cells transfected with the indicated siRNA were infected with SV40 and processed using the ER-to-cytosol transport assay as described in (). The resulting cytosol and membrane fractions were subjected to SDS-PAGE followed by immunoblotting with the indicated antibodies. ( E) The cytosol fraction from D was layered on a discontinuous sucrose gradient and centrifuged to generate individual fractions (see Materials and Methods). Fractions were subjected to SDS-PAGE followed by immunoblotting for VP1. The VP1 signal from fractions 1-7 (representing disassembled virus) and fraction 8 (representing assembled virus) was quantified and normalized to the Scr control. ** p ≤ 0.01; ns= not significant.

Article Snippet: Antibodies, along with the companies that were purchase from and the corresponding catalog numbers, are indicated: SUN1 (Invitrogen, MA5-47231), SUN2 (Proteintech, 27556-1-AP), β actin (Cell Signaling, 4967S), SV40 large T-antigen (Western blot: Santa Cruz Biotechnology, SC-53448; Immunofluorescence: Santa Cruz Biotechnology, SC-147), Nesprin-2 (IP and Immunofluorescence: Invitrogen, MA5-18075; Western blot: Bethyl, A305-393A), SV40 VP1 (Abcam, ab53977), SV40 VP2/3 (Abcam, ab53983), Bap31 (Invitrogen, MA 3002), Hsp90 (Santa Cruz Biotechnology, sc-13119), M2 FLAG (Millipore, F3165-1MG), FLAG (Millipore, F7425), BicD2 (Abcam, ab117818), KPNA1 (Proteintech, 18137-1-AP), KPNA2 (Invitrogen, 108191AP150UL), KPNA4 (Invitrogen, PA518239), Mab414 (Abcam, ab24609).

Techniques: Transfection, Control, Infection, Staining, Membrane, Transport Assay, SDS Page, Western Blot, Virus

Journal: bioRxiv

Article Title: SV40 exploits the Nesprin-2-SUN1-KPNA4 axis for stepwise targeting and entry into the host nucleus to promote infection

doi: 10.64898/2026.03.15.711898

Figure Lengend Snippet: (A) Endogenous KPNA4 was IPed from whole cell extracts derived from SV40-infected CV-1 cells. The precipitated samples were subjected to SDS-PAGE and immunoblotting. (B) As in A, except CV-1 cells were subjected to SUN1 KD. (C) CV-1 cells transfected with the indicated siRNA and SUN1-3xFLAG were infected with SV40. SUN1-3xFLAG was IPed from the resulting whole cell extracts, and the precipitated samples subjected to SDS-PAGE and immunoblotting. (D) The VP1 band intensity in C was quantified by the FIJI software. Data were normalized to the Scr condition. (E) As in , except cells were depleted of KPNA4 and stained for FLAG (red), VP2/3 (green), and counterstained with DAPI (blue). Scale bar: 10 µm. (F) Manders’ coefficient was used to quantify overlap of VP2/3+ signal on SUN1-3xFLAG. Each data point represents one cell. (G) CV-1 cells transfected with the indicated siRNA were infected with SV40. Cells were fixed and stained for mAb414 (red), VP2/3 (green), and counterstained with DAPI (blue). Scale bar: 10 µm. (H) The percent of cells with a discrete VP2/3+ signal in the nucleus in G was quantified and normalized to the Scr control condition. (I) Model: After SV40 reaches the cytosol from the ER (foci), the viral particle is partially disassembled and targeted to the Nesprin-2-SUN1 nuclear membrane protein complex (Step 1). The virus is then released to the KPNA4 importin α receptor, which translocates SV40 into the nucleus to promote infection (Step 2). Schematic created in biorender.com. * p ≤ 0.05, ** p ≤ 0.01

Article Snippet: Antibodies, along with the companies that were purchase from and the corresponding catalog numbers, are indicated: SUN1 (Invitrogen, MA5-47231), SUN2 (Proteintech, 27556-1-AP), β actin (Cell Signaling, 4967S), SV40 large T-antigen (Western blot: Santa Cruz Biotechnology, SC-53448; Immunofluorescence: Santa Cruz Biotechnology, SC-147), Nesprin-2 (IP and Immunofluorescence: Invitrogen, MA5-18075; Western blot: Bethyl, A305-393A), SV40 VP1 (Abcam, ab53977), SV40 VP2/3 (Abcam, ab53983), Bap31 (Invitrogen, MA 3002), Hsp90 (Santa Cruz Biotechnology, sc-13119), M2 FLAG (Millipore, F3165-1MG), FLAG (Millipore, F7425), BicD2 (Abcam, ab117818), KPNA1 (Proteintech, 18137-1-AP), KPNA2 (Invitrogen, 108191AP150UL), KPNA4 (Invitrogen, PA518239), Mab414 (Abcam, ab24609).

Techniques: Derivative Assay, Infection, SDS Page, Western Blot, Transfection, Software, Staining, Control, Membrane, Virus

Journal: bioRxiv

Article Title: SV40 exploits the Nesprin-2-SUN1-KPNA4 axis for stepwise targeting and entry into the host nucleus to promote infection

doi: 10.64898/2026.03.15.711898

Figure Lengend Snippet: (A) Endogenous SUN1 was immunoprecipitated (IPed) from whole cell extracts derived from SV40-infected CV-1 cells. Bound DNA was eluted from the precipitated material and subjected to PCR to detect the SV40 genomic DNA. (B) CV-1 cells expressing the indicated FLAG-tagged constructs were infected with SV40. FLAG-tagged proteins were IPed from the resulting whole cell extracts, the precipitated samples eluted and subjected to SDS-PAGE followed by immunoblotting with the indicated antibodies. * indicates degraded SUN proteins. (C) As in B, except the precipitated samples were subjected to PCR to detect the SV40 genome. (D) Nesprin-2 was IPed from whole cell extracts derived from CV-1 cells expressing the indicated constructs. The precipitated samples were subjected to SDS-PAGE and immunoblotting with the indicated antibodies. * indicates degraded SUN proteins (E) As in B, except SUN1 ΔLU-3xFLAG was also used. (F) CV-1 cells transfected with the indicated siRNA and construct were infected with SV40. FLAG-tagged proteins were IPed from the resulting whole cell extracts and processed as in B. * indicates degraded SUN1 protein. (G) CV-1 cells transfected with the indicated siRNA were infected with SV40. The resulting whole cell extracts were subjected to Nesprin-2 IP and the precipitated samples subjected to SDS-PAGE followed by immunoblotting with the indicated antibodies. (H) Endogenous BicD2 was IPed from whole cell extracts derived from CV-1 cells and the precipitated samples subjected to SDS-PAGE and immunoblotting. (I) Endogenous BicD2 was IPed from whole cell extracts derived from mock or SV40-infected CV-1 cells. The precipitated samples were subjected to SDS-PAGE and immunoblotting.

Article Snippet: Antibodies, along with the companies that were purchase from and the corresponding catalog numbers, are indicated: SUN1 (Invitrogen, MA5-47231), SUN2 (Proteintech, 27556-1-AP), β actin (Cell Signaling, 4967S), SV40 large T-antigen (Western blot: Santa Cruz Biotechnology, SC-53448; Immunofluorescence: Santa Cruz Biotechnology, SC-147), Nesprin-2 (IP and Immunofluorescence: Invitrogen, MA5-18075; Western blot: Bethyl, A305-393A), SV40 VP1 (Abcam, ab53977), SV40 VP2/3 (Abcam, ab53983), Bap31 (Invitrogen, MA 3002), Hsp90 (Santa Cruz Biotechnology, sc-13119), M2 FLAG (Millipore, F3165-1MG), FLAG (Millipore, F7425), BicD2 (Abcam, ab117818), KPNA1 (Proteintech, 18137-1-AP), KPNA2 (Invitrogen, 108191AP150UL), KPNA4 (Invitrogen, PA518239), Mab414 (Abcam, ab24609).

Techniques: Immunoprecipitation, Derivative Assay, Infection, Expressing, Construct, SDS Page, Western Blot, Transfection

Journal: bioRxiv

Article Title: SV40 exploits the Nesprin-2-SUN1-KPNA4 axis for stepwise targeting and entry into the host nucleus to promote infection

doi: 10.64898/2026.03.15.711898

Figure Lengend Snippet: (A) Schematic of full-length N-terminal FLAG-tagged SUN1 and SUN2, and of chimeric SUN proteins with swapped SUN domains. (B) CV-1 cells transfected with the indicated construct were fixed, stained for FLAG (green), and counterstained with DAPI (blue). Scale bar: 10 µm. (C) CV-1 cells transfected with the indicated construct and siRNA were infected with SV40, fixed, and stained for T-antigen, FLAG, and counterstained with DAPI. The percentage of transfected cells expressing T-antigen was quantified and normalized to the GFP+Scr control condition. (D) CV-1 cells transfected with the indicated construct were infected with SV40. FLAG-tagged proteins were IPed from the resulting whole cell extracts. The precipitated materials were subjected to PCR to detect SV40 genomic DNA or subjected to SDS-PAGE and immunoblotting. The dotted line indicates intervening lanes were removed with adjacent lanes spliced from the same immunoblot. ** p ≤ 0.01

Article Snippet: Antibodies, along with the companies that were purchase from and the corresponding catalog numbers, are indicated: SUN1 (Invitrogen, MA5-47231), SUN2 (Proteintech, 27556-1-AP), β actin (Cell Signaling, 4967S), SV40 large T-antigen (Western blot: Santa Cruz Biotechnology, SC-53448; Immunofluorescence: Santa Cruz Biotechnology, SC-147), Nesprin-2 (IP and Immunofluorescence: Invitrogen, MA5-18075; Western blot: Bethyl, A305-393A), SV40 VP1 (Abcam, ab53977), SV40 VP2/3 (Abcam, ab53983), Bap31 (Invitrogen, MA 3002), Hsp90 (Santa Cruz Biotechnology, sc-13119), M2 FLAG (Millipore, F3165-1MG), FLAG (Millipore, F7425), BicD2 (Abcam, ab117818), KPNA1 (Proteintech, 18137-1-AP), KPNA2 (Invitrogen, 108191AP150UL), KPNA4 (Invitrogen, PA518239), Mab414 (Abcam, ab24609).

Techniques: Transfection, Construct, Staining, Infection, Expressing, Control, SDS Page, Western Blot

Journal: bioRxiv

Article Title: SV40 exploits the Nesprin-2-SUN1-KPNA4 axis for stepwise targeting and entry into the host nucleus to promote infection

doi: 10.64898/2026.03.15.711898

Figure Lengend Snippet: (A) CV-1 cells transfected with the indicated siRNA were infected with SV40 and the resulting whole cell extracts were subjected to SDS-PAGE followed by immunoblotting. The extent of depletion by the KPNA1, KPNA2, and KPNA4 siRNA is shown in the Western blots below. Data were normalized to the Scr control. (B) The T-Ag band intensity in A was quantified by the FIJI software. Data were normalized to the Scr control. (C) CV-1 cells transfected with the indicated siRNA were infected with SV40 and the resulting whole cell extracts were subjected to SDS-PAGE and immunoblotting. (D) The T-Ag band intensity in C was quantified by the FIJI software. Data were normalized to the Scr control. (E) CV-1 cells were fixed and stained for Nesprin-2 (red) and either one of the KPNA1, KPNA2, or KPNA4 protein (green) and counterstained with DAPI (blue). Scale bar: 10 µm. (F) Pearson’s coefficient was used to quantify colocalization between KPNA1, KPNA2, or KPNA4 with Nesprin-2. Each data point represents one field of view with at least 10 cells. * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001

Article Snippet: Antibodies, along with the companies that were purchase from and the corresponding catalog numbers, are indicated: SUN1 (Invitrogen, MA5-47231), SUN2 (Proteintech, 27556-1-AP), β actin (Cell Signaling, 4967S), SV40 large T-antigen (Western blot: Santa Cruz Biotechnology, SC-53448; Immunofluorescence: Santa Cruz Biotechnology, SC-147), Nesprin-2 (IP and Immunofluorescence: Invitrogen, MA5-18075; Western blot: Bethyl, A305-393A), SV40 VP1 (Abcam, ab53977), SV40 VP2/3 (Abcam, ab53983), Bap31 (Invitrogen, MA 3002), Hsp90 (Santa Cruz Biotechnology, sc-13119), M2 FLAG (Millipore, F3165-1MG), FLAG (Millipore, F7425), BicD2 (Abcam, ab117818), KPNA1 (Proteintech, 18137-1-AP), KPNA2 (Invitrogen, 108191AP150UL), KPNA4 (Invitrogen, PA518239), Mab414 (Abcam, ab24609).

Techniques: Transfection, Infection, SDS Page, Western Blot, Control, Software, Staining