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