Use of E2F2 in inhibiting the proliferation of HIV-1 and HIV-2

Abstract

The application discloses application of E2F2 in inhibition of proliferation of HIV-1 and HIV-2, and specifically discloses application of any one of the following (1)-(3) in at least one of the following (a)-(c): (1) E2F2 protein; (2) nucleic acid molecule coding E2F2 protein; (3) expression cassette containing the nucleic acid molecule, recombinant vector or transgenic cell; (a) inhibition of proliferation of HIV-1 in cells, or preparation of products for inhibiting proliferation of HIV-1 in cells; (b) inhibition of proliferation of HIV-2 in cells, or preparation of products for inhibiting proliferation of HIV-2 in cells; (c) inhibition of proliferation of SIVnano in cells, or preparation of products for inhibiting proliferation of SIVnano in cells.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, specifically to the application of E2F2 overexpression in inhibiting or treating HIV infection. Background Technology

[0002] Untreated HIV replication leads to progressive loss of CD4+ T cells and widespread immune abnormalities, increasing the risk of infections and cancerous complications. HIV infection also causes several common diseases, including cardiovascular disease, bone disease, and kidney and liver dysfunction. Antiretroviral drugs are highly effective in suppressing HIV replication. For individuals with access to and use of antiretroviral drugs, consistent use of these drugs in combination with antiretroviral therapy can lead to durable (and potentially lifelong) suppression of viral replication. Viral suppression restores the immune system and nearly eliminates the risk of developing acquired immunodeficiency syndrome (AIDS).

[0003] E2F transcription factor 2 (E2F2) is a member of the E2F transcription factor family. The classic view is that some E2Fs act as "activators" of cell cycle gene expression, while others act as "inhibitors," playing seemingly contradictory roles in cell cycle, proliferation, apoptosis, inflammation, cell migration, and invasion. However, relatively little research has been done on the role of E2F2 in viral infection, and whether E2F2 possesses anti-HIV infection function and its mechanism remain unclear. Summary of the Invention

[0004] The purpose of this invention is to provide an application of E2F2 in inhibiting the proliferation of HIV-1 and HIV-2.

[0005] This invention claims protection for the use of any one of the substances shown in (1)-(3) in at least one of (a)-(c):

[0006] (1) E2F2 protein;

[0007] (2) Nucleic acid molecules encoding the E2F2 protein;

[0008] (3) An expression cassette, recombinant vector, or transgenic cell containing the nucleic acid molecule;

[0009] (a) Inhibit HIV-1 proliferation in cells, or prepare products for inhibiting HIV-1 proliferation in cells;

[0010] (b) Inhibit HIV-2 proliferation in cells, or prepare products for inhibiting HIV-2 proliferation in cells;

[0011] (c) Inhibit the proliferation of SIVnano in cells, or prepare a product for inhibiting the proliferation of SIVnano in cells.

[0012] Furthermore, the recombinant vector is psLenti-E2F2-3Flag or pcDNA3.1-E2F2-3Flag plasmid.

[0013] This invention claims protection for the use of substances capable of promoting the expression and / or activity of (1) or (2) in at least one of (a)-(c):

[0014] (1) E2F2 protein;

[0015] (2) Nucleic acid molecules encoding the E2F2 protein;

[0016] (a) Inhibit HIV-1 proliferation in cells, or prepare products for inhibiting HIV-1 proliferation in cells;

[0017] (b) Inhibit HIV-2 proliferation in cells, or prepare products for inhibiting HIV-2 proliferation in cells;

[0018] (c) Inhibit the proliferation of SIVnano in cells, or prepare a product for inhibiting the proliferation of SIVnano in cells.

[0019] Furthermore, the substance that promotes the expression and / or activity of (1) or (2) is a recombinant vector containing the nucleic acid molecule, wherein the recombinant vector is psLenti-E2F2-3Flag or pcDNA3.1-E2F2-3Flag plasmid.

[0020] Furthermore, the product is a pharmaceutical product.

[0021] This invention claims protection for the use of a substance capable of inhibiting the expression and / or reducing the activity of (1) or (2) below in at least one of (a)-(c):

[0022] (1) E2F2 protein;

[0023] (2) Nucleic acid molecules encoding the E2F2 protein;

[0024] (a) Promoting the proliferation of HIV-1 in cells, or preparing products for promoting the proliferation of HIV-1 in cells;

[0025] (b) Promote the proliferation of HIV-2 in cells, or prepare products for promoting the proliferation of HIV-2 in cells;

[0026] (c) Promote the proliferation of SIVnano in cells, or prepare products for promoting the proliferation of SIVnano in cells.

[0027] Furthermore, the product used to promote HIV-1 proliferation in cells is a cell model with enhanced HIV-1 proliferation capacity, or a substance used to prepare a cell model with enhanced HIV-1 proliferation capacity;

[0028] The product used to promote HIV-2 proliferation in cells is a cell model with enhanced HIV-2 proliferation capacity, or a substance used to prepare a cell model with enhanced HIV-2 proliferation capacity; the product used to promote SIVnano proliferation in cells is a cell model with enhanced SIVnano proliferation capacity, or a substance used to prepare a cell model with enhanced SIVnano proliferation capacity.

[0029] Furthermore, the substance capable of inhibiting (1) or (2) expression and / or reducing activity is the shRNA 124068 plasmid used to knock out or reduce E2F2 expression.

[0030] Furthermore, the E2F2 protein is a protein with an amino acid sequence as shown in Sequence Listing 1.

[0031] The beneficial effects of this invention are as follows: E2F2 overexpression can significantly inhibit HIV-1 infection, and this inhibitory effect is independent of the plasmid backbone used. Furthermore, this inhibitory effect is consistently observed at low to high HIV-1 infection doses. Increasing E2F2 expression levels can enhance its ability to inhibit HIV-1 infection to a certain extent, while decreasing its expression levels can promote HIV-1 infection to a certain extent. E2F2 also exhibits some inhibitory ability against other retroviruses such as HIV-2 and SIV. It has been further found that E2F2's function in inhibiting HIV-1 is based on its ability to prevent HIV-1 integration into the host genome. Overexpression of E2F2 may play an important role in anti-HIV-1 infection and treatment of HIV carriers. Attached Figure Description

[0032] Figure 1 To investigate the inhibitory effect of E2F2 overexpression on different infectious doses of HIV-1

[0033] Figure 2 To inhibit HIV-1 proliferation in a dose-dependent manner by overexpressing E2F2.

[0034] Figure 3 Knocking down E2F2 to some extent promotes HIV-1 proliferation;

[0035] Figure 4 This demonstrates the inhibitory effect of E2F2 on HIV-2 and SIVnano;

[0036] Figure 5 E2F2 prevents HIV-1 infection by inhibiting viral integration. Detailed Implementation

[0037] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0038] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0039] Unless otherwise specified, the quantitative experiments in the following examples are all repeated three times, and the results are averaged.

[0040] The cells, viruses, and plasmids used in the following examples are as follows: The 293T cells and TZM-bl cells used in the experiments were both obtained from ATCC. The 293T and TZM-bl cells were cultured in DMEM medium supplemented with 10% fetal bovine serum and 2% penicillin-streptomycin antibiotics, respectively, and RPMI 1640 medium. The plasmids pcDNA3.1-MCS-3Flag and pcDNA3.1-E2F2-3Flag were synthesized by mBio. The plasmids pCD4 and pCXCR4, and the virus strain NL4-3.Luc.VSVG / X4 Env / SIVnano / MLV were obtained from the Biotherapy Research Laboratory of China Medical University. The E2F2 shRNA plasmid used was purchased from Horizon Discovery, with clone IDs V2LHS_172943, V2LHS_403193, V2LHS_403192, V2LHS_403191, and V2LHS_324068.

[0041] The animal viruses involved in this application are available to the public from the applicant in accordance with the relevant national biosafety regulations. These biological materials are only for repeating the relevant experiments of this invention and may not be used for other purposes.

[0042] Biological reagents involved in the following examples: LipoD293 TM Transfection reagents were purchased from SignaGen; lipofectamine™ 3000 transfection reagents were purchased from Thermo Fisher Scientific; luciferase assay kit (Cat. E1500) was purchased from Promega; RNeasy Mini Kit (Cat. 74104) was purchased from Qiagen; PrimeScript... TMRTreagent Kit with gDNA Eraser (Cat.RR047Q) purchased from TAKARA; TB Green Premix ExTaq TM II (Cat.RR820A) was purchased from TaKaRa, Gibco. TM Fetal bovine serum (Cat. 10091148) was purchased from Thermo Fisher Scientific, Hyclone. TM Penicillin-Streptomycin Antibiotic Mixture (Cat. SV30010), Hyclone TM RPMI 1640 medium (Cat. SH30027.01), Hyclone TM DMEM medium (Cat. SH30243.01) was purchased from Cytiva.

[0043] Addressing the challenges of treating HIV latent infection and rebound after drug withdrawal, this research focuses on the key factors and mechanisms by which host cells regulate HIV latent infection and rebound after drug withdrawal. This invention demonstrates that E2F2 can inhibit HIV infection through E2F2 overexpression and silencing.

[0044] Example 1: Inhibitory effect of E2F2 overexpression on different infectious doses of HIV-1

[0045] The E2F2 protein (encoding sequence as shown in NM_004091.4, encoding amino acids as shown in sequence 1) was fused with a 3Flag tag and cloned into the psLenti-3Flag plasmid and pcDNA3.1-3Flag plasmid to obtain the psLenti-E2F2-3Flag / pcDNA3.1-E2F2-3Flag plasmid.

[0046] 1. Construction of overexpression plasmids

[0047] Replace the sequence between the EcoRI and XbaI sites on the psLenti-3Flag plasmid with the E2F2 coding sequence (as shown in NM_004091.4) and the 3Flag tag, leaving other sequences unchanged, to obtain the plasmid psLenti-E2F2-3Flag.

[0048] Replace the sequence between the BamHI and AgeI sites on the pcDNA3.1-3Flag plasmid with the coding sequence of E2F2 (as shown in NM_004091.4), leaving other sequences unchanged, to obtain the plasmid pcDNA3.1-E2F2-3Flag.

[0049] 2. Inhibitory effect of E2F2 overexpression on different infectious doses of HIV-1

[0050] 18 to 24 hours before transfection, apply 0.8-1 mg / well. 10 6 293T cells were seeded in 6-well plates at a volume of 1 mL. For each well, 1.0 μg of pcDNA3.1-E2F2-3Flag / psLenti-E2F2-3Flag / psLenti-3Flag / Mock DNA was diluted in 100 μL of serum-free DMEM preheated at 37 °C. Gently vortex to mix; add 3 times the plasmid volume of LipoD293 to each tube; gently vortex to mix; incubate at room temperature for 10 minutes to form the DNA-LipoD293™ transfection complex; add the LipoD293 / DNA mixture dropwise to the medium in each well, gently swirl the plate to homogenize the mixture; remove the medium containing the LipoD293 / DNA complex 4–6 hours after transfection and replace it with 1 mL of pre-warmed medium containing whole serum / antibiotics at 37°C; 24 hours later, infect cells with 1 / 5 / 10 / 25 / 50 / 100 ng p24 equivalent of NL4-3.Luc (VSV-G) strain, thawed at room temperature. After 24 hours of incubation, add 300 μL of Passive Lysis Buffer, shake at room temperature for 15 minutes, mix with Luciferase Assay Reagent at a 2:1 ratio, and read the luciferase value using a microplate reader.

[0051] The results are as follows Figure 1 As shown, after transfection with the E2F2 plasmid based on the psLenti backbone, the luciferase values ​​of HIV at all infection doses decreased significantly. Similarly, after transfection with the 1.0 μg E2F2 plasmid based on the pcDNA3.1 backbone, the Luc values ​​of HIV at all infection doses decreased significantly. This indicates that E2F2 overexpression strongly inhibits HIV-1 proliferation in cells, and its anti-HIV-1 infection effect is unrelated to the plasmid backbone, but rather due to its own antiviral function.

[0052] Example 2 illustrates the inhibitory effect of overexpressing different levels of E2F2 on HIV-1.

[0053] 18 to 24 hours before transfection, use 0.8-1 mg / well. 10 6293T cells were seeded at 1 mL per well in a 6-well plate. For transfection, pcDNA3.1-E2F2-3Flag, psLenti-E2F2-3Flag, psLenti-3Flag, and Mock DNA were diluted to 100 μL of serum-free DMEM at doses of 0.5, 1.0, and 1.5 μg, respectively. The DMEM was preheated to 37°C. The cells were gently vortexed to mix. Three times the plasmid volume of LipoD293 was added to each well; the cells were gently vortexed again; and the cells were incubated at room temperature for 10 minutes to form the DNA-LipoD293™ transfection complex. The LipoD293 / DNA mixture was added dropwise to the medium in each well, and the plate was gently rotated to ensure homogeneity. 4–6 hours after transfection, the medium containing the LipoD293 / DNA complex was removed and replaced with medium containing whole serum / antibiotics. 24 hours later, the cells were infected with 100 ng p24 equivalent of the NL4-3.Luc (VSV-G) strain. After incubation for 24 hours, add 300 μL of Passive Lysis Buffer, shake at room temperature for 15 minutes, mix with Luciferase AssayReagent at a ratio of 2:1, and read the luciferase value using a microplate reader.

[0054] The results are as follows Figure 2 As shown, after transfection with 1.0 and 2.0 μg E2F2 plasmids based on the psLenti backbone, the HIV Luc value decreased significantly, by 7.69-fold and 10.80-fold, respectively, while the HIV Luc value did not decrease after transfection with 0.5 μg E2F2 plasmid. Similarly, after transfection with 1.0 and 2.0 μg E2F2 plasmids based on the pcDNA3.1 backbone, the HIV Luc value decreased significantly, by 7.69-fold and 8.03-fold, respectively. This indicates that overexpression of E2F2 has a strong ability to inhibit HIV-1 proliferation, and its anti-HIV-1 proliferation effect is independent of the plasmid backbone. At high expression levels, the antiviral function of E2F2 is enhanced to a certain extent.

[0055] Example 3 demonstrates how E2F2 knockdown promotes HIV-1 proliferation in cells.

[0056] 1. Determine the optimal shRNA:

[0057] Considering the low endogenous level of E2F2 in 293 cells, this embodiment transfected shRNA into the gene overexpressing E2F2 to confirm the knockdown efficiency of E2F2.

[0058] 18 to 24 hours before transfection, use 0.8-1 mg / well. 10 6293T cells were seeded at 1 mL in a 6-well plate. For each well, 1.5 μg shRNA and 0.5 μg F2-3Flag DNA were diluted in 100 μL of preheated serum-free DMEM at 37°C. The mixture was gently vortexed. 6 μL of LipoD293 was added to each well; the mixture was gently vortexed again; and the plate was incubated at room temperature for 10 minutes to form the DNA-LipoD293™ transfection complex. 100 μL of the LipoD293 / DNA mixture was added dropwise to the medium in each well, and the plate was gently rotated to ensure homogeneity. 4–6 hours post-transfection, the medium containing the LipoD293 / DNA complex was removed and replaced with medium containing whole serum / antibiotics. 48 hours later, the knockdown efficiency of each candidate shRNA on E2F2 cells was assessed by Western blotting.

[0059] The shRNAs 403191, 403192, 403193, and 172943, and the control plasmid shRNA NT (corresponding to E2F2 shRNA plasmids with clone IDs V2LHS_403191, V2LHS_403192, V2LHS_403193, V2LHS_172943, and V2LHS_324068, respectively) were processed according to the above method, and the results are as follows. Figure 3 As shown.

[0060] 2. Reduced E2F2 expression indicates decreased resistance to HIV-1 infection:

[0061] 18 to 24 hours before transfection, use 0.8-1 mg / well. 10 6293T cells were seeded at 1 mL in a 6-well plate. For each well, 1.0 or 1.5 μg F2-3 Flag and 1.0 or 0.5 μg shRNA NT / shRNA124068 DNA were diluted in 100 μL of preheated serum-free DMEM at 37°C. The mixture was gently vortexed. 6 μL of LipoD293 was added to each well; the mixture was gently vortexed again; and the cells were incubated at room temperature for 10 minutes to form the DNA-LipoD293™ transfection complex. 100 μL of the LipoD293 / DNA mixture was added dropwise to the medium in each well, and the plate was gently rotated to ensure homogeneity. 4–6 hours after transfection, the medium containing the LipoD293 / DNA complex was removed and replaced with medium containing whole serum / antibiotics. 24 hours later, the cells were infected with NL4-3.Luc (VSV-G) (1000 ng p24). After incubation for 24 hours, add 300 μL of Passive Lysis Buffer, shake at room temperature for 15 minutes, mix with Luciferase Assay Reagent at a ratio of 2:1, and read the luciferase value using a microplate reader.

[0062] The results are as follows Figure 3 As shown, based on E2F2 overexpression, the E2F2 expression levels transfected with control plasmid shRNA NT and candidate plasmids shRNA 403191, 403192, 403193, and 172943 were basically the same, indicating that these candidate shRNAs did not have the function of inhibiting E2F2 expression; candidate shRNA 124068 significantly reduced the expression level of E2F2, therefore shRNA 124068 was used as the shRNA plasmid in subsequent HIV-1 Luc detection. The results showed that when the ratio of E2F2 plasmid to shRNA plasmid was 1:1, the HIV-1 Luc value in the shRNA124068 co-transfection group decreased to some extent compared to the shRNA NT co-transfection group. When the ratio of E2F2 plasmid to shRNA plasmid was 3:1, the HIV-1 Luc value in the shRNA124068 co-transfection group increased to some extent compared to the shRNA NT co-transfection group. Furthermore, compared to the 1:1 ratio, the HIV-1 Luc value in the 3:1 ratio shRNA NT co-transfection group increased significantly, by approximately 1-2 times. These results indicate that the anti-HIV-1 ability of E2F2 is positively correlated with its expression level, and that shRNA knockdown of E2F2 can promote HIV-1 proliferation in cells to some extent.

[0063] Example 4 illustrates the inhibitory effect of E2F2 on HIV-2 and SIVnano.

[0064] The above studies demonstrate the role of E2F2 in HIV-1 proliferation. This section investigates the role of E2F2 in the proliferation of other retroviruses.

[0065] 18 to 24 hours before transfection, use 0.8-1 mg / well. 10 6 293T cells were seeded at 1 mL per well in a 6-well plate. For each well, 0.5 / 1.0 / 2.0 μg pcDNA3.1-F2-3Flag / Mock DNA was diluted to 100 μL in preheated serum-free DMEM at 37°C. The mixture was gently vortexed. Three times the plasmid volume of LipoD293 was added to each well; the mixture was gently vortexed again; the plate was incubated at room temperature for 10 minutes to form the DNA-LipoD293™ transfection complex. The LipoD293 / DNA mixture was added dropwise to the medium in each well, and the plate was gently rotated to ensure homogeneity. 4–6 hours post-transfection, the medium containing the LipoD293 / DNA complex was removed and replaced with medium containing whole serum / antibiotics. 24 hours later, the cells were infected with HIV-2-Luc (VSV-G) / SIVnano (VSV-G) (3 mL viral supernatant per well). After incubation for 24 hours, add 300 μL of Passive Lysis Buffer, shake at room temperature for 15 min, mix with Luciferase Assay Reagent at a ratio of 2:1, and read the luciferase value using a microplate reader.

[0066] The results are as follows Figure 4 As shown, E2F2 overexpression has a certain inhibitory effect on HIV-2 and SIVnano, reducing them by 21%~38% and 7%~14%, respectively. This indicates that E2F2 has a broad-spectrum antiretroviral ability, but its inhibitory effect on HIV-1 is significantly stronger.

[0067] Example 5 illustrates how E2F2 inhibits HIV-1 infection by mediating viral integration.

[0068] 18 to 24 hours before transfection, use 0.8-1 mg / well. 10 6293T cells were seeded at a volume of 1 mL in a 6-well plate. For each well, 1.0 μg of pcDNA3.1-F2-3Flag / Mock DNA was diluted in 100 μL of serum-free DMEM preheated at 37 °C. Gently vortex to mix; add 3 times the plasmid volume of LipoD293 to each tube; gently vortex to mix; incubate at room temperature for 10 minutes to form the DNA-LipoD293™ transfection complex; add 10⁴ / 10⁶ μL of the LipoD293 / DNA mixture dropwise to the medium in each well, gently swirl the plate to homogenize the mixture; 4–6 hours after transfection, remove the medium containing the LipoD293 / DNA complex and replace it with medium containing whole serum / antibiotics; 24 hours later, cells are infected with NL4-3.Luc (VSV-G) (10 / 100 ng p24); and genomic DNA and circular DNA are extracted from the cells, and HIV total cDNA, integrated DNA, and 2-LTR are detected by quantitative polymerase chain reaction.

[0069] The results are as follows Figure 5 As shown, compared with the Mock transfection group, there was no significant difference in total cDNA and 2LTR in the E2F2 overexpression group, indicating that E2F2 does not affect the reverse transcription and nuclear translocation of HIV-1; on the other hand, E2F2 overexpression significantly reduced the amount of HIV integrated DNA, indicating that E2F2 overexpression prevents HIV-1 infection by inhibiting HIV-1 integration.

[0070] In summary, E2F2 overexpression significantly inhibits HIV-1 infection. This inhibitory effect is independent of the plasmid backbone used and remains stable across low to high HIV-1 infection doses. Increasing E2F2 expression levels can enhance its HIV-1 inhibitory capacity to some extent, while decreasing its expression levels can promote HIV-1 infection to some extent. E2F2 also exhibits some inhibitory activity against other retroviruses such as HIV-2 and SIV. Furthermore, it was found that E2F2's HIV-1 inhibitory function is based on its ability to prevent HIV-1 integration into the host genome. Overexpression of E2F2 may play an important role in anti-HIV-1 infection and treatment of HIV carriers.

[0071] The present invention has been described in detail above. For those skilled in the art, the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. Although specific embodiments have been given, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein. Some of the essential features can be applied within the scope of the following appended claims.

Claims

1. The use of any one of the substances shown in (1)-(3) below in at least one of (a)-(c) below: (1) E2F2 protein, wherein the E2F2 protein is a protein with an amino acid sequence as shown in Sequence Listing 1; (2) The nucleic acid molecule encoding the E2F2 protein as shown in (1); (3) Expression cassettes, recombinant vectors or transgenic cells containing nucleic acid molecules as shown in (2); (a) Preparation of products for inhibiting HIV-1 proliferation in cells; (b) Prepare products for inhibiting HIV-2 proliferation in cells; (c) Prepare a product for inhibiting the proliferation of SIVnano in cells.

2. The application according to claim 1, characterized in that: The recombinant vector is either psLenti-E2F2-3Flag or pcDNA3.1-E2F2-3Flag plasmid.

3. The use of a substance capable of promoting the expression and / or activity of (1) or (2) in at least one of (a)-(c): (1) an E2F2 protein, said E2F2 protein having an amino acid sequence as shown in Sequence Listing 1; (2) a nucleic acid molecule encoding the E2F2 protein as shown in (1); (a) the preparation of a product for inhibiting the proliferation of HIV-1 in cells; (b) Prepare products for inhibiting HIV-2 proliferation in cells; (c) Prepare products for inhibiting the proliferation of SIVnano in cells; The substance that promotes the expression and / or activity of (1) or (2) is a recombinant vector containing the nucleic acid molecule, wherein the recombinant vector is psLenti-E2F2-3Flag or pcDNA3.1-E2F2-3Flag plasmid.

4. The application according to any one of claims 1-3, characterized in that: The product in question is a pharmaceutical product.

Images