CN108721646B

CN108721646B - Method for inhibiting virus infection and antiviral drug

Info

Publication number: CN108721646B
Application number: CN201710249051.1A
Authority: CN
Inventors: 王荣福; 崔隽; 杜阳
Original assignee: Sun Yat Sen University
Current assignee: Sun Yat Sen University
Priority date: 2017-04-17
Filing date: 2017-04-17
Publication date: 2021-10-01
Anticipated expiration: 2037-04-17
Also published as: CN108721646A

Abstract

The invention discloses an antiviral drug for inhibiting virus infection, which is characterized in that the virus infection is inhibited by targeting LRRC25 protein rich in leucine repeat. Through research and a large number of experiments on LRRC25 in the aspect of antivirus, LRRC25 is finally selected as a new target of treatment or medicine, and LRRC25 silencing or deletion through the means of medicine can play a role in resisting virus infection.

Description

Method for inhibiting virus infection and antiviral drug

Technical Field

The invention relates to the field of treatment of virus infection, in particular to a method for inhibiting virus infection and an antiviral drug.

Background

Viral infection is a common clinical disease, and some viral infections have high transmission speed and high mortality rate and seriously endanger human health. Currently, there are two main approaches to intervention in viral infections. One is to prevent viral infection by vaccination and one is to reduce the activity and replication capacity of the virus by antiviral drugs. Despite the existence of numerous drugs and therapeutic approaches, the search for safer and more effective targets for the treatment of viral infections has been a focus of health research.

The natural immunity is the first defense line of organisms against pathogenic microorganisms, and the mode recognition receptor plays a significant role in the process of recognizing pathogen-related molecular modes, activating natural immune response and effectively removing pathogens. As a central force in the antiviral immune response, the regulation of the type I interferon signaling pathway is particularly important for antiviral immunity.

Type I interferons are polypeptides secreted by infected cells and have three major functions: (1) activating the inherent antiviral state of the cell in the infected cell or nearby cells, limiting the spread of infectious agents, particularly viruses; (2) the I-type interferon can limit the release of proinflammatory factor signal paths and cytokines in a balanced manner, so that the natural immune response is regulated and controlled, and the functions of antigen presentation and natural killer cells are promoted; (3) type I interferons activate the adaptive immune system, thereby promoting the generation of T, B lymphocyte immune responses and immunological memory with high antigen specificity. Type I interferons have important protective effects against acute viral infections. IFN-alpha (with multiple homologous genes) and IFN-beta (with only one gene) in the type I interferon family are two members which are clearly studied and have relatively wide expression.

The cell surface receptors to which IFN- α and IFN- β bind are known as IFN- α receptors (IFNAR). IFNAR is a heterodimeric transmembrane receptor consisting of two subunits, IFNAR1 and IFNAR 2. IFNAR is involved in activating receptor-associated protein tyrosine kinases Janus kinase 1(JAK1) and tyrosine kinase 2(TYK2), which in turn phosphorylate intracellular transcription factor signal transducers and activators STAT1 and STAT 2. Tyrosine phosphorylated STATs 1 and STAT2 dimerize and then enter the nucleus, recruiting IRF9 in the nucleus to form a three-molecule complex called interferon-stimulated gene factor 3(ISGF 3). ISGF3 binds to the cognate DNA sequence interferon-stimulated response elements (ISREs), thus activating transcription of ISGs. Classical type I interferon signals induce the expression of hundreds of ISGs, establishing an antiviral state. ISG-encoded proteins inhibit pathogens by a variety of mechanisms, including inhibiting viral transcription, translation and replication, promoting viral nucleic acid degradation or altering cellular lipid metabolism responses.

The RLR receptor recognizes a variety of viruses and induces the production of interferon. RIG-I and MDA5 signal through a common linker molecule, MAVS. The interaction of RIG-I and MDA5 with MAVS allows RLRs to be recruited to the MAVS-binding membrane and then bind to downstream signaling molecules to form MAVS signalizers, inducing the production of type I interferons. The RIG-I signal path is divided into two paths at the level of TRAFs, and TRAF2 and TRAF6 activated by MAVS activate classical IKK alpha/beta kinase, induce phosphorylation of I kappa B alpha and degrade through a proteasome pathway. The activated p65/p50 NF-. kappa.B dimer was released and incorporated into the nucleus, initiating transcription of NF-. kappa.B-dependent genes. While upon activation of TRAF3 by MAVS, TRAF3 activates the non-canonical IKK-related kinases TBK1 and IKKi, which in turn phosphorylates the C-terminus of IRF3/IRF7, induces IRF3 dimerization and incorporation into the nucleus to activate transcription of IRF-dependent genes.

Disclosure of Invention

In view of the above drawbacks, the present invention aims to provide a safer and more effective method for inhibiting viral infection.

In order to solve the above problems the present invention proposes a method for inhibiting viral infection, characterized in that viral infection is inhibited by targeting LRRC25 protein rich in leucine repeat.

The method for inhibiting virus infection is characterized in that the effect of LRRC25 is inhibited by using siRNA for silencing LRRC25 expression or using CRISPR/Cas9 system to knock out LRRC25 gene; the virus is an RNA virus.

The method for inhibiting the virus infection is characterized in that when the siRNA is selected to inhibit the THP-1 cell from expressing LRRC25, the siRNA is transferred into the THP-1 cell to silence the expression of LRRC25, then VSV-eGFP is added to enable the virus to infect the THP-1 cell, and the effect of LRRC25 on promoting the virus infection is inhibited.

The method for inhibiting virus infection is characterized in that when the CRISPR/Cas9 system is selected to knock out LRRC25 gene so as to inhibit the expression of LRRC25, HEK 293T cells are used for preparing lentivirus targeting LRRC25 gene; then concentrating the virus by means of ultracentrifugation; infecting the concentrated virus into THP-1 cells; then, screening the LRRC25 monoclonal THP-1 cells through puromycin resistance; LRRC25KO THP-1 cells were then infected with VSV-eGFP to inhibit the effect of LRRC 25.

The method for inhibiting virus infection is characterized in that the component for inhibiting LRRC25 is siRNA targeting LRRC25 mRNA.

The method for inhibiting the virus infection is characterized in that the component for inhibiting the LRRC25 is sgRNA targeting LRRC25 and lentivirus prepared by the sgRNA.

An antiviral agent characterized by inhibiting RNA viral infection by targeting LRRC25 protein rich in leucine repeat.

The antiviral drug is characterized in that the effect of LRRC25 is inhibited by knocking out LRRC25 gene by using siRNA for silencing LRRC25 expression or using CRISPR/Cas9 system.

The antiviral drug is characterized in that when the siRNA is selected to inhibit the THP-1 cell from expressing LRRC25, the siRNA is transferred into the THP-1 cell to silence the expression of LRRC25, then VSV-eGFP is added to enable the virus to infect the THP-1 cell, and the effect of inhibiting LRRC25 to promote virus infection is achieved; when the CRISPR/Cas9 system is selected to knock out the LRRC25 gene so as to inhibit the expression of LRRC25, HEK 293T cells are used for preparing lentiviruses targeting the LRRC25 gene; then concentrating the virus by means of ultracentrifugation; infecting the concentrated virus into THP-1 cells; then, screening the LRRC25 monoclonal THP-1 cells through puromyc i n resistance; LRRC25KO THP-1 cells were then infected with VSV-eGFP to inhibit the effect of LRRC 25.

The antiviral drug is characterized in that the component for inhibiting LRRC25 is siRNA targeting LRRC25 mRNA; the component for inhibiting LRRC25 is sgRNA targeting LRRC25 and lentivirus prepared from the sgRNA.

According to the invention, through research and a large number of experiments on LRRC25 in the aspect of antivirus, LRRC25 is finally selected as a new target of treatment or medicine, and LRRC25 is silenced or deleted by means of medicine, so that the function of resisting virus infection is achieved.

Drawings

FIG. 1 is the expression of LRRC25 after infection of THP-1 with VSV-eGFP;

FIG. 2 is a graph showing the results of over-expressing LRRC25 in HEK 293T cells, inhibiting the type I interferon signaling pathway, promoting viral infection;

FIG. 3 is a graph of the results of enhancing antiviral immune responses by silencing expression of LRRC25 using siRNA targeting LRRC 25;

fig. 4 is a graph of the results of enhancing antiviral immune response after knocking out LRRC25 at the gene level using CRISPR/Cas9 system: wherein,

FIG. 4A, at the gene level, demonstrates the successful construction of the LRRC25KO THP-1 cell line;

FIG. 4B, at the protein level, demonstrates the success of construction of the LRRC25KO THP-1 cell line;

figure 4C, LRRC25 deletion significantly upregulated IFN- β transcription following infection with RNA virus;

figure 4D, LRRC25 deletion significantly upregulated IFN- β release following infection with RNA virus;

figure 4E, deletion of LRRC25 significantly upregulated transcription of downstream ISGs, e.g., IFIT2 and IFIT1, following infection with RNA virus;

figure 4F, LRRC25 deletion significantly upregulated the antiviral ability of THP-1 cells after infection with RNA virus.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Specific example 1: expression of LRRC25 after infection of THP-1 with VSV-eGFP. When THP-1 cells were infected with vesicular stomatitis virus (VSV-eGFP), the protein level of LRRC25 was elevated.

The specific experimental procedure is that 2ml of THP-1 cells with the density of 10^6/ml are paved into a 6-well plate, then VSV-eGFP (MOI ═ 0.1) is used for infecting THP-1 cell lines, cell lysates are collected after the THP-1 cells are infected at the designated time, and the expression of LRRC25 and beta-actin (internal reference) in the cell lysates is detected through an immunoblotting experiment. FIG. 1 shows the expression of LRRC25 after infection of THP-1 with VSV-eGFP, and it can be seen that the protein level of LRRC25 is increased when THP-1 cells are infected with VSV-eGFP. It was suggested that LRRC25 was involved in the regulation of antiviral responses.

Specific example 2: over-expression of LRRC25 can inhibit the type I interferon signaling pathway and promote viral infection.

100ul HEK 293T cells at a density of 4 x 10^5/ml were plated in 96-well plates and transfected with EV (200ng) or LRRC25(200ng) expression plasmids after 12h adherence, with concomitant ISRE-luc (25ng) or IFN- β -luc (25 ng). After 12h, the cells were stimulated with either IC poly (I: C) LMW (5. mu.g/ml)) or SeV (MOI ═ 0.1) for 24h or 14h, respectively. The fluorogenic reporter enzyme activity was then detected by a microplate reader. Luciferase reporter gene assay results show that LRRC25 can inhibit IC poly (I: C) LMW and SeV-induced activation of ISRE and IFN-beta. FIG. 2 is a graph showing the results of over-expression of LRRC25 in HEK 293T cells, inhibition of type I interferon signaling pathway, and promotion of viral infection, and FIG. 2A is a graph showing in part the results of luciferase reporter assay that LRRC25 is able to inhibit IC poly (I: C) LMW (a synthetic RIG-I ligand) and SeV-induced activation of ISRE and IFN- β. LRRC25 was shown to have a negative regulatory function on RLR-mediated type I interferon signaling pathways. FIG. 2B shows that expression of LRRC25 significantly suppresses the antiviral response of cells, both when LRRC25 is overexpressed in HEK 293T cells and VSV-eGFP is used to infect the cells, whether the infection of the cells is observed under a mirror or the percentage of infection of the cells is detected by flow. This result demonstrates that LRRC25 has a negative regulatory function on the RLR-mediated type I interferon signaling pathway. In addition, 500ul HEK 293T cells at a density of 4 x 10^5/ml were plated in 24-well plates and transfected with EV (200ng) or Myc-LRRC25(200ng) after 12h of attachment. After 24h, the cells were infected with VSV-eGFP (MOI ═ 0.001) for the indicated times, and then the cell infection was observed under a microscope and the percentage of infected cells was analyzed by flow analysis. The results show that whether the infection of the cells is observed under a mirror or the infection percentage of the cells is detected by flow, the over-expression of LRRC25 can obviously inhibit the antiviral response of the cells.

Specific example 3: silencing expression of LRRC25 with siRNA targeting LRRC25 enhances antiviral immune responses.

FIG. 3 results of the enhancement of antiviral immune response by silencing the expression of LRRC25 with LRRC 25-targeting siRNA are shown by spreading 500ul of THP-1 cells at a density of 10^6/ml in 24 well plates to transfect LRRC 25-specific siRNA (30nM) or control siRNA (30nM) not targeting any protein in THP-1 cells. After 30h, cell lysates were collected. Compared with a control group, the expression level of LRRC25 in the THP-1 cell line transfected with the siRNA specific to LRRC25 is obviously reduced through an immunoblotting experiment. FIG. 3A, silencing of expression of LRRC25 by siRNA targeting LRRC25, demonstrates that our synthetic siRNA has good silencing effect. Further, 100ul HEK 293T cells at a density of 4 x 10^5/ml were plated in 96-well plates and transfected with siRNA (30nM) of LRRC25 specific sequence or control siRNA (30nM) not targeting any protein after 12h of attachment. ISRE-luc (25ng) reporter plasmid was transfected 12h later. Cells were stimulated for 24h after 12h by transfection with poly (I: C) (5. mu.g/ml). The activity of the fluorescent reporter enzyme was detected by a microplate reader. The results show that silencing of expression of LRRC25 with siRNA targeting LRRC25 significantly promotes endogenous Poly I: C-induced ISRE activation. To further demonstrate the role of LRRC25 in host antiviral immune responses, 500ul of THP-1 cells at a density of 10^6/ml were plated in 24-well plates and either LRRC25 specific siRNA (30nM) or control siRNA (30nM) not targeting any protein were transfected into THP-1 cells. Cells were infected with VSV-eGFP (MOI ═ 0.01) after 24h for the indicated times. FIG. 3C is a graph in which an LRRC 25-targeting siRNA silences expression of LRRC25, significantly inhibits infection of THP-1 cells by VSV-eGFP, and compared with a control group, silencing of LRRC25 can substantially weaken infection of cells by viruses.

Example 4: anti-viral immune response experiments were enhanced after knocking out LRRC25 at the gene level using CRISPR/Cas9 system.

It is currently believed that IFN- β in the type I interferon signaling pathway is the most important cytokine of the host against viruses. Once released by upstream signal induced expression, the protein binds to its receptor IFNAR, activates STAT signal pathway to induce and express more ISGs molecules, and establishes stronger antiviral immune response. To further confirm the function of LRRC25 in antiviral immune response, LRRC25KO THP-1 stable transformant was constructed using CRISPR/Cas9 system. Genomic DNA and proteins of the LRRC25KO THP-1 stable transformant were extracted, and the constructed cell line was the LRRC25KO THP-1 cell line (FIGS. 4A and 4B) by gene level sequencing and protein level verification. 2ml of THP-1 cells at a density of 10^6/ml were plated in 6-well plates and control and LRRC25KO THP-1 cells were infected with VSV-eGFP (MOI ═ 0.01) for the indicated time. Real-time quantitative PCR experiments and enzyme-linked immunosorbent assay show that the transcription and release of IFN-beta in LRRC25KO THP-1 cells are greatly improved (FIG. 4C, 4D). Furthermore, it was confirmed using the same method that the transcription of IFIT2 and IFIT1 was also significantly improved in LRRC25KO THP-1 cells (FIG. 4E). More importantly, using the same approach, i.e., infecting control and LRRC25KO THP-1 cells for 0-18h with VSV-eGFP (MOI ═ 0.01), it was observed microscopically that GFP signal was significantly weaker in LRRC 25-depleted cells (VSV-eGFP infected cells) than in control (fig. 4F). Quantitative analysis of infected cells by flow cytometry demonstrated that the deletion of LRRC25 reduced the infection rate of the cells from 63.8% to 14.5% (fig. 4F). These experimental results fully demonstrate that targeting LRRC25 to silence or delete LRRC25 functions to combat viral infection.

As noted above, it should be noted that the use of particular terminology when describing certain features or aspects of the invention should not be taken to imply that the terminology is being re-defined herein to be restricted to certain specific characteristics, features, or aspects of the invention with which that terminology is associated. In general, the terms used in the following claims should not be construed to limit the invention to the specific embodiments disclosed in the specification, unless the above detailed description section explicitly defines such terms. Accordingly, the actual scope of the invention encompasses not only the disclosed embodiments, but also all equivalent ways of practicing or implementing the invention under the claims.

Claims

1. Use of a siRNA that silences LRRC25mRNA rich in leucine repeat or a sgRNA that knocks down LRRC25 gene using CRISPR/Cas9 system or a lentivirus prepared from the foregoing sgRNA in the preparation of an antiviral medicament for inhibiting RNA virus infection.

2. The use according to claim 1, characterized in that when selecting the use of siRNA to inhibit expression of LRRC25 in THP-1 cells, the siRNA is transferred into THP-1 cells to silence expression of LRRC25, and then VSV-eGFP is added to infect THP-1 cells with virus, inhibiting the effect of LRRC25 in promoting viral infection; when the LRRC25 gene knockout by using a CRISPR/Cas9 system is selected so as to inhibit the expression of LRRC25, preparing a lentivirus targeting the LRRC25 gene by using HEK 293T cells; then concentrating the virus by means of ultracentrifugation; infecting the concentrated virus into THP-1 cells; then, screening the LRRC25 monoclonal THP-1 cells through puromycin resistance; followed by infection with VSV-eGFPLRRC25The THP-1 knockout cell inhibits the action of LRRC 25.