A method for screening antiviral targets by targeting viral DNA

Abstract

The application discloses a method for screening antiviral target points by targeting viral DNA, and effective sgRNA sequences targeting key DNA regions of varicella-zoster virus, herpes simplex virus type I and Kaposi's sarcoma-associated herpes virus are obtained respectively; dCas9-sgRNA containing viral DNA plasmid is co-transfected, and DNA of the varicella-zoster virus, the herpes simplex virus type I and the Kaposi's sarcoma-associated herpes virus is immunoprecipitated and enriched respectively to screen proteins regulating viral replication; the proteins are identified by mass spectrometry, and it is verified that the method is also applicable to screening proteins regulating viral replication by targeting HIV DNA, so that the method for screening proteins regulating viral replication by targeting viral DNA is established.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a method for screening antiviral targets by targeting viral DNA. Background Technology

[0002] Herpesviruses are a family of DNA viruses widely found in nature, capable of causing a variety of acute and chronic diseases. This virus family includes several types, such as herpes simplex virus (HSV-1 and HSV-2), varicella-zoster virus (VZV), human herpesvirus 6 (HHV-6), and human herpesvirus 8 (HHV-8). Because herpesviruses have a latent infectivity characteristic, they can remain infectious for life once infected, making research on this class of viruses of great significance.

[0003] Herpes simplex virus (HSV) and varicella-zoster virus (VZV) can cause severe infectious skin diseases, seriously endangering the health and quality of life of adults and immunocompromised children, and placing a heavy burden on families. Kaposi's sarcoma-associated herpesvirus (KSHV) is a human herpesvirus belonging to the Gammaherpesviridae family. KSHV is associated with the development of various diseases, most notably Kaposi's sarcoma, a type of vascular tumor that typically occurs in individuals with compromised immune systems, such as those with HIV / AIDS. Furthermore, KSHV is also associated with primary lymphomas and multiple plasma cell diseases (such as multiple myeloma), severely impacting patients' health and quality of life. However, due to the current lack of in vitro viral research models, it is difficult to explore the interaction between the host and the virus, and the key regulatory mechanisms by which the host regulates the virus.

[0004] Currently, we can explore ways of interaction between the host and the virus by using high-throughput analysis methods such as various omics to compare the differences in host protein expression before and after viral infection, or by using sgRNA library screening to explore the knockdown of host genes and then discover related regulatory factors for in-depth mechanism exploration.

[0005] Therefore, exploring efficient research methods to discover key targets in order to control viral latency and replication is a research hotspot in the field and also a need for clinical treatment.

[0006] The main methods currently used to study protein regulation of viral gene transcription are as follows:

[0007] (1) Starting from a single protein: After analyzing the sequence of the viral promoter region, conserved binding sites of potential transcription factor binding proteins are found, and then transcriptional function is studied; the disadvantage of this method is that the transcriptional protein being studied needs to be a widely studied and known conserved binding sequence [1].

[0008] (2) Based on high-throughput analysis methods such as proteomics and transcriptomics, compare the differences in protein expression before and after viral infection to find proteins that may be directly or indirectly involved in viral transcription regulation. The disadvantage of this method is that most differentially expressed proteins are not directly involved in the process of viral gene transcription regulation, but may be the overall response of the host after viral infection, and are not specific for studying viral gene transcription regulation [2].

[0009] (3) sgRNA / shRNA library screening: After knocking down or knocking out the host gene through the library, the protein that affects the transcription of the viral gene is screened, and then the transcription function is verified. The disadvantage of this method is that the deletion of many key genes will affect cell growth, such as essential genes for cell survival. The noise background of these gene knockdowns often affects the screening results, and there is an obvious indirect effect [3].

[0010] (4) Using biotin probes to bind to viral promoter segments, for example, using streptomycin-conjugated beads to isolate and purify proteins that interact with the segment. Although the regulatory factors found by this method are relatively direct, the proteins enriched by this method have poor specificity due to the high non-specificity of biotin-labeled probes binding to fragmented DNA [4].

[0011] The patent application No. 202010131159.2, entitled "Screening of Viral Transcription Targets Based on Viral LTR Immunoprecipitation Using Cas9-sgRNA", discloses that: for the first time, Cas9-sgRNA combined with immunoprecipitation is used to detect proteins that interact with the HIV promoter region LTR. It is shown that this method can be used to screen proteins that bind to the viral LTR and regulate HIV-1 gene transcription. However, this method has limitations, as it can only be applied to the HIV LTR region, and the method is inefficient, with a very limited number of proteins obtained by mass spectrometry.

[0012] Establishing a method that simultaneously applies to the LTR regions of both herpesvirus and HIV can more efficiently expand the range of viruses that can be screened and can also achieve efficient screening of antiviral targets, but there are many difficulties, for the following reasons:

[0013] The activation system requires the promoter to be in a well-activated state, but the openness of chromatin affects the participation of transcriptional regulatory factors;

[0014] The difficulty in selecting sgRNA lies in the fact that, based on the effective targeting effect of sgRNA, we must fully consider the coverage of the target DNA, while also taking into account the lowest possible nonspecificity, so as to avoid excessive nonspecific signals that would affect specificity.

[0015] CHIP experiments typically require large sample sizes and long experimental cycles, involving multiple complex steps. The experimental procedure includes cell fixation, DNA shearing, immunoprecipitation, rinsing, decrosslinking, and DNA purification, each of which requires careful operation and appropriate optimization. Due to the influence of different experimental conditions (such as antibody selection, shearing conditions, etc.) on the results, multiple rounds of experiments are needed to determine the optimal conditions.

[0016] The complex life cycle and latent reactivation mechanism of herpesviruses make them a major challenge in virological research. Extending the technology of screening proteins that interact with viral promoters to herpesvirus research is beneficial for developing more effective prevention and control strategies to reduce the spread and impact of herpesvirus infection, and has promising clinical applications.

[0017] References:

[0018] [1]Jayaram, N., D. Usvyat, and RM AC, Evaluating tools fortranscription factor binding site prediction. BMC Bioinformatics, 2016. 17(1): p. 547.

[0019] [2]Cui, M., C. Cheng, and L. Zhang, High-throughput proteomics: amethodological mini-review. Lab Invest, 2022. 102(11): p. 1170-1181.

[0020] [3]Joung, J., et al., Genome-scale CRISPR-Cas9 knockout and transcriptional activation screening. Nat Protoc, 2017. 12(4): p. 828-863.

[0021] [4]Cheah, JS and S. Yamada, A simple elution strategy for biotinylated proteins bound to streptavidin conjugated beads using excessbiotin and heat. Biochem Biophys Res Commun, 2017.493(4): p. 1522-1527. Summary of the Invention

[0022] To address the current limitations in research on protein regulation of key viral gene transcription, this application establishes a method for screening antiviral targets by targeting viral DNA. This method yields an effective sgRNA sequence targeting the promoter region of the critical immediate early protein (ORF4) of varicella-zoster virus (VZV). Co-transfection with plasmids containing the ORF4 promoter region and dCas9-sgRNA yields an effective sgRNA sequence targeting the ICP27 promoter region of herpes simplex virus type I (HSV-1). Co-transfection with plasmids containing the ICP27 promoter region and dCas9-sgRNA yields an effective sgRNA sequence targeting the RTA promoter region of Bosi sarcoma-associated herpesvirus (BSA). Co-transfection with plasmids containing the RTA promoter region and dCas9-sgRNA, followed by immunoprecipitation enrichment of VZV, HSV-1, and BSA DNA to screen for proteins regulating viral replication. Mass spectrometry identification of the proteins is performed, and the method is verified to be applicable to screening for proteins regulating viral replication by targeting HIV DNA. This establishes a method for screening proteins regulating viral replication by targeting viral DNA. To achieve the above objectives, the specific technical solution of this invention is as follows:

[0023] First aspect: sgRNA design and plasmid construction

[0024] Ten different sgRNA sequences (SEQ ID NO.1-SEQ ID NO.10) were designed targeting the ORF4 promoter region of varicella-zoster virus (VZV).

[0025] Eight sgRNAs with different sequences (SEQ ID NO.11-SEQ ID NO.18) were designed targeting the ICP27 promoter region of herpes simplex virus type I (HSV-1).

[0026] For the LTR promoter region of HIV, a mixture of sgRNAs with three different sequences (SEQ ID NO.19, SEQ ID NO.20, and SEQ ID NO.21) was used as a pool of sgRNAs for subsequent interaction with the dCas9 protein.

[0027] Eight different sgRNA sequences (SEQ ID NO.11-SEQ ID NO.18) were designed targeting the RTA promoter region of Kaposi's sarcoma-associated herpesvirus (KSHV).

[0028] Different sgRNA sequences were constructed into PX459 vector plasmids containing Cas9.

[0029] Secondly, screening for effective sgRNAs targeting viral DNA.

[0030] The effectiveness of sgRNA targeting was verified using a report system, and effective sgRNA sequences were obtained.

[0031] sgRNAs of SEQ ID NO.3, SEQ ID NO.7, and SEQ ID NO.10 were screened for the ORF4 promoter of varicella-zoster virus (VZV) and used as a pool of sgRNAs for subsequent interaction with dCas9 protein.

[0032] Targeting the ICP27 promoter region of herpes simplex virus type I (HSV-1), a mixture of sgRNAs of SEQ ID NO.12, SEQ ID NO.15, and SEQ ID NO.18 was screened and used as a pool of sgRNAs for subsequent interaction with the dCas9 protein;

[0033] Targeting the RTA promoter region of Kaposi's sarcoma-associated herpesvirus (KSHV), a mixture of sgRNAs of SEQ ID NO.24, SEQ ID NO.27, and SEQ ID NO.29 was screened and used as a pool of sgRNAs for subsequent interaction with the dCas9 protein.

[0034] Thirdly, Chip-qPCR was used to verify the binding of dCas9 to the VZV ORF4 promoter region, HSV1 ICP27 promoter region, and KSHV RTA promoter region.

[0035] To further verify the effectiveness of the second experimental procedure, i.e. whether dCas9 effectively binds to the region, a portion of the protein product to be identified in the second aspect was subjected to q-PCR verification.

[0036] Results: Compared with the negative control group with added sg-Gal4, sg-mix can significantly guide 3×Flag dCas9 to the promoter region, verifying the effectiveness of the second experimental procedure.

[0037] Fourthly, immunoprecipitation preparation of proteins interacting with the VZV ORF4 promoter region / HSV1 ICP27 promoter region / HIVLTR promoter region / KSHV RTA promoter region.

[0038] Following the second aspect, the mixed sgRNA was used to prepare proteins that interact with the VZV ORF4 promoter region / HSV1 ICP27 promoter region / HIV LTR promoter region / KSHV RTA promoter region by immunoprecipitation, thus obtaining the protein product to be identified.

[0039] Fifthly, mass spectrometry identification of enriched interacting proteins.

[0040] Mass spectrometry analysis was performed on the protein product to be identified obtained in the fourth aspect, and the results are as follows:

[0041] For the VZV ORF4 promoter, several proteins that can regulate varicella-zoster virus (VZV) were identified, and their gene names are: KRT86, MIF, RNF20, AASDHPPT, DUS3L, YY1, ILKAP, FLYWCH2, YBX1, NUP50, and CPSF6.

[0042] For the HSV1 ICP27 promoter, several proteins that can regulate herpes simplex virus type I (HSV1) were identified, and their gene names are: H3C1, RNH1, WDR18, PAK1IP1, WARS1, PAICS, NOMO3, IRS4, CIRBP, CPNE3, and RHOC.

[0043] For the KSHV RTA promoter, several proteins that can regulate Kaposi's sarcoma-associated herpesvirus (KSHV) were identified, and their gene names are: POLR2B, CLIC4, STAU2, PPP1CB, TPR, WARS1, IRS4, PAICS, RAB8A, NOMO3, RAB1B, and TMEM33.

[0044] Sixth aspect: The screening process of the method in this application

[0045] (i) Targeting of target DNA using single sgRNA and mixed sgRNA

[0046] Results: While individual sgRNAs showed high efficiency, complete coverage of the target DNA could not be guaranteed. Using mixed sgRNAs ensured better targeting of each region. Therefore, for varicella-zoster virus, a mixed sgRNA of SEQ ID NO.3, SEQ ID NO.7, and SEQ ID NO.10 was used; for herpes simplex virus type I, a mixed sgRNA of SEQ ID NO.12, SEQ ID NO.15, and SEQ ID NO.18 was used; and for Kaposi's sarcoma-associated herpesvirus, SEQ ID NO.24, SEQ ID NO.27, and SEQ ID NO.29 was used.

[0047] (ii) The targeting ability of magnetic beads or agarose beads to target DNA

[0048] Results: Magnetic beads were easy to use for elution, but the nonspecific signal was too high. Agarose beads were easier to use and had a lower nonspecific signal, so agarose beads were chosen.

[0049] (III) The targeting effect of different amounts of agarose beads on the target DNA

[0050] Results: The targeting efficiency of the experimental group with 15 μL of agarose beads was good, but the signal of the control group was too high. With agarose beads of 11 μL, a slight adjustment made the signal of the control group decrease significantly. Therefore, the agarose bead dosage of 11 μL was selected.

[0051] Seventhly, a comparison between the method of this application and the patent application number 202010131159.2.

[0052] (a) Comparison of the targeting efficiency of this application and patent application number 202010131159.2 on HIV viral DNA

[0053] Results: The targeting efficiency of the method in this application for HIV viral DNA is much higher than that of the patent application number 202010131159.2. Therefore, the method in this application is also applicable to screening targets that regulate HIV transcription. Moreover, the targeting efficiency of the method in this application for HIV viral DNA is significantly improved compared with the method of the patent application number 202010131159.2.

[0054] (II) Comparison of the efficiency of this application and the patent application No. 202010131159.2 in targeting herpesvirus

[0055] Results: The targeting efficiency of the method in this application for varicella-zoster DNA is significantly improved compared with the method of patent application number 202010131159.2; the method of patent application number 202010131159.2 is not suitable for screening new antiviral targets for varicella-zoster DNA due to its low efficiency.

[0056] Compared with the prior art, the beneficial effects of this application are as follows:

[0057] 1. For the first time, a method for screening antiviral targets by simultaneously targeting herpesvirus and HIV DNA has been established, which can expand the range of viruses to be screened more efficiently.

[0058] 2. The targeting efficiency of the method in this application for HIV DNA is significantly improved compared with the method of patent application 202010131159.2;

[0059] 3. Multiple proteins that can regulate varicella-zoster virus (VZV), herpes simplex virus type I (HSV1), and Kaposi's sarcoma-associated herpesvirus (KSHV) were identified, which helps to elucidate the pathogenic mechanism of herpesviruses and lays the foundation for revealing new anti-herpesvirus therapeutic targets, thereby promoting the development of new drugs. Attached Figure Description

[0060] Figure 1 Schematic diagram of varicella-zoster virus sgRNA design;

[0061] Figure 2 Schematic diagram of herpes simplex virus type I sgRNA design;

[0062] Figure 3 Schematic diagram of Kaposi's sarcoma-associated herpesvirus sgRNA design;

[0063] Figure 4 The luciferase assay was used to verify the targeting efficiency of sgRNA in the ORF4 promoter region of varicella-zoster virus.

[0064] Figure 5 The luciferase assay was used to verify the targeting efficiency of sgRNA in the ICP27 promoter region of herpes simplex virus type I.

[0065] Figure 6 Luciferase assays were used to verify the targeting efficiency of sgRNA in the RTA promoter region of Kaposi's sarcoma-associated herpesvirus.

[0066] Figure 7 Chromatin immunoprecipitation assays were used to verify the targeting of sgRNAmix to the target DNA in the ORF4 promoter region of varicella-zoster virus.

[0067] Figure 8 Chromatin immunoprecipitation assays were used to verify the targeting of sgRNAmix to the target DNA in the ICP27 promoter region of herpes simplex virus type I.

[0068] Figure 9Chromatin immunoprecipitation assays were used to verify the targeting of sgRNAmix to the target DNA in the RTA promoter region of Kaposi's sarcoma virus.

[0069] Figure 10 Mass spectrometry was used to obtain protein maps of interaction with varicella-zoster virus DNA;

[0070] Figure 11 Mass spectrometry was used to obtain a protein map of interaction with herpes simplex virus type I DNA;

[0071] Figure 12 Mass spectrometry was used to obtain a protein map of DNA interactions with Kaposi's sarcoma-associated herpesvirus.

[0072] Figure 13 The targeting of varicella-zoster virus DNA using a single sgRNA;

[0073] Figure 14 The targeting of varicella-zoster virus DNA using mixed sgRNAs;

[0074] Figure 15 The targeting of herpes simplex virus DNA using a single sgRNA;

[0075] Figure 16 The targeting of herpes simplex virus DNA using mixed sgRNAs;

[0076] Figure 17 Using magnetic beads + mixed sgRNA to target herpes simplex virus DNA;

[0077] Figure 18 Using agarose beads + mixed sgRNA to target herpes simplex virus DNA;

[0078] Figure 19 Using magnetic beads + mixed sgRNA to target varicella-zoster virus DNA;

[0079] Figure 20 Using agarose beads + mixed sgRNA to target varicella-zoster virus DNA;

[0080] Figure 21 The targeting of varicella-zoster virus DNA was studied using 15 μL of a mixture of sgRNA and agarose beads.

[0081] Figure 22 The targeting of varicella-zoster virus DNA was studied using 11 μL of a mixture of sgRNA and agarose beads.

[0082] Figure 23The targeting of herpes simplex virus DNA was studied using 15 μL of a mixture of sgRNA and agarose beads.

[0083] Figure 24 The targeting of herpes simplex virus DNA was studied using 11 μL of a mixture of sgRNA and agarose beads.

[0084] Figure 25 The targeting efficiency of the method in this application for HIV viral DNA is compared with that of the method in the patent application number 202010131159.2, "Viral LTR Immunoprecipitation Screening for Targets Regulating Viral Transcription Based on Cas9-sgRNA";

[0085] Figure 26 Mass spectrometry was used to obtain protein maps of interactions with HIV viral DNA.

[0086] Figure 27 According to the patent application number 202010131159.2, "Viral LTR Immunoprecipitation Screening of Targets Regulating Viral Transcription Based on Cas9-sgRNA", the efficiency of targeting varicella-zoster virus DNA.

[0087] Figure 28 The efficiency of targeting varicella-zoster virus DNA according to the method of this application.

[0088] Figure 1-28 middle,

[0089] 1-250bp, 251-500bp, 501-750bp, and 751-1000bp refer to the fragment regions of the target DNA;

[0090] *, **, ***, and **** represent P<0.05, P<0.01, P<0.001, and P<0.0001, respectively, indicating that the difference between the two groups is statistically significant. Detailed Implementation

[0091] The following embodiments are used to illustrate the present invention, but are not intended to limit the scope of the invention. Unless otherwise specified, the technical means used in the embodiments are conventional means well known to those skilled in the art.

[0092] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0093] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0094] Reagents and Materials

[0095]

[0096]

[0097] instrument

[0098]

[0099] Definitions of some terms

[0100] Unless otherwise defined below, all technical and scientific terms used in the specific embodiments of this invention are intended to have the same meaning as commonly understood by those skilled in the art. While it is believed that the following terms will be well understood by those skilled in the art, the following definitions are set forth to better explain the invention.

[0101] The "immunoprecipitation (IP)" described in this invention refers to the process where an antibody targeting a specific target protein forms an immune complex with the target protein in a sample (such as cell lysates). This immune complex is then precipitated from the mixture using Protein A agarose beads or Anti-Flag agarose beads. Finally, the target protein is eluted from the agarose beads, and the eluted protein is analyzed using techniques such as SDS-PAGE, Western blotting, and mass spectrometry.

[0102] The "Cas9-sgRNA-based viral LTR immunoprecipitation (dCas9 / sgRNA-guidedviral LTR IP)" described in this invention refers to a process based on the CRISPR-Cas9 principle. By designing specific sgRNAs that target the LTR region, tagged Cas9 is guided to the LTR region. Since the Cas9 carries a tag for subsequent immunoprecipitation enrichment, all proteins that interact with the LTR can be enriched, thus achieving the purpose of screening viral LTR interacting proteins through immunoprecipitation.

[0103] The “dCas9” mentioned in this invention refers to deactivated Cas9, which retains the ability to recognize and bind to sgRNA but loses the ability to cut DNA.

[0104] The ORF4 promoter region mentioned in this invention refers to the promoter region of the varicella-zoster virus immediate early protein ORF4.

[0105] The ICP27 promoter region described in this invention refers to the promoter region of the immediate early protein of herpes simplex virus type I.

[0106] The RTA promoter region described in this invention refers to the promoter region of the immediate early protein of Kaposi's sarcoma-associated herpesvirus.

[0107] Example 1: Design of sgRNA and construction of plasmids

[0108] This invention designs 10 different sgRNA sequences targeting the ORF4 promoter region of varicella-zoster virus (VZV), as shown in the schematic diagram. Figure 1 The specific sequence is as follows:

[0109]

[0110] This invention designs eight different sgRNA sequences targeting the ICP27 promoter region of herpes simplex virus type I (HSV1), as shown in the schematic diagram. Figure 2 The specific sequence is as follows:

[0111]

[0112] This invention uses three different sgRNA sequences targeting the LTR promoter region of HIV, as follows:

[0113]

[0114] This invention designs eight different sgRNA sequences targeting the RTA promoter region of Kaposi's sarcoma-associated herpesvirus (KSHV), as shown in the schematic diagram. Figure 3 The specific sequence is as follows:

[0115]

[0116] This invention establishes a rapid reporter system by constructing different sgRNA sequences into a PX459 vector plasmid containing Cas9. This plasmid, along with a luciferase reporter plasmid containing the ORF4 promoter, is co-transfected into 293T cells. Upon addition of the VZV IE62 protein, transcription of this promoter is activated. Effective sgRNAs specifically target this region to disrupt luciferase expression, resulting in decreased luciferase expression levels.

[0117] Luciferase assay results showed that among the 10 sgRNAs targeting the VZV ORF4 promoter region, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.7, and SEQ ID NO.10 significantly downregulated Luciferase expression. Figure 4 This indicates that the sgRNAs of SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.7, and SEQ ID NO.10 can effectively target three different regions of the target DNA.

[0118] To further increase the enrichment efficiency of DNA sequences and eliminate factors such as the possibility that a single sgRNA / dCas9 protein may occupy a certain region of the target DNA, resulting in low protein-protein interaction binding efficiency, this invention creatively selects a combination of sgRNA sequences, namely the mixture of sgRNAs of SEQ ID NO.3, SEQ ID NO.7, and SEQ ID NO.10, as the sgRNA pool for subsequent interaction with the dCas9 protein, thereby minimizing other interfering factors in the experimental design.

[0119] The same method was used for the HSV1 ICP27 promoter. Results showed that among the eight sgRNAs targeting the HSV1 ICP27 promoter region, SEQ ID NO.11-SEQ ID NO.18 all significantly downregulated Luciferase expression. (See attached image) Figure 5 This indicates that these sgRNAs can effectively target different regions of the target DNA.

[0120] To minimize the impact of dCas9 protein occupancy and to fully cover the target DNA fragment, the final choice was a combination of sgRNA sequences, namely the sgRNA mixture of SEQ ID NO.12, SEQ ID NO.15, and SEQ ID NO.18, as the sgRNA pool for subsequent interaction with dCas9 protein, thus minimizing other interfering factors in the experimental design.

[0121] For the LTR promoter region of HIV, a mixture of sgRNAs with three different sequences (SEQ ID NO.19, SEQ ID NO.20, and SEQ ID NO.21) was used as a pool of sgRNAs for subsequent interaction with the dCas9 protein.

[0122] The same method was used for the KSHV RTA promoter. Results showed that among the eight sgRNAs targeting the KSHV RTA promoter region, SEQ ID NO.22-SEQ ID NO.29 all significantly downregulated Luciferase expression. (See attached image.) Figure 6 This indicates that these sgRNAs can effectively target different regions of the target DNA.

[0123] To minimize the impact of dCas9 protein occupancy and to fully cover the target DNA fragment, the final choice was a combination of sgRNA sequences, namely the mixture of sgRNAs from SEQ ID NO.24, SEQ ID NO.27, and SEQ ID NO.29, as the pool of sgRNAs for subsequent interaction with the dCas9 protein. This approach aimed to eliminate other interfering factors as much as possible from the experimental design.

[0124] Example 2: Targeting viral DNA to screen for novel proteins regulating viral replication

[0125] I. Targeting the DNA of varicella-zoster virus (VZV) to screen for novel proteins regulating viral replication

[0126] 1. Transfected samples

[0127] Plasmids containing the VZV ORF4 promoter region, an empty vector (as a negative control), and a dCas9-sgRNA pool consisting of sgRNA sequences of SEQ ID NO.3, SEQ ID NO.7, and SEQ ID NO.10 were transfected into 293T cells.

[0128] 2. Cell cross-linking

[0129] Each sample contained 8 trays of 15cm-dish cells. 48 hours after transfection, the cells were fixed with 1% methanol-free formaldehyde for 15 minutes. After adding glycine and reacting for 5 minutes, the supernatant was discarded, and the cells were washed twice with pre-cooled phosphate-buffered saline (PBS).

[0130] 3. Cell lysis

[0131] Add 2 mL of PBS containing protease inhibitors to each dish to collect cells. After centrifuging at 1600 rpm to collect cells, add sodium dodecyl sulfate (SDS) lysis buffer containing protease inhibitors and dithiothreitol (DTT) to resuspend the cells to ensure homogeneity. Perform thorough lysis on ice.

[0132] 4. Obtaining DNA fragments by ultrasound

[0133] DNA fragments ranging in size from 200bp to 1000bp were obtained by using an ultrasonic instrument to break down the DNA; after repeated centrifugation at 15000rpm at least 3 times, 1mL of fragmented DNA was taken and diluted to 3mL.

[0134] 5. Pretreatment to remove non-specific binding

[0135] Pretreatment with protein A agarose beads and incubation with the sample for 2 hours reduces non-specific binding.

[0136] 6. Immunoprecipitation

[0137] Discard the protein A beads, then add 11 μL of agarose beads and incubate overnight at 4°C.

[0138] 7. Rinse and remove impurities to obtain the protein product to be identified.

[0139] Agarose beads containing DNA interaction products were repeatedly washed with wash buffer in the following order: 11 min with low-salt buffer, 5 min with high-salt buffer, 11 min twice with low-salt buffer, TE twice with high-salt buffer, and finally PBS three times. All washing was done at low speed on a shaker to obtain the protein product to be identified. Mass spectrometry samples were directly frozen at -80°C. The low-salt buffer consisted of: 0.1% sodium dodecyl sulfate, 1% Triton X-100, 2 mM ethylenediaminetetraacetic acid, 20 mM tris(hydroxymethyl)aminomethane hydrochloride, and 150 mM sodium chloride; the high-salt buffer consisted of: 0.1% sodium dodecyl sulfate, 1% Triton X-100, 2 mM ethylenediaminetetraacetic acid, 20 mM tris(hydroxymethyl)aminomethane hydrochloride, and 300 mM sodium chloride; and the TE buffer consisted of: 10 mM tris(hydroxymethyl)aminomethane hydrochloride and 1 mM ethylenediaminetetraacetic acid.

[0140] II. DNA screening targeting herpes simplex virus type I (HSV1) to identify novel proteins regulating viral replication.

[0141] 1. Transfected samples

[0142] In 293T cells, a plasmid containing the ICP27 promoter region of HSV1 was transfected, along with an empty vector (as a negative control) and a pool of dCas9-sgRNAs consisting of the sgRNA sequences of SEQ ID NO.12, SEQ ID NO.15 and SEQ ID NO.18.

[0143] The remaining steps (2-7) are the same as in "I. Screening for novel proteins that regulate viral replication by targeting the DNA of varicella-zoster virus (VZV).

[0144] III. Screening for novel proteins regulating viral replication using HIV DNA targeting.

[0145] 1. Transfected samples

[0146] Plasmids containing the HIV LTR promoter region, an empty vector (as a negative control), and a dCas9-sgRNA pool consisting of sgRNA sequences of SEQ ID NO.19, SEQ ID NO.20, and SEQ ID NO.21 were transfected into 293T cells.

[0147] The remaining steps 2-7 are the same as "I. Screening for novel proteins that regulate viral replication by targeting the DNA of varicella-zoster virus (VZV)".

[0148] IV. DNA screening for novel proteins regulating viral replication targeting Kaposi's sarcoma-associated herpesvirus (KSHV).

[0149] 1. Transfected samples

[0150] Plasmids containing the RTA promoter region, an empty vector (as a negative control), and a dCas9-sgRNA pool consisting of sgRNA sequences of SEQ ID NO.24, SEQ ID NO.27, and SEQ ID NO.29 were transfected into 293T cells.

[0151] The remaining steps 2-7 are the same as "I. Screening for novel proteins that regulate viral replication by targeting the DNA of varicella-zoster virus (VZV)".

[0152] Example 3: q-PCR verification of dCas9 binding in the VZV ORF4 promoter region and the HSV1 ICP27 promoter region

[0153] I. Methods

[0154] To further verify the effectiveness of the experimental procedure in Example 2, i.e. whether dCas9 effectively binds to the region, a portion of the protein product to be identified in Example 2 was taken, decrosslinked overnight at 65°C, and then treated with RNase and protease. The DNA was then purified using a PCR fragment recovery kit. The binding of dCas9 to the promoter region was detected by q-PCR.

[0155] Primers were designed to amplify different regions of the ORF4 promoter (the promoter is 1000 bp in total, divided into four regions of 250 bp each: 1-250 bp, 251-500 bp, 501-750 bp, and 751-1000 bp). Here, HSV1 represents the ICP27 promoter region, and KSHV represents the RTA promoter region, also 1000 bp, divided into four regions of 250 bp each.

[0156] II. Results

[0157] The qPCR results for VZV are shown below. Figure 7 The results for HSV1 can be found in [link to HSV1 results]. Figure 8 The results of KSHV can be found in [link to KSHV results]. Figure 9 This indicates that, compared to the negative control group with added sg-Gal4, sg-mix can significantly guide 3×Flag dCas9 to the promoter region, verifying the effectiveness of the experimental procedure in Example 2.

[0158] Example 4: Mass Spectrometry Analysis

[0159] The protein products targeting VZV DNA prepared in Example 2 were sent to the Hangzhou Jingjie Biomass Spectrometry Platform for identification and analysis. The results showed that multiple proteins capable of binding to varicella-zoster virus (VZV) DNA were screened, including YY1 and YBX1, which are known proteins in the prior art that can regulate VZV. This result also demonstrates the accuracy and effectiveness of the method. The results are shown in Table 1 and... Figure 10 .

[0160] Table 1. Partial list of DNA proteins that can bind to VZV, screened by the method of this application.

[0161]

[0162] The protein products targeting HSV1 DNA prepared in Example 2 were sent to the Hangzhou Jingjie Biomass Spectrometry Platform for identification and analysis. Multiple interacting proteins were screened and found to bind to herpes simplex virus type I (HSV1) DNA. The results are shown in Table 2. Figure 11 .

[0163] Table 2. Partial list of proteins that can bind to HSV1 DNA, screened by the method of this application.

[0164]

[0165] The protein products targeting KSHV DNA prepared in Example 2 were sent to the Hangzhou Jingjie Biomass Spectrometry Platform for identification and analysis. Multiple interacting proteins were screened and found to bind to Kaposi's sarcoma (KSHV) DNA. The results are shown in Table 3. Figure 12 .

[0166] Table 3. Some DNA proteins that can bind to KSHV, screened by the method of this application.

[0167]

[0168] Example 5: Targeting of target DNA using single sgRNA and mixed sgRNA

[0169] I. Targeting of varicella-zoster DNA using single sgRNA and mixed sgRNA

[0170] 1. Method

[0171] The sgRNAs of SEQ ID NO.3, SEQ ID NO.7, and SEQ ID NO.10 were mixed as a pool of sgRNAs interacting with dCas9, or individual sgRNAs of SEQ ID NO.2, SEQ ID NO.5, and SEQ ID NO.8 were used as sgRNAs interacting with dCas9. Proteins (varicella-zoster virus) interacting with the ORF4 promoter were prepared by immunoprecipitation. The binding of dCas9 to the ORF4 promoter region was verified by qPCR, referring to the methods in Examples 1-3. In this example, 15 μL of magnetic beads were used as Flag beads.

[0172] 2. Results

[0173] While individual sgRNAs can be highly efficient, complete coverage of the target DNA cannot be guaranteed. Figure 13 As shown;

[0174] Using mixed sgRNAs can ensure better targeting of each region, such as Figure 14 As shown.

[0175] 3. Conclusion

[0176] In summary, a mixture of sgRNAs containing SEQ ID NO.3, SEQ ID NO.7, and SEQ ID NO.10 was selected for use.

[0177] II. Targeting of Herpes Simplex Virus Type I DNA Using Single sgRNA and Mixed sgRNA

[0178] 1. Method

[0179] The sgRNAs of SEQ ID NO.12, SEQ ID NO.15, and SEQ ID NO.18 were mixed as a pool of sgRNAs interacting with dCas9, or individual SEQ ID NO.12, SEQ ID NO.15, and SEQ ID NO.18 were used as sgRNAs interacting with dCas9. Proteins (herpes simplex virus type I) interacting with the ICP27 promoter were prepared by immunoprecipitation. The binding of dCas9 to the ICP27 promoter region was verified by qPCR, referring to the methods in Examples 1-3. In this example, 15 μL of magnetic beads were used as Flag beads.

[0180] 2. Results

[0181] While individual sgRNAs can be highly efficient, complete coverage of the target DNA cannot be guaranteed. Figure 15 As shown;

[0182] Using mixed sgRNAs can ensure better targeting of each region, such as Figure 16 As shown.

[0183] 3. Conclusion

[0184] In summary, a mixture of sgRNAs containing SEQ ID NO.12, SEQ ID NO.15, and SEQ ID NO.18 was selected for use.

[0185] Example 6: Targeting of magnetic beads or agarose beads to target DNA

[0186] I. Targeting ability of magnetic beads or agarose beads to varicella-zoster virus DNA

[0187] 1. Method

[0188] The sgRNAs of SEQ ID NO.3, SEQ ID NO.7, and SEQ ID NO.10 were mixed to form a pool of sgRNAs that interact with dCas9; proteins (varicella-zoster virus) that interact with the ORF4 promoter were prepared by immunoprecipitation; qPCR was used to verify the binding of dCas9 to the ORF4 promoter region, as described in Examples 1-3. In this example, the amount of Flag beads used was 15 μL.

[0189] 2. Results and Conclusions

[0190] Magnetic beads are convenient to use for elution, but the non-specific signal is too high, such as... Figure 17 As shown;

[0191] Agarose beads are easy to handle and have low nonspecific signal, such as Figure 18 As shown, agarose beads were therefore chosen.

[0192] II. Targeting ability of magnetic beads or agarose beads to herpes simplex virus type I DNA

[0193] 1. Method

[0194] The sgRNAs of SEQ ID NO.12, SEQ ID NO.15, and SEQ ID NO.18 were mixed as a pool of sgRNAs interacting with dCas9, or individual SEQ ID NO.12, SEQ ID NO.15, and SEQ ID NO.18 were used as sgRNAs interacting with dCas9. Proteins (herpes simplex virus type I) interacting with the ICP27 promoter were prepared by immunoprecipitation. The binding of dCas9 to the ICP27 promoter region was verified by qPCR, referring to the methods in Examples 1-3. In this example, 15 μL of magnetic beads were used as Flag beads.

[0195] 2. Results and Conclusions

[0196] Magnetic beads are convenient to use for elution, but the non-specific signal is too high, such as... Figure 19 As shown;

[0197] Agarose beads are easy to handle and have low nonspecific signal, such as Figure 20 As shown, agarose beads were therefore chosen.

[0198] Example 7: The targeting effect of different amounts of agarose beads on target DNA

[0199] I. The targeting effect of different amounts of agarose beads on varicella-zoster virus DNA

[0200] 1. Method

[0201] The sgRNAs of SEQ ID NO.3, SEQ ID NO.7, and SEQ ID NO.10 were mixed to form a pool of sgRNAs interacting with dCas9; proteins (varicella-zoster virus) interacting with the ORF4 promoter were prepared by immunoprecipitation; qPCR was used to verify the binding of dCas9 to the ORF4 promoter region. See the methods in Examples 1-3.

[0202] 2. Results

[0203] Before adjustment, the agarose bead dosage was 15 μL. The target efficiency of the experimental group was good, but the signal of the control group was too high, such as... Figure 21 As shown;

[0204] The adjusted agarose bead dosage was 11 μL. This slight adjustment resulted in a significant decrease in the signal in the control group, such as... Figure 22 As shown.

[0205] 3. Conclusion

[0206] In summary, the optimal amount of agarose beads added was 11 μL.

[0207] II. Targeting effect of different agarose bead dosages on herpes simplex virus type I DNA

[0208] 1. Method

[0209] The sgRNAs of SEQ ID NO.12, SEQ ID NO.15, and SEQ ID NO.18 were mixed to form a pool of sgRNAs interacting with dCas9; proteins (herpes simplex virus type I) interacting with the ICP27 promoter were prepared by immunoprecipitation; qPCR was used to verify the binding of dCas9 to the ICP27 promoter region. See the methods in Examples 1-3.

[0210] 2. Results

[0211] Before adjustment, the agarose bead dosage was 15 μL. The target efficiency of the experimental group was good, but the signal of the control group was too high, such as... Figure 23 As shown;

[0212] The adjusted agarose bead dosage was 11 μL. This slight adjustment resulted in a significant decrease in the signal in the control group, such as... Figure 24 As shown.

[0213] 3. Conclusion

[0214] In summary, the optimal amount of agarose beads added was 11 μL.

[0215] Example 8: Comparison of the targeting efficiency of this application and patent application number 202010131159.2 on HIV viral DNA.

[0216] I. Methods

[0217] SgGal4: The sgRNA / dCas9 complex prepared according to Examples 1-4 of the patent application No. 202010131159.2 entitled "Screening of Viral Transcription Targets Based on Cas9-sgRNA Immunoprecipitation".

[0218] SgLTR: The sgRNA mixture used in the patent application No. 202010131159.2, entitled "Viral LTR Immunoprecipitation Screening of Viral Transcription Targets Based on Cas9-sgRNA", is used as a pool of sgRNAs that interact with dCas9 in the future.

[0219] Proteins interacting with LTR were prepared by immunoprecipitation according to Example 2 of this application;

[0220] q-PCR was used to verify the binding of dCas9 to the HIV-1 LTR region according to Example 3 of this application.

[0221] II. Results

[0222] like Figure 25 and Figure 26 As shown, the targeting efficiency of the method in this application for HIV viral DNA is much higher than that of the patent application number 202010131159.2, P < 0.001.

[0223] III. Conclusion

[0224] 1. The method of this application is also applicable to screening targets that regulate HIV transcription;

[0225] 2. The targeting efficiency of the method in this application for HIV viral DNA is significantly improved compared with the method of the patent application 202010131159.2.

[0226] Example 9: Efficiency comparison of this application with that of patent application number 202010131159.2 targeting herpesvirus

[0227] I. Methods

[0228] Using the method of "Screening Viral Transcription Targets Based on Viral LTR Immunoprecipitation Using Cas9-sgRNA" in application number 202010131159.2, we screened for novel targets that regulate viral replication by targeting varicella-zoster virus DNA.

[0229] The method of this application targets a new target for screening and regulating viral replication of varicella-zoster virus DNA, as described in Examples 1-3.

[0230] II. Results

[0231] Following the method described in application number 202010131159.2, "Screening of Viral Transcription Targets Based on Cas9-sgRNA-based Viral LTR Immunoprecipitation," targeting varicella-zoster virus DNA showed very low efficiency. (See results below.) Figure 27 ;

[0232] The method described in this application targets varicella-zoster virus DNA with high efficiency, and the results are shown in [see figure]. Figure 28 .

[0233] III. Conclusion

[0234] 1. The targeting efficiency of the method in this application for varicella-zoster DNA is significantly improved compared with the method of patent application 202010131159.2;

[0235] 2. The method of “screening viral transcription targets based on Cas9-sgRNA-based viral LTR immunoprecipitation” in application number 202010131159.2 is not applicable to screening new targets for varicella-zoster virus replication regulation by targeting varicella-zoster DNA due to its low efficiency.

[0236] Although the present invention has been described in detail above with general descriptions and specific embodiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention.

Claims

1. A method for targeting viral DNA to screen for proteins that regulate viral replication, characterized in that, The virus is varicella-zoster virus VZV, and the method includes the following steps: (1) Transfected samples The plasmid containing the VZV ORF4 promoter region was transfected into 293T cells, along with an empty vector and a dCas9-sgRNA pool as a negative control, the dCas9-sgRNA pool consisting of the sgRNA sequences of SEQ ID NO.3, SEQ ID NO.7 and SEQ ID NO.10; (2) Cell cross-linking Each sample contained 8 trays of 15cm-dish cells. 48 hours after transfection, the cells were fixed with 1% methanol-free formaldehyde for 15 minutes. After adding glycine and reacting for 5 minutes, the supernatant was discarded, and the cells were washed twice with pre-cooled phosphate-buffered saline (PBS). (3) Cell lysis Add 2 mL of PBS containing protease inhibitors to each dish to collect cells. After centrifuging at 1600 rpm to collect cells, add sodium dodecyl sulfate SDS lysis buffer containing protease inhibitors and dithiothreitol (DTT) to resuspend the cells to ensure homogeneity. Perform thorough lysis on ice. (4) Obtaining DNA fragments by ultrasound DNA fragments ranging in size from 200bp to 1000bp were obtained by using an ultrasonic instrument to break down the DNA; after centrifuging at 15000rpm at least 3 times, 1mL of fragmented DNA was diluted to 3mL. (5) Pretreatment to remove non-specific binding Pretreatment with protein A agarose beads and incubation with the sample for 2 hours reduces non-specific binding. (6) Immunoprecipitation Discard the protein A beads and then add 11 μL of agarose beads (Flag beads) and incubate overnight at 4°C. (7) Rinse and remove impurities to obtain the protein product to be identified. Agarose beads containing DNA interaction products were repeatedly washed with rinsing buffer in the following order: 11 min with low-salt buffer, 5 min with high-salt buffer, 11 min twice with low-salt buffer, TE buffer twice with 5 min, and finally washed three times with PBS. All washing was performed at low speed on a shaker to obtain the protein product to be identified. Mass spectrometry samples were directly frozen at -80°C. The low-salt buffer consisted of: 0.1% sodium dodecyl sulfate, 1% Triton X-100, 2 mM ethylenediaminetetraacetic acid (EDTA), 20 mM tris(hydroxymethyl)aminomethane hydrochloride, and 150 mM sodium chloride. The high-salt buffer consisted of: 0.1% sodium dodecyl sulfate, 1% Triton X-100, 2 mM EDTA, 20 mM tris(hydroxymethyl)aminomethane hydrochloride, and 300 mM sodium chloride. The TE buffer consisted of: 10 mM tris(hydroxymethyl)aminomethane hydrochloride and 1 mM EDTA. (8) Mass spectrometry analysis of the protein product to be identified.

2. The method as described in claim 1, characterized in that, The names of the protein genes that interact with the varicella-zoster virus (VZV) ORF4 promoter region include: KRT86, MIF, RNF20, AASHPPT, DUS3L, YY1, ILKAP, FLYWCH2, YBX1, NUP50, and CPSF6.

3. The method as described in claim 1, characterized in that, The virus also includes herpes simplex virus type I HSV1. The (1) transfection sample refers to the plasmid containing the ICP27 promoter region of HSV1 transfected in 293T cells, as well as the empty vector and dCas9-sgRNA pool as a negative control group. The dCas9-sgRNA pool consists of the sgRNA sequences of SEQ ID NO.12, SEQ ID NO.15 and SEQ ID NO.

18.

4. The method as described in claim 3, characterized in that, The names of protein genes that interact with the ICP27 promoter region of herpes simplex virus type I (HSV1) include: H3C1, RNH1, WDR18, PAK1IP1, WARS1, PAICS, NOMO3, IRS4, CIRBP, CPNE3, and RHOC.

5. The method as described in claim 1, characterized in that, The virus also includes Kaposi's sarcoma-associated herpesvirus (KSHV). The (1) transfection sample refers to a plasmid containing the RTA promoter region of KSHV transfected into 293T cells, as well as an empty vector and a dCas9-sgRNA pool as a negative control group. The dCas9-sgRNA pool consists of the sgRNA sequences of SEQ ID NO.24, SEQ ID NO.27 and SEQ ID NO.

29.

6. The method as described in claim 5, characterized in that, The names of the protein genes that interact with the Kaposi's sarcoma-associated herpesvirus (KSHV) RTA promoter region include: POLR2B, CLIC4, STAU2, PPP1CB, TPR, WARS1, IRS4, PAICS, RAB8A, NOMO3, RAB1B, and TMEM33.

7. The method as described in claim 1, characterized in that, The virus also includes HIV, and the (1) transfection sample refers to a plasmid containing the LTR promoter region of HIV transfected in 293T cells, an empty vector as a negative control group, and a dCas9-sgRNA pool consisting of the sgRNA sequences of SEQ ID NO.19, SEQ ID NO.20 and SEQ ID NO.21.

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