阅读说明：本技术 靶向逆转录引物结合位点从而抑制hiv病毒复制的方法 (Method for inhibiting HIV virus replication by targeting reverse transcription primer binding site ) 是由 杨鹏 王译萱 杨博 房璐 于 2020-12-30 设计创作，主要内容包括：本发明属于基础研究领域,尤其涉及一种靶向逆转录引物结合位点从而抑制HIV病毒复制的方法。首先公开了一种从数据库中筛选出HIV抑制因子的方法,包括：S1：从数据库提取锌指蛋白ChIP结合峰的记录数据文件；S2：整理HIV靶点序列,利用MEME工具获得靶点基序Motif；获得人类基因组上含有的HIV靶点的位置信息；S3：将锌指蛋白ChIPseq结合峰文件与人类基因组上HIV靶点的位置信息文件进行比对,得到潜在的HIV靶向抑制的锌指蛋白。本发明提供了一种寻找HIV限制因子的方法,找到了HIV的两个限制性锌指蛋白ZNF417和ZNF587,填补了人体对于HIV的天然免疫作用这一领域的空白。(The invention belongs to the field of basic research, and particularly relates to a method for inhibiting HIV virus replication by targeting reverse transcription primer binding sites. First, a method for screening HIV inhibitors from a database is disclosed, comprising: s1: extracting a record data file of a zinc finger protein ChIP binding peak from a database; s2: arranging HIV target sequences, and obtaining a target Motif Motif by utilizing a MEME tool; obtaining positional information of HIV targets contained on a human genome; s3: and comparing the zinc finger protein ChIPseq binding peak file with a position information file of an HIV target on a human genome to obtain the potential HIV target-inhibited zinc finger protein. The invention provides a method for searching HIV restriction factors, finds two restrictive zinc finger proteins ZNF417 and ZNF587 of HIV, and fills the gap of the field of natural immunity of human bodies to HIV.)

1. A method of screening a database for HIV inhibitory factors comprising:

s1: obtaining ChIPseq data of zinc finger proteins of a human KRAB-ZFPs family from a GEO database, and extracting recorded data BED files of ChIP binding peaks of the zinc finger proteins;

s2: finishing HIV target sequences and outputting FASTA files; obtaining a target Motif Motif by utilizing a MEME tool according to a FATSA file; finding the position containing the motif in the human genome by using an FIMO tool, and comparing the position with an annotation information BED file of the human endogenous retrovirus; obtaining position information of HIV targets contained in a human genome, and outputting the position information as a BED file;

s3: and comparing the zinc finger protein ChIPseq binding peak BED file with the position information BED file of the HIV target on the human genome by using a BEDTools tool to obtain the potential zinc finger protein for HIV targeted inhibition, namely the HIV inhibiting factor.

2. The method of claim 1, wherein in S1, the binding site information file of human KRAB-ZFPs is compiled from two sets of high-throughput zinc finger protein ChIPseq data of GSE78099 and GSE76496 of GEO database.

3. The method of claim 1, wherein in S2-S3, HIV target PBS-Lys is determined, motif of PBS-Lys and location information on genome are obtained;

and comparing the PBS-Lys site information with the data of KRAB-ZFPs to obtain the zinc finger proteins ZNF417 and ZNF587 of potential HIV inhibiting factors.

4. The method according to claim 3, further comprising step S4: the inhibitory effect of ZNF417 and ZNF587 on HIV virus was verified.

5. The method according to claim 4, wherein step S4 includes:

s41: constructing ZNF417 and ZNF587 expression vectors, and determining the combination effect of ZNF417 and ZNF587 on PBS-Lys through ChIP experiments and reporter gene experiments;

s42: constructing ZNF417 and ZNF587 knockout cell lines by using a CRSPR-cas9 system, and infecting HIV pseudoviruses in the cell lines; or

HIV pseudovirus was infected in cells overexpressing ZNF417 and ZNF587, and the inhibitory effect of ZNF417 and ZNF587 on HIV virus was confirmed.

6. Application of zinc finger proteins ZNF417 and ZNF587 in preparing medicine for treating HIV.

7. The use according to claim 6, wherein the medicament is a medicament for inhibiting replication of the HIV virus.

8. The application of zinc finger proteins ZNF417 and ZNF587 in preparing target reverse transcription HIV primer binding site medicine.

9. A method of inhibiting replication of an HIV virus, comprising binding HIV PBS-Lys via a zinc finger protein, thereby inhibiting replication of the HIV virus.

10. An agent for inhibiting replication of an HIV virus, comprising zinc finger proteins ZNF417 and/or ZNF 587.

Technical Field

The invention belongs to the field of basic research, and particularly relates to a method for inhibiting HIV virus replication by targeting reverse transcription primer binding sites.

Technical Field

Retroviruses (retroviruses) have long been attached to humans and other higher animals, have evolved with their co-survival, and are closely related to various biological phenomena. Retroviruses can be classified as endogenous and exogenous depending on their relationship to the host. Endogenous viruses (ERVs) are derived from the invasion of mammalian germ cells by ancient exogenous retroviruses and accumulate gradually as part of the genome over long-term evolution. Approximately 10% of the sequences in the mouse and human genomes belong to ERVs, which are mostly transcriptionally silent to maintain genome stability. Exogenous retroviruses are more pathogenic than ERVs. For example, the well-known retrovirus, the human immunodeficiency virus, hiv (human immunodeficiency virus), is still one of the important threats to public health and safety as the causative agent of Acquired Immune Deficiency Syndrome (AIDS).

The reverse transcription process of a retrovirus is the core of its replication cycle. The retroviral genome has a sequence matching the 3' end of the host tRNA. Therefore, retroviruses can use tRNA as a primer to synthesize DNA using genomic RNA as a template to complete the reverse transcription process. This sequence that binds to tRNA is PBS (primer binding site). PBS of different viruses are matched to different tRNA's, and human immunodeficiency virus HIV utilizes lysine (Lys) tRNA. At present, the mechanism of action of reverse transcriptase has been studied preliminarily. The structure of the tRNA was preliminarily resolved in 2018, and the tRNA is proved to be capable of being directly combined with PBS to initiate the reverse transcription process of retrovirus. Therefore, PBS is very important for replication of retrovirus, and the sequence of PBS is relatively conserved, so that PBS is suitable for target research.

KRAB-ZFPs are the largest subset of the C2H2 tandem Zinc Finger Protein (ZFP) family, the largest family of transcription factors in the mouse and human genomes. KRAB-ZFPs contain two domains: an N-terminal KRAB domain and a C-terminal C2H2 zinc finger tandem domain. The KRAB domain mediates recruitment of TRIM28 (also known as KAP1, Tif1 β or KRIP-1) to serve as a scaffold for a histone modification complex, including histone H3K9me3 methyltransferase SETDB1, histone deacetylase HDAC, and heterochromatin protein 1(HP1), catalyzing heterochromatin formation and transcriptional repression. KRAB/TRIM 28-induced gene silencing during early embryonic development may also promote heritable epigenetic silencing by inducing DNA methylation, which may be maintained even after TRIM28 has been depleted. Furthermore, in some cases, KRAB/TRIM 28-induced heterochromatin silencing may be transmitted at longer genomic distances along the chromosome, probably facilitated by the H3K9me3 recognition protein HP1, and H3K9me3 may be further transmitted by recruitment of SETDB 1.

KRAB-ZFPs have been reported to be involved in a number of biological processes including embryonic development, genomic imprinting, cell differentiation, metabolic control and sex determination. Molecular biology studies find that the KRAB-ZFPs mostly have ERVs in the genome-wide binding sites. Knock-out of the scaffold protein TRIM28 or its chaperone histone methyltransferase SETDB1, which interacts with the KRAB domain, activates a variety of ERVs including those specifically expressed in mouse and human Embryonic Stem Cells (ESCs). Among KRAB-ZFPs that inhibit the expression of ERVs, the gene ZFP809 was studied more extensively. ZFP809 and TRIM28 were first identified by analyzing the binding proteins of the mouse leukemia virus (MuLV) proline primer binding sequence (PBS-Pro), and were found to inhibit ERVs expression with PBS-Pro elements in mouse embryonic stem cells. However, it has not been reported how the zinc finger protein has an effect on HIV virus and its primer binding sequence PBS-Lys.

Disclosure of Invention

In a recent research, the inventor finds potential zinc finger proteins binding to a primer binding site PBS-Lys of a lysine Lys tRNA through a zinc finger protein database published in a large-scale analysis literature, and the discovery provides a research basis for further exploring the effect of the zinc finger proteins in inhibiting HIV virus.

Specifically, the technical scheme of the invention is as follows:

in a first aspect, the present invention discloses a method for screening HIV inhibitors from a database, comprising:

s1: obtaining ChIPseq data of zinc finger proteins of a human KRAB-ZFPs family from a GEO database, and extracting recorded data BED files of ChIP binding peaks of the zinc finger proteins;

s2: finishing HIV target sequences and outputting FASTA files; obtaining a target Motif Motif by utilizing a MEME tool according to a FATSA file; finding the position containing the motif in the human genome by using an FIMO tool, and comparing the position with an annotation information BED file of the human endogenous retrovirus; obtaining position information of HIV targets contained in a human genome, and outputting the position information as a BED file;

s3: and comparing the zinc finger protein ChIPseq binding peak BED file with the position information BED file of the HIV target on the human genome by using a BEDTools tool to obtain the potential zinc finger protein for HIV targeted inhibition, namely the HIV inhibiting factor.

Preferably, in S1, the binding site information file of human KRAB-ZFPs is collated from two groups of high-flux zinc finger protein ChIPseq data of GSE78099 and GSE76496 of the GEO database.

Preferably, in S2-S3, HIV target PBS-Lys is determined, and motif of the PBS-Lys and positioning information on a genome are obtained;

the PBS-Lys site information and the KRAB-ZFPs data are compared to obtain the zinc finger proteins ZNF417 (NP-689688.2) and ZNF587 (NP-116217.1) of potential HIV inhibitors.

More preferably, the method further comprises step S4: the inhibitory effect of ZNF417 and ZNF587 on HIV virus was verified.

Preferably, step S4 includes:

s41: constructing ZNF417 and ZNF587 expression vectors, and determining the combination effect of ZNF417 and ZNF587 on PBS-Lys through ChIP experiments and reporter gene experiments;

s42: constructing ZNF417 and ZNF587 knockout cell lines by using a CRSPR-cas9 system, and infecting HIV pseudoviruses in the cell lines; or

HIV pseudovirus was infected in cells overexpressing ZNF417 and ZNF587, and the inhibitory effect of ZNF417 and ZNF587 on HIV virus was confirmed.

The second aspect of the invention discloses an application of zinc finger proteins ZNF417 and ZNF587 in preparing a medicament for treating HIV.

Preferably, the drug is a drug that inhibits HIV viral replication.

The third aspect of the invention discloses an application of zinc finger proteins ZNF417 and ZNF587 in preparing a target reverse transcription HIV primer binding site medicament.

In the fourth aspect of the invention, the invention discloses a method for inhibiting the replication of HIV virus, which inhibits the replication of HIV virus by combining zinc finger protein with HIV PBS-Lys.

In the fifth aspect of the invention, the invention discloses a medicament for inhibiting the replication of HIV virus, which comprises zinc finger proteins ZNF417 and/or ZNF 587.

In some preferred embodiments of the invention, a method of identifying an HIV inhibitor comprises the steps of:

1. ChIPseq data of zinc finger proteins of the human KRAB-ZFPs family are obtained from a GEO database (Gene Expression Omnibus https:// www.ncbi.nlm.nih.gov/GEO /), and record data BED files of the ChIP binding peaks of the zinc finger proteins are extracted.

2. And (5) finishing HIV target sequences and outputting a FASTA file. The target Motif Motif was obtained from the FATSA file using the MEME (http:// me-suite. org /) tool. The FIMO (http:// me-suite. org /) tool was used to find the location in the human genome that contained the motif and aligned with the annotated information BED file of Human Endogenous Retrovirus (HERV). And obtaining the position information of HIV targets contained in the human genome and outputting the position information as a BED file.

3. The zinc finger protein ChIPseq binding peak BED file was aligned with the location information BED file of HIV targets on the human genome using the BEDTools (http:// dx. doi. org/10.1093/bioinformatics/btq033) tool. Obtaining the zinc finger protein of potential HIV target inhibition.

In other preferred embodiments of the present invention, a method of inhibiting HIV using the zinc finger proteins ZNF417 and ZNF587 identified by the above method comprises the steps of:

1. through two groups of high-flux zinc finger protein ChIPseq data of GSE78099 and GSE76496 of a GEO database, a KRAB-ZFPs binding site information file of a human is arranged.

2. By determining HIV target point PBS-Lys, motif of PBS-Lys and positioning information on genome are obtained.

3. And comparing the PBS-Lys site information with the data of KRAB-ZFPs to obtain zinc finger proteins ZNF417 and ZNF587 of potential HIV-targeted PBS.

4. ZNF417 and ZNF587 expression vectors are constructed, and the combination effect of ZNF417 and ZNF587 on PBS-Lys is determined through ChIP experiments and reporter gene experiments.

5. Constructing ZNF417 and ZNF587 knockout cell lines by using a CRISPR-cas9 system, and infecting HIV pseudovirus in the cell lines; or infecting HIV pseudovirus in the cell over expressing ZNF417 and ZNF587, and verifying the inhibiting effect of ZNF417 and ZNF587 on HIV virus. And further verifies the correctness and practicability of the method for identifying the HIV restriction factor provided by the first aspect of the invention.

The innovation points of the invention are as follows: 1) the method initially focuses on the PBS regulatory element of retrovirus and the characteristic of zinc finger protein specificity recognition DNA sequence, and screens the zinc finger protein as a potential HIV virus inhibiting factor. 2) At present, the mechanism of how HIV is recognized and defended by host cells remains unclear. The invention starts from endogenous retroviruses, combines more than two generations of deep sequencing and bioinformatics analysis means at the whole genome level, and screens out the zinc finger protein of the PBS targeting HIV from the database of the interaction of the zinc finger protein and the endogenous retroviruses.

Compared with the prior art, the invention has at least the following beneficial effects:

(1) provides a method for searching HIV restriction factors, and finds two restrictive zinc finger proteins ZNF417 and ZNF587 of HIV. Provides an evidence for the mechanism that the zinc finger protein targets the reverse transcription primer binding site to inhibit the retrovirus.

(2) The invention helps to fill the gap in the field of natural immunization of humans against retroviruses, in particular HIV.

(3) The invention provides an important content for researching the zinc finger protein as a transcription factor family which is evolved by mammals and inhibits retrovirus, and provides an action mechanism of HIV which threatens human life health in recent decades; a new possible method for inhibiting the HIV virus is provided.

Drawings

In FIG. 1, A represents the statistics of the number of binding peaks of zinc finger proteins in GEO database with respect to PBS-Lys sites. B shows that luciferase reporter genes verify the binding effect of ZNF417 and ZNF587 to PBS-Lys. "peak counts" in the figure refers to "ChIP binding peak count"; "relative luciferase activity" means "relative luciferase activity".

FIG. 2A shows ChIPseq verification of ZNF417 and ZNF587 after expression of fusion with GFP, and the binding motif of ZNF417 and ZNF587 is matched with PBS-Lys. B shows the locus heatmap verifying that ChIPseq analysis ZNF417 and ZNF587 bind to the genome. C and D represent schematic diagrams of typical binding targets of two ZNF417 and ZNF 587. In the figure, "PBS-Lys sites" refers to "PBS-Lys sites".

FIG. 3 shows the inhibition of the LTR (promoter) transcription ability of HIV by ZNF417 and ZNF587 verified by dual luciferase reporter genes.

FIG. 4, panel AB, shows the different degrees of infection of two different HIV pseudoviruses in cells overexpressing (above) or knocking out (below) the zinc finger proteins ZNF 417/587. Viral infection was shown by the degree of expression of RFP, as detected by flow cytometry. In the figure, "ZNFS overexpression" means "ZNFS overexpression".

FIG. 5 shows HIV infection following knockout of ZNF417 and ZNF587 in human CD4+ T cells. Viral infection was detected by flow cytometry analysis.

FIG. 6 is a schematic representation of the inhibition of ZNF417 and ZNF587 in one replication cycle of HIV.

Detailed Description

The present application is further illustrated by the following detailed examples, which should be construed to be merely illustrative and not limitative of the remainder of the disclosure.

The instruments, equipment, reagents used in the examples are available from various sources, for example, purchased, or may be prepared.

Example 1: high throughput sequencing databases of binding zinc finger proteins identified ZNF417 and ZNF587 as potential zinc finger proteins binding PBS-Lys.

Firstly, the purpose is as follows: identification of zinc finger proteins binding to HIV target PBS-Lys

Secondly, the method comprises the following steps:

1. candidate zinc finger proteins are screened out by comparing two groups of high-flux zinc finger protein ChIPseq data of GSE78099 and GSE76496 of a GEO database with PBS-Lys sites.

2. The reporter gene activity is detected by inserting PBS-Lys sites in front of a reporter gene vector promoter and co-expressing zinc finger proteins ZNF417 and ZNF587 expression plasmids, and the binding capacity of ZNF417 and ZNF587 on PBS-Lys is verified by detecting the reporter gene activity.

3. The inhibitory effect of ZNF417 and ZNF587 on the PBS-Lys site was verified by ChIP experiments and high throughput sequencing after overexpression of GFP-tag ZNF417 and ZNF587 in HEK293T cells.

Third, result and discussion

By the data analysis of GEO, we obtained zinc finger proteins ZNF417 and ZNF587 (FIG. 1A) which potentially bind to PBS-Lys. In reporter gene assays, we observed significant binding capacity of ZNF417 and ZNF587 to PBS-Lys (FIG. 1B). A second chupseq validation of ZNF417 and ZNF587 we found that the binding motifs of ZNF417 and ZNF587 matched PBS-Lys (fig. 2A) and that significant binding of ZNF417 and ZNF587 was found at the endogenous retroviral sites of PBS-Lys containing humans (fig. 2B) and at these sites ZNF417 and ZNF587 could recruit the inhibitor KAP1 where heterochromatin modified H3K9me3 was formed to form inhibitory effects on PBS-Lys (fig. 2B-D).

Example 2: HIV pseudovirus infection experiment verifies that ZNF417 and ZNF587 have inhibiting effect on HIV

Firstly, the purpose is as follows: verification of the ability of ZNF417 and ZNF587 to inhibit HIV

Secondly, the method comprises the following steps:

1. the ability of ZNF417 and ZNF587 to inhibit transcription of HIV was verified by co-expression of a luciferase reporter gene driven by LTR (similar promoter) of HIV with ZNF417 and ZNF 587.

2. Whether ZNF417 and ZNF587 can inhibit HIV infection or not is judged by designing HIV pseudovirus (here, the full-length HIV virus pNL4.3 and the skeletonvirus pSicoR are used for expressing RFP to indicate the infection efficiency of HIV virus according to the expression condition of RFP) to infect in the cells of ZNF417 and ZNF587 overexpression or gene knockout based on the CRSIPCRas 9 system and detecting by flow cytometry.

3. The role of ZNF417 and ZNF587 in real HIV challenge to host cells was simulated by infecting the HIV pseudovirus after knocking out ZNF417 and ZNF587 in human CD4+ T cells.

Third, result and discussion

By reporter gene system, ZFP961 as mouse PBS-Lys binding zinc finger protein as positive control and empty plasmid (control) as negative control, we confirmed the ability of ZNF417 and ZNF587 to target PBS-Lys to inhibit HIV transcription (fig. 3). By infecting the HIV virus after overexpression of ZNF417 and ZNF587 in HEK293T, we verified the inhibitory effect of ZNF417 and ZNF587 on HIV (fig. 4A, B) and demonstrated a stronger viral infection effect in a series of ZNF417 and ZNF587 gene knock-out cell lines (fig. 4A, B). In ZNF417 and ZNF587 knockout CD4+ T cells, HIV was more susceptible to infection (FIG. 5), demonstrating the function of zinc finger proteins ZNF417 and ZNF587 as inhibitors in the body facing HIV viral infection. FIG. 6 is a schematic representation of the inhibition of ZNF417 and ZNF587 in one replication cycle of HIV.

The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.