Enhanced expression of polo-like kinase 3 (PLK3) in human immunodeficiency virus (HIV)-infected cells

Abstract

Biomarker of HIV or SIV infected cells and its application are provided. The marker is PLK (polo-like kinase). By inhibiting the activity of a PLK protein or clearing the same, the purpose of releasing viruses without activating a repository is achieved, such that the viruses can be detected in a physiological state and can also be recognized and cleared by an immune system or a drug in vivo. Enhancing the activity of the PLK protein directly inhibits the release of viruses in an HIV and/or SIV-infected cell. The present invention provides a new target for diagnosis and antiviral therapy of HIV and/or SIV infection, provides medication basis and guarantee for the early rapid detection and extremely early treatment of virus infection, and has important clinical value.

Claims

1. An in vitro method of enhancing an expression of a PLK3 protein in an HIV-infected cell comprising the step of transfecting the HIV-infected cell with a PLK3 protein expression vector; wherein the PLK3 protein is a PLK3-201 protein, and the PLK3-201 protein has the amino acid sequence shown in SEQ ID NO: 1.

2. The in vitro method according to claim 1, wherein the HIV-infected cell comprises: peripheral blood cells, CD4+T cells, natural killer cells, macrophages, dendritic cells, and neurogliocytes.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 is a diagram showing comparison of the infection rate of HIV in a supernatant after transfection with a PLK3 protein expression vector.

(2) FIG. 2 is a diagram showing comparison of the content of p24 after transfection with a PLK3 protein expression vector.

(3) FIG. 3 is a diagram showing comparison of the content of genomic RNA associated with virus particles after transfection with a PLK3 protein expression vector.

(4) FIG. 4 is a diagram showing comparison of the content of HIV Gag RNA after transfection with a PLK3 protein expression vector.

(5) FIG. 5 is a diagram showing comparison of the contents of a wild type Gag protein and a codon optimized Gag protein after transfection with a PLK3 protein expression vector.

(6) FIG. 6 is a diagram showing comparison of the content of HBV in a supernatant after transfection with a PLK3 protein expression vector.

(7) FIG. 7 is a diagram showing comparison of the content of HIV-1 in a supernatant after transfection of 293T cells with PLK1, PLK2, PLK3, PLK4 and PLK5 protein expression vectors.

(8) FIG. 8 is a diagram showing comparison of the content of HIV-1 in a supernatant after transfection with a PLK3 protein expression vector.

(9) FIG. 9 is a diagram showing comparison of the contents of different envelope (VSV-G, HIV-1.sub.DoL, HIV-1.sub.SF162, HIV-1.sub.89.6 and HIV-1.sub.AD8) viruses in a supernatant after transfection with a PLK3 protein expression vector.

(10) FIG. 10 is a diagram showing comparison of the content of HIV Gag RNA after transfection with a PLK3 protein expression vector.

(11) FIG. 11 is a diagram showing comparison of the content of CCR5 tropic HIV-1.sub.AD8 in a supernatant after transfection with PLK1, PLK2, PLK3, PLK4 and PLK5 protein expression vectors.

(12) FIG. 12 is a diagram showing comparison of the content of CCR5 and CXCR4 (bitropic) HIV-1.sub.89.6 in a supernatant after transfection with PLK1, PLK2, PLK3, PLK4 and PLK5 protein expression vectors.

(13) FIG. 13 is a diagram showing comparison of the content of CCR5 tropic HIV-1.sub.BaL in a supernatant after transfection with PLK1, PLK2, PLK3, PLK4 and PLK5 protein expression vectors.

(14) FIG. 14 is a diagram showing comparison of the content of HIV-2.sub.ROD in a supernatant after transfection with PLK1, PLK2, PLK3, PLK4 and PLK5 protein expression vectors.

(15) FIG. 15 is a diagram showing comparison of the content of SIV.sub.agm in a supernatant after transfection with PLK1, PLK2, PLK3, PLK4 and PLK5 protein expression vectors.

(16) FIGS. 16A-16B are diagrams showing effect of a PLK3 kinase inhibitor, GW843682X, on HIV-1 infection.

(17) FIG. 17 is a diagram showing effect of a PLK3 kinase defective mutant expression vector on HIV-1 infection after transfection.

DETAILED DESCRIPTION OF THE EMBODIMENTS

(18) The present invention is described in detail below in combination with specific embodiments, for the purpose of making technical contents better understood by the public, rather than limiting the technical contents. Actually, improvements made according to same or similar principles are within the scope of protection of claims of the present invention.

Example 1

(19) A human PLK3 protein in HIV repository cells has the effect of inhibiting translation of an HIV-1 virus protein and has a specific inhibition effect on HIV-1.

(20) In order to verify the effects of PLK3, 293T cells with different PLK3 protein expression levels were treated with an HIV-1 virus vector, and it was found that compared with 293T cells without PLK3 protein expression, the HIV infectivity of the 293T cells with PLK3 protein expression was reduced to a maximum of about 1/150 of an original level. Specific test steps are as follows. 293T cells were transfected with a 3 FLAG-labeled PLK3 protein expression vector, and including a negative control group, were treated with pNL4.3. After the transfection was performed for 48 hours, a supernatant was used for infecting TZM-bl reporter cells to obtain virus infection levels. Data measured each time were obtained by subtracting background RLU (FIG. 1).

(21) In the case of overexpression of the PLK3 protein, the contents of a core antigen p24 of virus particles and genomic RNA of the virus particles in a cultured supernatant were also greatly decreased. Specific test steps are as follows. Similar to pretreatment steps shown in FIG. 1, p24 enzyme-linked immunosorbent assay (ELISA) was performed (FIG. 2), and qPCR based on a specific probe was performed to measure genomic RNA associated with virus particles (FIG. 3).

(22) However, the transcription level of a virus Gag in cells was not affected by the overexpression of the PLK3 protein. It was indicated that the overexpression of the PLK3 protein obviously reduced the expression of the HIV-1 protein without affecting the transcription of HIV-1. Meanwhile, the virus Gag protein associated with the PLK3 protein was also obviously reduced, indicating that the PLK3 was involved in the process of specifically inhibiting the translation of the HIV protein. Specific test steps are as follows. All RNAs were extracted for qPCR to measure the Gag transcription level of HIV-1 (FIG. 4). Cells were collected for a flow cytometry test, and the expression level of an HIV-1 p24 protein was detected by using specific antibodies (FIG. 5).

(23) Effects of the overexpression of the PLK3 protein in 293T cells on a hepatitis B virus (HBV) driven by a CMV promoter were detected to determine whether the PLK3 protein acted specifically on HIV-1. Results show that complete HBV replication is found, and that is to say, replication and release of the HBV are not affected by the PLK3 protein. Specific test steps are as follows. 293T cells in an experimental group were transfected with a 3 FLAG-labeled PLK3 expression vector, and including a negative control group, were treated with an HBV replicator (CMV promoter). After 48 hours, HBV virus particles in a cultured supernatant were measured by HBs enzyme-linked immunosorbent assay (FIG. 6).

Example 2

(24) In a human PLK (PLK1, PLK2, PLK3, PLK4 and PLK5) protein family, only a PLK3 protein has the effect of inhibiting the translation of an HIV-1 protein and has a specific inhibition effect on HIV-1. Meanwhile, a chimpanzee PLK3 protein also has the effect of inhibiting the translation of an HIV-1 protein and has a specific inhibition effect on HIV-1.

(25) Human PLK proteins specifically include the following five types: a PLK1 protein, a PLK2 protein, a PLK3 protein, a PLK4 protein and a PLK5 protein. The anti-HIV-1 activity of all the five human PLK proteins and primate PLK3 proteins was detected. Compared with a negative control group, the HIV-1 infectivity was reduced by human PLK3 with different expression amounts to 1/11 and 1/115 of that of the negative control group. The human PLK1 protein, the PLK2 protein, the PLK4 protein and the PLK5 protein did not show anti-HIV-1 activity. A chimpanzee PLK3 protein and a macaca PLK3 protein can also reduce the HIV-1 infectivity, but have slightly different reduction degrees compared with the human PLK3 protein. It is indicated that all primate PLK3 family proteins may have anti-HIV-1 activity. Specific test steps are as follows: 293T cells were transfected with 3 FLAG-labeled human PLK1, PLK2, PLK3, PLK4 and PLK5 protein expression vectors, a 3 FLAG-labeled chimpanzee PLK3 protein expression vector and a 3 FLAG-labeled macaca PLK3 protein expression vector, respectively, and including a negative control group, were treated with pNL4.3. After the transfection was performed for 48 hours, a supernatant was used for infecting TZM-bl reporter cells to obtain virus infection levels. Data measured each time were obtained by subtracting background RLU (FIG. 7 and FIG. 8).

Example 3

(26) A PLK3 protein has an inhibition effect on HIV-1, which is reflected in an inhibition effect on HIV wrapped by CXCR4 and CCR5 tropic envelope proteins without affecting the transcription of Gag.

(27) A human PLK3 protein has an extremely strong ability to block HIV-1 infection without affecting the transcription of Gag Specific test steps are as follows. 293T cells were transfected with a 3 FLAG-labeled PLK3 protein expression vector, and including a negative control group, were treated with pNL4.3E-GFP and vectors expressing VSV-G, pDoL-gp160, pSF162-gp160, pAD8-gp160 or p89.6-gp160, respectively. After the transfection was performed for 48 hours, a supernatant was used for infecting TZM-bl reporter cells to obtain virus infection levels. Data measured each time were obtained by subtracting background RLU (FIG. 9). All RNAs extracted were subjected to qPCR to quantify a Gag transcript, followed by normalization with phosphoglyceraldehyde dehydrogenase (FIG. 10).

Example 4

(28) A PLK3 protein has an inhibition effect on HIV-1, HIV-2 and SIV, which is reflected in an inhibition effect on wild type CCR5 tropic viruses (AD8 and BaL), a bitropic virus (89.6), HIV-2.sub.ROD and SIV.sub.agm.

(29) The expression of human PLK3 also has an ability to limit infection with HIV-1.sub.AD8, HIV-1.sub.BaL, HIV-1.sub.89.6, HIV-2.sub.ROD and SIV.sub.agm. Specific test steps are as follows. 293T cells were transfected with a 3 FLAG-labeled PLK3 protein expression vector, and including a negative control group, were treated with virus vectors pAD8 (FIG. 11), p89.6 (FIG. 12), pBaL (FIG. 13), pHIV-2.sub.ROD (FIG. 14) and pSIV.sub.agm (FIG. 15), respectively. After the transfection was performed for 48 hours, a supernatant was used for infecting TZM-bl reporter cells to obtain virus infection levels. Data measured each time were obtained by subtracting background RLU.

Example 5

(30) PLK3 requires the kinase activity to achieve an inhibition effect on HIV-1. PLK3 kinase inhibitors or kinase domain defective mutants can inhibit the inhibition effect of PLK3 on HIV-1.

(31) Human PLK3 is Polo-like kinase. Through treatment with a PLK3 kinase inhibitor, GW843682X, although the expression level of PLK3 is not changed, the antiviral ability is inhibited. Treatment was performed with GW843682X or a PLK3 kinase domain mutant vector, respectively, and results were observed. The GW843682X or the PLK3 kinase domain mutant can inhibit the anti-HIV-1 ability of human PLK3. Specific test steps are as follows. Cells were transfected with a 3 FLAG-labeled PLK3 protein expression vector, and including a negative control group, were treated with NL4.3.Luc (VSV-G) after 24 hours. Meanwhile, the cells were also treated with GW843682X. After the transfection was performed for 48 hours, the cells were collected for a flow cytometry test (FIGS. 16A-16B). Cells were transfected with a 3 FLAG-labeled PLK3 kinase domain defective mutant protein expression vector, and including a negative control group, were treated with NL4.3.Luc (VSV-G) after 24 hours. After the transfection was performed for 48 hours, the cells were collected for a flow cytometry test (FIG. 17).