Method for inhibiting replication of human parvovirus B19 in cells

Abstract

The invention belongs to the technical field of cell replication inhibition, and particularly relates to a method for inhibiting replication of human parvovirus B19 in cells. According to the method, the aim of inhibiting the replication of the human parvovirus B19 in the cells can be remarkably achieved by inhibiting CRM1 or mutating any one of the 255th to 263th amino acids and / or the 285th to 297th amino acids in an NS1 protein amino acid sequence. For example, by taking an NS1 defective genome M20-NS1 as a core and combining with pHelper through a functional complementation experiment, it is found that wild type NS1 (3 * FlagB19-NS1) can restore virus DNA replication, RNA transcription and generation of filial generation virus particles; and the mutant blocks the CRM1-mediated nuclear output pathway, so that the virus genome replication level is reduced by 30%-95%, the RNA transcription is reduced by 20%-80%, and the generation efficiency of filial generation virus particles is reduced by 86%.

Description

Technical Field

[0001] This invention belongs to the field of cell replication inhibition technology, specifically relating to a method for inhibiting the replication of human parvovirus B19 in cells. Background Technology

[0002] Human parvovirus B19 (B19V), a major pathogen of the genus *Erythrovirus* in the family Parvoviridae, is widely distributed globally, and its infection is closely associated with a variety of serious clinical conditions. Epidemiological data show that B19V can cause erythema infectiosum (disease 5), fetal hydrops, recurrent miscarriage, and oligoarthritis, especially leading to fatal complications in immunocompromised patients and pregnant women. Currently, there are no specific antiviral drugs available clinically, and treatment is limited to symptomatic support, highlighting the urgent need to develop targeted therapies for B19V.

[0003] The B19V genome is a single-stranded DNA, encoding the non-structural protein NS1, which plays a core regulatory role in the viral life cycle. Current research indicates that the NS1 protein (671 amino acids) mediates viral DNA replication through its N-terminal nuclease domain and induces G2 / M phase arrest in host cells through its C-terminal functional domain, thus facilitating viral proliferation. Notably, NS1 exhibits a dynamic subcellular distribution in infected cells: early studies observed its enrichment in the nucleus using immunofluorescence, while subsequent studies detected significant cytoplasmic localization in CD36+ erythroid progenitor cells, UT7 / Epo cell lines, and transfection models, suggesting nucleocytoplasmic shuttle behavior. Nucleocytoplasmic transport of proteins is typically regulated by the synergistic nuclear localization signal (NLS) and nuclear export signal (NES). Although the NLS motifs of NS1 (177-KKPR-180 and 316-KKCGKK-321) have been previously identified, its nuclear export mechanism has long been unknown. In particular, among other members of the Parvoviridae family (such as mouse parvovirus MVM and canine parvovirus CPV), functional NES have only been found in the NS2 protein, while the nuclear export pathway of the NS1 protein has not yet been elucidated. This knowledge gap severely restricts the development of targeted intervention strategies against the NS1 nucleocytoplasmic transport mechanism.

[0004] Current research on the function of B19V NS1 has significant limitations: First, traditional understanding holds that the parvovirus NS1 protein lacks the classic CRM1-dependent nuclear export signal, and its cytoplasmic distribution mechanism remains unclear. Second, while some studies have observed cytoplasmic localization of NS1, it is unclear whether this is achieved through active nuclear export, and key NES motifs and binding residues have not been identified. Third, the functional link between NS1 nucleoplasmic transport and viral replication has not been established, particularly lacking direct evidence on the impact of nuclear export obstruction on viral DNA replication, transcription, and progeny virus assembly. More critically, antiviral drugs targeting the nuclear export pathway (such as the CRM1 inhibitor leprosycin B) have shown potential in treating other viral infections (such as HIV and influenza viruses), but due to the unclear nuclear export mechanism of B19V NS1, such strategies cannot be specifically applied to B19V infection. Therefore, systematically analyzing the nuclear export mechanism of the NS1 protein, identifying key functional domains and core residues, and elucidating the regulatory role of this pathway in viral replication have become core scientific issues in the development of novel anti-B19V therapies. This invention is based on this unmet major clinical need and technological gap. Summary of the Invention

[0005] Based on this, the present invention provides a method for inhibiting the replication of human parvovirus B19 in cells. This method achieves the purpose of inhibiting the replication of human parvovirus B19 in cells by inhibiting CRM1 or causing mutations in any one or more amino acids from amino acids 255 to 263 and / or amino acids 285 to 297 in the amino acid sequence of the NS1 protein.

[0006] To achieve the above objectives, the present invention can adopt the following technical solutions: This invention provides a method for inhibiting the replication of human parvovirus B19 in cells in vitro, wherein the method is selected from one or more of the following: (i) The purpose of co-culturing CRM1 inhibitor with cells carrying human parvovirus B19 is to inhibit the replication of human parvovirus B19 in vitro. (ii) To inhibit the replication of human parvovirus B19 in vitro by co-culturing a gene mutation reagent with cells carrying human parvovirus B19; wherein the gene mutation reagent is a reagent that causes mutation of any one or more amino acids in the amino acid sequence of NS1 protein from amino acid position 255 to 263 and / or amino acid position 285 to 297.

[0007] Preferably, in method (i) above, the CRM1 inhibitor is leprosycin B.

[0008] Preferably, in method (ii) above, the reagent that causes mutation of any one or more amino acids in amino acids 255 to 263 and / or 285 to 297 of the NS1 protein amino acid sequence includes an oligonucleotide primer pair, wherein the upstream and downstream primers of the oligonucleotide primer pair form complementary pairs with the two complementary strands of the DNA encoding the NS1 protein, respectively; wherein most of the sequence of the upstream primer is completely complementary to the corresponding strand, and only at the preset mutation site does it carry a mutated base that is not complementary to the base of the DNA encoding the NS1 protein.

[0009] Preferably, in method (ii) above, the gene mutation reagent is a reagent that causes a mutation in leucine at position 259 and / or position 261 of the amino acid sequence of the NS1 protein.

[0010] More preferably, in method (ii) above, the reagent that causes the leucine at position 259 and / or 261 of the amino acid sequence of the NS1 protein to mutate includes an oligonucleotide primer pair, wherein the upstream primer and the downstream primer in the oligonucleotide primer pair form complementary pairings with the two complementary strands of the DNA encoding the NS1 protein, respectively; wherein most of the sequence of the upstream primer is completely complementary to the corresponding strand, and only at the preset mutation site does it carry a mutated base that is not complementary to the base of the DNA encoding the NS1 protein.

[0011] Another aspect of the present invention provides the use of lepromycin B in the preparation of a medicament for treating human parvovirus B19 infection.

[0012] Preferably, in the above applications, human parvovirus B19 infection causes erythema infectiosum, fetal hydrops, recurrent miscarriage, or oligoarthritis.

[0013] The beneficial effects of this invention include: (1) In this invention, HEK-293T cells expressing NS1 were treated with the CRM1-specific inhibitor Leptomycin B (LMB). Immunofluorescence combined with confocal microscopy analysis showed that after 6 hours of LMB treatment, the nucleoplasmic distribution of NS1 was significantly reversed and the proportion of NS1 accumulation in the nucleus was increased, confirming that its nuclear export depends on the CRM1 pathway, thereby achieving the purpose of inhibiting the replication of human parvovirus B19 in the cells.

[0014] (2) This invention uses the NS1 defective genome M20-NS1 as the core and combines it with the packaging helper plasmid pHelper to evaluate the rescue ability of wild-type and mutant NS1 through functional complementation experiments. The results showed that wild-type NS1 (3×FlagB19-NS1) could restore viral DNA replication, RNA transcription and progeny virus particle generation; while the mutant, due to blocking the CRM1-mediated nuclear export pathway, resulted in a 30%-95% decrease in viral genome replication level, a 20%-80% decrease in RNA transcription, and a reduction in progeny virus particle generation efficiency of up to 86%. Among them, the Φ2 (L259) mutant (m259) had the most significant inhibitory effect. Attached Figure Description

[0015] Figure 1 This section describes the NS1 truncation strategy and its construction. A shows a schematic diagram of the NS1 truncation strategy; B shows the enzyme digestion verification of each truncated NS1 recombinant plasmid; M: DL10000 DNA marker; 1: full-length NS1 recombinant plasmid; 2-15: BamHI / NdeI double enzyme digestion verification of the recombinant plasmid. Figure 2 To detect the localization of NS1 after transfection of HEK-293T cells with 3×FlagB19NS1 using immunofluorescence; where A represents the NS1 protein mainly located in the cytoplasm without LMB treatment, with only a small amount distributed in the nucleus; B represents the NS1 localization significantly changed after LMB treatment: nuclear accumulation increased significantly, but some cytoplasmic distribution was still retained, ultimately showing a typical nucleoplasmic co-distribution pattern. Figure 3 The identification of NESs signal in the parvovirus B19 NS1 protein; where A shows the localization of the NS1 truncated form in HEK-293T; and B shows that NES1 and NES2 can mediate the nuclear export and translocation of NS1. Figure 4 A schematic diagram showing the mutations of key hydrophobic residues in the full-length NS1 protein and the truncated NES1 and NES2 proteins; Figure 5 To identify key amino acid residues of the NESs motif that mediate B19VNS1-dependent nuclear export from CRM1; where A represents the impact of key hydrophobic amino acid mutations in NES1 on nuclear export of NS1 protein; and B represents the identification of key amino acid residues in NES1 that mediate full-length nuclear export of NS1. Figure 6To detect the localization of NS1 after co-transfection of HEK-293T cells using immunofluorescence; (A) co-transfection with pHelper, M20-NS1(−), and 3×FlagB19-NS1(WT); (B) co-transfection with pHelper, M20-NS1(−), and 3×FlagB19-NS1mNES1; (C) co-transfection with pHelper, M20-NS1(−), and empty pcDNA3.1 vector; compared with 3×FlagB19-NS1(WT) and 3×FlagB19-NS1mNES1 plasmid transfection, co-transfection with 3×FlagB19-NS1(WT) allowed NS1 protein to enter the cell nucleus, compensating for replication defects; co-transfection with 3×FlagB19-NS1mNES1 showed no significant difference. Figure 7 This is the standard curve for quantitative PCR B19; where A represents the NS1 region: the slope is -3.4917, and the coefficient of determination R0 is 1. 2 =0.9936, the established equation is y=-3.4917x+39.428; B is the 11kDa region: the slope is -3.0613, and the coefficient of determination R 2 =0.9953, the established equation is y=-3.0613x+34.384; C is the VP region: the slope is -3.6444, and the coefficient of determination R 2 =0.9978, the established equation is y=-3.6444x+37.926; D is the region 2210-2363: the slope is -3.1063, and the coefficient of determination R 2 =0.9903, and the established equation is y=-3.1063x+37.764; in addition, x represents the copy number and y represents the Ct value; Figure 8 This is a qPCR analysis of the effect of LMB treatment on RNA transcription levels; Figure 9 This section presents the qPCR analysis of the effects of the B19NS1 mutant on viral replication and transcription in a co-transfection model. In this model, A represents the effect of primers from different regions on the total genomic DNA replication level, and B represents the effect of primers from different regions on the RNA transcription level. Note that ns: P>0.05. P<0.05, P<0.01, P < 0.001 P < 0.0001; Figure 10 This is a qPCR analysis of the effect of the B19NS1 mutant on the proliferation of primary viral particles in a co-transfection model; where ns: P>0.05, P<0.05, P<0.01, P < 0.001 P < 0.0001. Detailed Implementation

[0016] The embodiments described are provided to better illustrate the present invention, but are not intended to limit the scope of the invention to the embodiments described. Therefore, non-essential improvements and adjustments made to the embodiments by those skilled in the art based on the above description are still within the scope of protection of the present invention.

[0017] The terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit this disclosure. Singular expressions include plural expressions unless they have a distinct meaning in the context. As used herein, it should be understood that terms such as “comprising,” “having,” “including,” are intended to indicate the presence of features, numbers, operations, components, parts, elements, materials, or combinations thereof. The terminology of the invention is disclosed in the specification and is not intended to exclude the possibility that one or more other features, numbers, operations, components, parts, elements, materials, or combinations thereof may be present or added. As used herein, “ / ” may be interpreted as “and” or “or,” depending on the context.

[0018] In a first aspect, embodiments of the present invention provide a method for inhibiting the replication of human parvovirus B19 in cells in vitro, wherein the method is selected from one or more of the following: (i) The purpose of co-culturing CRM1 inhibitor with cells carrying human parvovirus B19 is to inhibit the replication of human parvovirus B19 in vitro. (ii) To inhibit the replication of human parvovirus B19 in vitro by co-culturing a gene mutation reagent with cells carrying human parvovirus B19; wherein the gene mutation reagent is a reagent that causes mutation of any one or more amino acids in the amino acid sequence of NS1 protein from amino acid position 255 to 263 and / or amino acid position 285 to 297.

[0019] It should be noted that this invention utilizes the CRM1-specific inhibitor Leptomycin B (LMB) to treat HEK-293T cells expressing NS1. Immunofluorescence combined with confocal microscopy analysis showed that after 6 hours of LMB treatment, the nucleoplasmic distribution of NS1 was significantly reversed, and the proportion of NS1 accumulation in the nucleus increased, confirming that its nuclear export depends on the CRM1 pathway, thereby achieving the purpose of inhibiting the replication of human parvovirus B19 in cells.

[0020] It should also be noted that this invention uses the NS1-deficient genome M20-NS1 as the core, combined with the packaging helper plasmid pHelper, and evaluates the rescue capabilities of wild-type and mutant NS1 through functional complementation experiments. The results show that wild-type NS1 (3×FlagB19-NS1) can restore viral DNA replication, RNA transcription, and progeny virus particle generation; while the mutant, due to blocking the CRM1-mediated nuclear export pathway, leads to a 30%-95% decrease in viral genome replication, a 20%-80% decrease in RNA transcription, and a reduction in progeny virus particle generation efficiency of up to 86%, among which the Φ2 (L259) mutant (m259) has the most significant inhibitory effect.

[0021] It should also be noted that the co-culture conditions are well known in the art, and preferably, the cells are cultured at 37°C in a 5% CO2 cell incubator for 6 hours.

[0022] In some specific examples, in method (i) above, the CRM1 inhibitor is leprosycin B.

[0023] It should be noted that the CRM1 inhibitor in this invention is known in the art, and leprosycin B is preferred.

[0024] In some specific examples, in method (ii) above, the reagent that causes mutations in any one or more amino acids from amino acids 255 to 263 and / or 285 to 297 of the NS1 protein amino acid sequence includes an oligonucleotide primer pair, wherein the upstream and downstream primers of the oligonucleotide primer pair form complementary pairs with the two complementary strands of the DNA encoding the NS1 protein, respectively; wherein most of the sequence of the upstream primer is completely complementary to the corresponding strand, and only at the preset mutation site does it carry a mutated base that is not complementary to the base of the DNA encoding the NS1 protein.

[0025] In some specific examples, in method (ii) above, the gene mutation agent is a reagent that causes a mutation in leucine at position 259 and / or 261 of the amino acid sequence of the NS1 protein.

[0026] It should be noted that, In some specific examples, in method (ii) above, the reagents that cause mutations in leucines at positions 259 and / or 261 of the NS1 protein amino acid sequence include oligonucleotide primer pairs, wherein the upstream and downstream primers of the oligonucleotide primer pairs form complementary pairings with the two complementary strands of the DNA encoding the NS1 protein, respectively; wherein most of the sequence of the upstream primer is completely complementary to the corresponding strand, and only at the preset mutation site does it carry a mutated base that is not complementary to the base of the DNA encoding the NS1 protein.

[0027] Secondly, embodiments of the present invention provide the use of lepromycin B in the preparation of a medicament for treating human parvovirus B19 infection.

[0028] In some specific examples, in the above applications, human parvovirus B19 infection causes erythema infectivity, fetal hydrops, recurrent miscarriage, or oligoarthritis.

[0029] To better understand the present invention, specific examples are provided below to further illustrate the content of the present invention, but the content of the present invention is not limited to the examples below.

[0030] Example 1 This invention provides the process and results of discovering and verifying CRM1-dependent NES signals.

[0031] (a) Construction of NS1 truncated body (1) The NS1 protein (SEQ ID NO: 1 nucleotide encoding) was analyzed by NetNES1.1Server, and three candidate NES were identified: NES1 (255-IQSALKLAI-263), NES2 (285-MCIKDNKIVKLLL-297), and NES3 (435-LKERMVKLNFTV-446). (2) To verify the functionality of these candidate NESs, a series of 3×Flag tag fusion NS1 truncated bodies were constructed (see Table 1), such as Figure 1 As shown in (A), using the p3×Flag-CMV-7 vector and the B19V infectious clone pB19-M20 as templates, PCR amplification was performed using the primers in Table 1 to obtain the full-length vector 3×Flag-CMV, the non-structural protein NS1, and its truncated fragments. The PCR amplification system and PCR amplification program are shown in Table 2 below.

[0032] Table 1 Primers required for amplification

[0033] Table 2 PCR amplification system and PCR amplification procedure

[0034] (3) After verifying the correctness of the bands by 1% agarose gel electrophoresis, the amplified products were then used... DH5α The competent cells were transformed. Specifically, the vector and the target fragment (1:3) were mixed, T5 exonuclease (2 units) was added, and the mixture was incubated on ice for 5 min. The competent cells were then added, and the mixture was incubated on ice for 30 min. The cells were then heat-shocked at 42°C for 45 s, followed by an ice incubation for 2 min. NZY medium was then added, and the cells were incubated at 37°C for 1 h. Finally, the bacterial culture was spread on solid plates (containing antibiotics) and incubated overnight at 37°C. Single colonies were picked, inoculated into liquid medium, and plasmids were extracted.

[0035] (4) BamHI and NdeI Enzyme digestion verification, the results are as follows Figure 1 As shown in (B), it is demonstrated that each truncated variant has been successfully constructed into the 3×Flag-CMV plasmid, and sequencing verifies the correctness of the inserted truncated variant sequence; (ii) Determining that nuclear export of the NS1 protein depends on CRM1 mediation The eukaryotic expression vector was constructed and transfected into HEK-293T cells. Analysis was performed using immunofluorescence combined with laser confocal microscopy. The results are as follows: Figure 2 As shown in (A), NS1 protein is mainly enriched in the cytoplasm under uninterrupted conditions, with only a small amount diffusely distributed in the nucleus, indicating that it may achieve dynamic regulation of subcellular localization through an active nuclear export mechanism. The pathway mediated by the nuclear export receptor CRM1 (chromosome region maintenance 1 / exportin-1) is the core mechanism of protein nucleoplasmic transport.

[0036] Furthermore, transfected cells were treated with the CRM1-specific inhibitor leptomycin B (LMB) to verify the molecular mechanism of NS1 nuclear export, and the results were as follows: Figure 2 As shown in (B), after 6 hours of 20 nM LMB treatment, the distribution pattern of NS1 was significantly reversed: the proportion of signal intensity in the nucleus was increased. This phenomenon is highly consistent with the nucleus retention effect after the function of CRM1 was suppressed, directly proving that the nuclear output of NS1 depends on the CRM1-mediated transport system.

[0037] (III) NES motif identification By transfecting the truncated variant from (I) above into HEK-293T cells and combining immunofluorescence and laser confocal microscopy, it was found that NS1-1-254, which contains NLS (positions 171-180) but lacks NESs, exhibits nucleocytoplasmic colocalization, while NS1-181-671, which lacks NLS but retains NES1 / 2 / 3, shows significant cytoplasmic enrichment, consistent with the localization pattern of full-length NS1. Figure 3 As shown in (A).

[0038] Furthermore, it was confirmed that the predicted NESs in the truncated forms NS1-456-671, NS1-298-434, and NS1-1-176 were not present in the nucleoplasm. However, as expected, the truncated form NS1-264-434 containing NES2, the truncated form NS1-298-455 containing NES3, and the truncated form NS1-264-671 containing NES2 / 3 were mainly located in the cytoplasm, indicating that the predicted export motifs NES2 and NES3 are involved in the nuclear export of NS1 protein.

[0039] Furthermore, the truncated form NS1-1-264 containing NES1, while retaining NLS, exhibits cytoplasmic localization, indicating that NES1 can facilitate nuclear export of NS1 compared to NS1-1-254.

[0040] In addition, to clarify whether the aforementioned NESs depend on the CRM1-mediated classical nuclear export pathway, HEK-293T cells transfected with the CRM1 inhibitor lepromycin B (LMB, 20 nM) were further treated. The results were as follows: Figure 3 As shown in (B), after 6 hours of LMB treatment, both the NES1-containing truncated variant NS1-1-264 and the NES2-containing truncated variant NS1-264-434 exhibited nuclear retention, while the NES3-containing truncated variant NS1-298-455 was insensitive to LMB treatment. This result confirms that NES1 and NES2, rather than NES3, mediate nuclear output through a CRM1-dependent pathway.

[0041] Example 2 The embodiments of the present invention provide the identification process and results of key amino acid residues.

[0042] (I) Construction of NS1 mutant The NES1 common sequence is similar to that of classic PKINES (LALKLAGLDI) (the typical nuclear output signal follows a leucine-rich common sequence, Φ1-(x)2-3-Φ2-(x)2-3-Φ3-x-Φ4, where Φ represents a hydrophobic residue and x represents any preferentially charged, polar, or small amino acid). In this invention, all key hydrophobic residues Φ1-Φ4 in NES1 and NES2 are mutated, as shown in Table 3.

[0043] Table 3 Comparison of the NES motif of the B19 virus NS1 protein with known NES motifs.

[0044] In addition, full-length and truncated NS1 fusion 3×Flag mutants (mNES1-NS1, mNES2-NS1, mNES1-NS1-1-264, and mNES2-NS1-264-434) were constructed, in which all hydrophobic residues were mutated to alanine, such as Figure 4 As shown above, using the 3×Flag-CMV-NS1 recombinant plasmid constructed above as a template, and based on the primer pairs in Table 4, PCR amplification was performed using high-fidelity DNA polymerase (PCR amplification system and amplification procedure are the same as in Table 2). After confirming the correct bands by 0.8% agarose gel electrophoresis, the amplification products and residual supercoiled plasmids in the amplification template were digested with DpnI, and then an equal amount of the digested products were used for transformation culture.

[0045] Table 4 Primers required for the experiment

[0046] In addition, after digestion, the amplified template was transformed into a plate with no colonies. Single colonies on the plate were picked from the deactivated product and transformed into a plate. Plasmids were extracted and verified by digestion with BamHI and NdeI (double digestion system is shown in Table 5 below). After inactivation treatment at 37℃ for 2 h, samples were taken for agarose electrophoresis detection and EB staining for imaging observation. The results were then sent to Shanghai Sangon Biotech for sequencing verification.

[0047] Table 5 Double enzyme digestion system

[0048] (II) Identification of key amino acid residues HEK-293T cells were transfected, and 42 hours post-transfection, they were treated with LMB (LMB was added to the culture medium to a final concentration of 20 nM, and the cells were cultured at 37°C in a 5% CO2 incubator for another 6 hours). Subcellular localization was then detected by immunofluorescence combined with laser confocal microscopy. The results are as follows: Figure 5 As shown in (A), both mNES1-NS1 and mNES1-NS1-1-264 exhibited nucleoplasmic distribution regardless of LMB treatment, losing their nuclear export function. In contrast, wild-type NS1 was retained in the nucleus only after LMB treatment. These results indicate that the key hydrophobic residues Φ1-Φ4 in NES1 are crucial amino acid residues for NS1, sufficient to mediate nuclear export of the NS1 protein.

[0049] Furthermore, mNES2-NS1 and mNES2-NS1-264-434 retained nuclear export activity and were distributed in the cytoplasm without LMB treatment, and in both the cytoplasm and nucleus with LMB treatment. These data indicate that NES1 plays a crucial role in mediating B19VNS1-dependent CRM1 nuclear export.

[0050] To further identify the key amino acid residues involved in NES1-mediated nuclear export of NS1, alanine scanning mutagenesis was performed on hydrophobic residues within NES1. The cellular localization of these single-point mutations at each Φ residue within the NES1 region of the NS1 protein was then determined. The results showed that the NES1 Φ2 (L259) and Φ3 (L261) mutations almost eliminated nuclear export activity, while NES1 Φ1 (I255) and Φ4 (I263) were not critical. Figure 5 (B).

[0051] Example 3 (I) Co-localization study of mutants in co-transfection model By constructing a safe and controllable viral packaging system, the regulatory role of the nucleocytoplasmic transport mechanism of the non-structural protein NS1 in parvovirus B19 on viral replication was systematically elucidated. The core components of the experimental system consist of two parts: the pHelper plasmid provides the capsid proteins VP1 / VP2 and cofactors necessary for viral packaging, but it does not carry the viral genome itself, thus ensuring that the generated viral particles meet biosafety standards due to the lack of autonomous replication elements; M20-NS1(−) is a viral genome with a defective ns1 gene, retaining the origin of replication (ori) but unable to express the functional NS1 protein, leading to early termination of the viral replication cycle. Specifically, pHelper and M20-NS1(−) were co-transfected into HEK-293T cells with 3×Flag-labeled B19-NS1 (WT), a nuclear export signal (NESs) mutated B19-NS1mNES1, and the empty vector pcDNA3.1, respectively. Results are as follows: Figure 6 As shown, the B19-NS1 (WT) protein can specifically rescue the defective phenotype of M20-NS1(−) by restoring the viral genome's replication capacity. However, NS1 carrying the NES1 domain mutation (mNES1) cannot complete its functional localization through the CRM1 (Chromosome Region Maintenance 1)-mediated nuclear export pathway, thus affecting viral replication efficiency. In the control experiment, no viral replication activation was observed in the pcDNA3.1 empty vector group, effectively excluding the potential influence of endogenous protein interference or vector self-effects on the experimental results, further verifying the functional specificity of the exogenous NS1 protein.

[0052] (II) Establishment of the B19V standard curve for real-time PCR Using the B19V infectious cloning plasmid pB19-M20 (containing the full-length B19V genome) as a standard template, after determining the concentration, the copy number of 0.879 ng of plasmid was calculated to be 1 × 10⁻⁶ according to the copy number formula. 8Subsequently, the pB19-M20 plasmid was diluted to 10⁻¹⁰ using a serial dilution method. 1 -10 8 The copy number / μL range was used to design and synthesize specific primers as shown in Table 6 for key functional regions of the B19V genome. Specifically, the NS1 region targets the non-structural protein coding region, the 11kDa region targets the small capsid protein coding region, and the VP region targets the major capsid protein VP1 / VP2 coding region to reflect the expression level of replication-related genes. The 2210-2363 region targets conserved regions of the genome to characterize the total DNA level. RT-PCR and qPCR reactions were performed according to the systems in Tables 7 and 8 to detect the CT values ​​of templates at each dilution gradient. Linear regression analysis was performed with the logarithm of plasmid copy number as the x-axis and the CT value as the y-axis to obtain standard curves for each target region, as shown in Table 6. Figure 7 As shown. The coefficient of determination R for all standard curves. 2 All values ​​were greater than 0.99, which meets the requirements of the international qPCR standardization guidelines (MIQE).

[0053] Table 6 Primers required for the experiment

[0054] Table 7. Real-time PCR reaction system

[0055] Table 8. Quantitative Real-Time PCR Reaction Procedure

[0056] (III) Determining that nuclear export of the NS1 protein depends on CRM1 mediation The pHelper plasmid was co-transfected into HEK-293T cells with the B19V infectious clone pB19-M20 (positive control) and the NS1-deficient genome M20-NS1(-) (negative control), respectively. The experimental groups were treated with pHelper and M20-NS1(-) co-transfected cells, followed by treatment with the CRM1-specific inhibitor Leptomycin B (LMB) (20 nM LMB was added to the culture medium, and the cells were cultured at 37°C in a 5% CO2 incubator for 6 hours). Cell samples were collected after transfection, and cDNA templates were prepared for each group. RNA transcription levels were detected by real-time quantitative PCR (qPCR). Each experiment was performed in triplicate. The results are shown below. Figure 8As shown in the figure, the results showed that, compared with the positive control group, the RNA transcription level in the experimental group treated with LMB decreased by 40%. This result indicates that inhibiting the CRM1-mediated nuclear export pathway significantly reduces viral transcriptional activity, thus supporting the conclusion that the nuclear export function of the NS1 protein depends on CRM1.

[0057] (iv) Effects of the B19VNS1 mutant on viral replication and transcription in a co-transfection model To investigate the regulatory role of the nucleocytoplasmic transport domain of the B19VNS1 protein in its replication and transcription functions, 3×FlagB19-NS1 (WT), nuclear export signal mutants (mNES1, mNES2), and nuclear localization key leucine residue mutants (m259, m261) were used as templates. PCR amplification was performed using high-fidelity DNA polymerase based on the primer pairs in Table 4. After confirming the correct bands by 0.8% agarose gel electrophoresis, the amplified products were first digested with DpnI. The amplified product and residual supercoiled plasmid in the amplification template were digested and transformed into culture in equal amounts. The culture plates transformed with the digested amplification template were sterile. Single colonies on the culture plates transformed with the digested product were picked, and plasmids were extracted. After verification by BamHI and NdeI restriction enzymes, the samples were sent to Shanghai Sangon Biotech for sequencing verification. The pcDNA3.1 empty vector, along with the pHelper plasmid and the NS1-deficient genome M20-NS1(-), were co-transfected into HEK-293T cells. After sample processing, whole-genome DNA and cDNA templates of each component were obtained. Viral genomic DNA replication and RNA transcription levels were detected by real-time quantitative PCR (qPCR). Each experiment had three biological replicates, with co-transfection of the pcDNA3.1 empty vector as a negative control and co-transfection of 3×FlagB19-NS1 (WT) as a positive control.

[0058] Experimental results are as follows Figure 9As shown, the results indicated that, compared to the positive control group, all mutants significantly inhibited viral replication and transcription. Nuclear export signal mutants (mNES1, mNES2) resulted in a 30%-60% decrease in genomic DNA replication and a 20%-60% decrease in RNA transcription; the leucine residue mutant (m261) reduced genomic DNA replication by 50% and RNA transcription by 40%-70%; the leucine residue mutant (m259) showed even more significant inhibition of replication and transcription, reducing genomic DNA levels by 60%-95% and RNA levels by 60%-80%. This result is consistent with the previous findings; Φ2 (L259) and Φ3 (L261) are core residues of the NS1 protein nuclear export signal, and their mutations almost completely blocked the nucleocytoplasmic transport activity of NS1. Further analysis revealed that NS1 nuclear export depends on the CRM1 pathway, and mutations in the NES domain or leucine residues disrupt the interaction between NS1 and CRM1, causing NS1 to remain in the nucleus and unable to coordinate viral genome replication and capsid assembly in the cytoplasm. Furthermore, the reduced RNA transcription level suggests that NS1 may regulate viral mRNA stability or translation efficiency through a nuclear export-dependent mechanism.

[0059] (v) Effect of B19V NS1 mutant on viral particle proliferation in co-transfection model To systematically evaluate the impact of the functional domains of the B19VNS1 protein on the assembly and release of progeny viral particles, 3×Flag-labeled B19-NS1 (WT), nuclear export signal mutants (mNES1, mNES2), and nuclear localization key leucine residue mutants (m259, m261) were used as templates in the constructed 3×Flag-CMV-NS1 recombinant plasmid. PCR amplification was performed using high-fidelity DNA polymerase based on the primer pairs in Table 4. The amplified products were confirmed by 0.8% agarose gel electrophoresis. After successful transfection, the amplification product and residual supercoiled plasmid in the amplification template were first digested with DpnI. Then, an equal amount of the digested product was used for transformation culture. The transformed culture plate containing the digested amplification template was sterile. Single colonies on the transformed culture plate containing the digested product were picked, and plasmids were extracted. After verification by BamHI and NdeI digestion, the plasmids were sent to Shanghai Sangon Biotech for sequencing verification. The empty pcDNA3.1 vector, pHelper plasmid, and NS1-deficient genome M20-NS1(-) were co-transfected into HEK-293T cells. Forty-eight hours after transfection, the samples were processed to obtain progeny viral particles from each component, and the viral genome copy number was detected using real-time quantitative PCR (qPCR). Three biological replicates were set up for each group, with co-transfection of the empty pcDNA3.1 vector as a negative control and co-transfection of 3×FlagB19-NS1 (WT) as a positive control.

[0060] Experimental results are as follows Figure 10As shown, the results indicate that all NS1 mutants significantly inhibited the generation efficiency of progeny viral particles. The nuclear export signal mutants (mNES1 and mNES2) resulted in a maximum reduction of 75% in the progeny viral genome copy number, indicating that NES1 and NES2 influence viral capsid assembly or genome packaging by mediating the nuclear export of the NS1 protein. The leucine residue mutant (m261) reduced the progeny viral copy number by 50%. The leucine residue mutant (m259) showed the most significant inhibitory effect, reducing the progeny viral copy number by 86%, confirming the function of Φ2 (L259) as a core residue for the nuclear export signal.

[0061] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.

Claims

1. A method for inhibiting the replication of human parvovirus B19 in cells in vitro, characterized in that, The method is selected from any one or more of the following: (i) The purpose of co-culturing CRM1 inhibitor with cells carrying human parvovirus B19 is to inhibit the replication of human parvovirus B19 in vitro. (ii) To inhibit the replication of human parvovirus B19 in vitro by co-culturing a gene mutation reagent with cells carrying human parvovirus B19; wherein the gene mutation reagent is a reagent that causes mutation of any one or more amino acids in the amino acid sequence of NS1 protein from amino acid position 255 to 263 and / or amino acid position 285 to 297.

2. The method according to claim 1, characterized in that, In method (i), the CRM1 inhibitor is leprosycin B.

3. The method according to claim 1, characterized in that, In method (ii), the reagent that causes mutations in any one or more amino acids from amino acids 255 to 263 and / or 285 to 297 of the NS1 protein amino acid sequence includes an oligonucleotide primer pair, wherein the upstream and downstream primers of the oligonucleotide primer pair form complementary pairs with the two complementary strands of the DNA encoding the NS1 protein, respectively; wherein most of the sequence of the upstream primer is completely complementary to the corresponding strand, and only at the predetermined mutation site, it carries a mutant base that is not complementary to the base of the DNA encoding the NS1 protein.

4. The method according to claim 1, characterized in that, The gene mutation reagent is a reagent that causes a mutation in leucine at position 259 and / or 261 of the amino acid sequence of the NS1 protein.

5. The method according to claim 4, characterized in that, The reagents that cause mutations in leucines at positions 259 and / or 261 of the NS1 protein amino acid sequence include oligonucleotide primer pairs. The upstream and downstream primers in the oligonucleotide primer pairs form complementary pairs with the two complementary strands of the DNA encoding the NS1 protein, respectively. The upstream primer has most of its sequence completely complementary to the corresponding strand, and only at the predetermined mutation site does it carry a mutated base that is not complementary to the DNA encoding the NS1 protein.

6. Application of Lepromycin B in the preparation of drugs for treating human parvovirus B19 infection.

7. The application according to claim 6, characterized in that, Human parvovirus B19 infection can cause erythema infectivity, fetal hydrops, recurrent miscarriage, or oligoarthritis.

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