In-vitro effect of human cathelicidin antimicrobial peptide LL-37 on dengue virus type 2
In-vitro effect of human cathelicidin antimicrobial peptide LL-37 on dengue virus type 2
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DOI：
10.1016/j.peptides.2017.04.002
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发表时间：
2017-06-01
期刊：
影响因子：
3
通讯作者：
Salunke, A.
中科院分区：
文献类型：
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作者：
Alagarasu, K.;Patil, P. S.;Salunke, A.
已知的人盲肠毒素抗菌肽LL-37对多种病毒具有抗病毒活性。在本研究中，我们研究了IL-37对登革2型病毒(DENV-2)在Vero E6细胞中感染和复制的体外作用。为了研究LL-37对病毒或细胞的预处理效果，用不同浓度的LL-37(2.5 mM~15 mM)或混杂(SCR)LL-37(5 mM~15 mM)对病毒或细胞进行预感染，或先用LL-37处理细胞再感染。为研究IL-37感染后(PI)的作用，首先感染细胞，然后在感染后24小时向培养液中加入IL-37。在所有条件下，培养上清液用实时荧光定量RT-PCR检测病毒RNA拷贝数，用焦点形成单位法(FFU)检测感染性病毒颗粒，用ELISA法检测非结构蛋白1(NS1)抗原水平。用免疫荧光法(IFA)评估感染百分率。结果表明，用10~15MU的L L-37对病毒进行预处理，与病毒对照组相比，其传染性显著降低(P<；0.0001)。此外，用10~15 mU的L L-37对病毒进行预处理，可显著降低病毒基因组核糖核酸和N S1抗原水平(P<；0.0001)。用10-15 PM的LL-37处理病毒，与病毒对照组相比，平均对数下降2-3个对数FFU/ml(P<；0.0001)。与病毒对照培养物相比，用混杂的LL-37处理病毒对感染率和病毒载量没有影响(P>；0.05)。细胞感染前的预处理和加入IL-37对病毒载量均无影响。分子对接研究表明，LL-37可能与DENV包膜(E)蛋白二聚体的两个单元结合。总之，体外实验和电子分析表明，LL-37通过与E蛋白结合，在进入细胞的阶段抑制DENV-2。这一结果可能对预防DENV感染有意义，需要进一步的体内研究。
Human Cathelicidin antimicrobial peptide LL-37 is known to have antiviral activity against many viruses. In the present study, we investigated the in-vitro effect of LL-37 on dengue virus type 2 (DENV-2) infection and replication in Vero E6 cells. To study the effect of pretreatment of virus or cells with LL-37, the virus was pretreated with different concentrations of LL-37 (2.5 mu M-15 mu M) or scrambled (Scr) LL-37(5 mu M-15 mu M) and used for infection or the cells were first treated with LL-37 and infected. To study the effect of LL-37 post infection (PI), the cells were infected first followed by addition of LL-37 to the culture medium 24 h after infection. In all conditions, after the incubation, the culture supernatant was assessed for viral RNA copy number by real time RT-PCR, infectious virus particles by focus forming unit assay (FFU) and non structural protein 1 (NS1) antigen levels by ELISA. Percentage of infection was assessed using immunoflourescence assay (IFA). The results revealed that pretreatment of virus with 10-15 mu M LL-37 significantly reduced its infectivity as compared to virus control (P < 0.0001). Moreover, pretreatment of virus with 10-15 mu M LL-37 significantly reduced the levels of viral genomic RNA and NS1 antigen (P < 0.0001). Treatment of virus with 10-15 pM LL-37 resulted in two to three log reduction of mean logio FFU/ml as compared to virus control (P < 0.0001). Treatment of the virus with scrambled LL-37 had no effect on percentage of infection and viral load as compared to virus control cultures (P > 0.05). Pretreatment of cells before infection or addition of LL-37 to the culture 24 h PI had no effect on viral load. Molecular docking studies revealed possible binding of LL-37 to both the units of DENV envelope (E) protein dimer. Together, the in-vitro experiments and in-silico analyses suggest that LL-37 inhibits DENV-2 at the stage of entry into the cells by binding to the E protein. The results might have implications for prophylaxis against DENV infections and need further in-vivo studies.