基于寨卡病毒复制子的抗病毒药物筛选系统

刘 娜,胡媛媛,陈凯丽,赵 莹,易文富,朱春玉,郑方亮

(辽宁大学生命科学学院,中国辽宁沈阳110036)

References:

Zika virus(ZIKV)was first isolated in 1947 in Uganda from a macaque monkey(Macaca mulatta)in the Zika Forest,from which the name of the virus is derived.ZIKV belongs to the genus Flavivirus of the family Flaviviridae[1～3].It is a mosquito-borne virus like other members of Flavivirus,such as dengue virus(DENV),Japanese encephalitis virus(JEV),and yellow fever virus(YFV)[4～6].The first reported human case of ZIKV occurred in Nigeria in 1952.ZIKV infection in humans causes mild symptoms with a slight fever.Only a few cases of ZIKV were reported until an outbreak of ZIKV in the Yap islands in Federated States of Micronesia in 2007,and in French Polynesia in 2013.ZIKV was subsequently identified in America in 2015.As of 2016,ZIKV has spread to at least 33 countries[3,7～9].ZIKV can be transmitted by vectors other than mosquitoes,including blood transfusion,sexual activity,and placental transplants[2].

The latent period of ZIKV is 3～10 days,during which the classical symptoms of rash,fever,headache,arthralgia,myalgia,and conjunctivitis would appear[10].Many ZIKV infections are self-limited and take less than one week to recover.Some acute ZIKV infections,however,produce severe neurological and ophthalmological complications,including microcephaly in infants and newborns[11～12]and Guillain-Barre syndrome(GBS)in adults[10].In 2016,the World Health Organization deemed ZIKV a public health emergency and emphasized the link between ZIKV infections and the devastating birth defect,microcephaly[13].No vaccines or drugs are presently available that can be effectively used to treat ZIKV infection.Therefore,development of effective vaccines and antiviral drugs to treat ZIKV infection is urgently needed due to the great threat this disease poses to the world at large.Many treatment options have been recommended,including drug repurposing,ZIKV neutralization,plasma protein therapy,and vaccines[10].Antiviral drugs have been successfully used for the treatment of hepatitis C[14]and several FDA-approved drugs have exhibited good inhibitory activity against ZIKV[12].

ZIKV is an enveloped and icosahedral virus with a non-segmented,positive single-stranded RNA(around 10.7 kb in size)that encodes a multimeric protein[10].The viral genome consists of three parts,the 5′-UTR and 3′-UTR,and an open reading frame(ORF).The ORF includes genes that encode three structural proteins(C,prM,and E)and seven non-structural proteins(NS1,NS2A,NS2B,NS3,NS-4A,NS4B,and NS5)[10,15～16].Structural proteins are mainly responsible for viral adsorption and the assembly of virus particles.Non-structural proteins play an important role in viral replication[17].

Replicon is a subgenomic,self-replicating sequence that contains cis-acting elements in the 5′and 3′ends and the coding sequence of non-structural viral proteins required for RNA replication and translation.Replicons have the ability to replicate but lack the essential genes that encode viral structural proteins.They cannot be assembled into virus particles and therefore are non-infectious.A non-infectious replicon provides a useful tool for studying viral replication and screening antivirals[17～18].Several Flavivirus replicons,including DENV replicon[6],YFV replicon[19],West Nile virus(WNV)replicon[17,20],and the JEV replicon[21],have been very useful for screening antivirals.In the present study,a DNA-based replicon of Zika virus was constructed for this purpose.DNA-based replicons have a number of advantages compared to RNA-based replicons.For example,they can be directly transfected into eukaryotic cells and are stable in host cells[15,17～18].

The present report describes the construction of a DNA-based replicon of Zika virus(Rluc-ZIKV-Rep).The replicon can be directly transfected and stably express viral proteins in host cells.The replication of the replicon can be easily monitored by RT-qPCR and/or Renilla luciferase activity in transfected cells.Furthermore,the Rluc-ZIKV-Rep was treated with two compounds manidipine and cilnidipine,and it was found that the Renilla luciferase activity was reduced in a dose-dependent manner,indicating that the Rluc-ZIKV-Rep can be used for screening antiviral drugs.

1 Materials and methods

1.1 Cells and antiviral drugs

Vero cells were cultured in Modified Eagle’s Medium(MEM)with 10%fetal bovine serum(FBS)(Gibco,USA),100 U/mL penicillin,and 100 μg/mL streptomycin.Cells were maintained at 37℃in 5%CO2.The ZIKV infectious cDNA clone,and the two compounds manidipine and cilnidipine were kindly provided by Dr.Wang Han-zhong and Dr.Xiao Geng-fu(Wuhan Institute of Virology,Chinese Academy of Sciences),respectively.

1.2 Plasmid construction

An infectious cDNA clone of ZIKV was used to construct a Renilla luciferase ZIKV replicon(Rluc-ZIKV-Rep).In the Rluc-ZIKV-Rep,most sequences of structural genes in the ZIKV were replaced with the Renilla luciferase(Rluc)gene and the 2A cleavage site(FMDV 2A)from the foot-and-mouth disease virus.The sequences encoding the 22 N-terminal amino acids of the C protein and the 30 C-terminal amino acids of the E protein,however,were preserved.Primers used in the construction of the replicon and subsequent analysis are listed in Table 1.

The Rluc-ZIKV-Rep was constructed in three steps.Overlap PCR was first performed to create encoding cassettes that included the KpnⅠ-CMV promoter-5′UTR-C22,Rluc2A,and the E30 to the unique ClaⅠsite in the NS3 protein.The KpnⅠ-CMV promoter-5′UTR-C22 was amplified with primers 1 and 2(Table 1)using the ZIKV infectious clone as a template.The fragment Rluc2A was amplified with primers 3 and 4(Table 1).Then,the KpnⅠ-CMV promoter-5′UTR-C22-Rluc2A was fused using primers 1 and 4(Table 1).The E30 to the unique ClaⅠsite in the NS3 protein was amplified with primers 5 and 6(Table 1),using the infectious clone of ZIKV as a template.The three fragments were fused together using primers 1 and 6(Table 1)and then digested with KpnⅠand ClaⅠ,and with ClaⅠand Xho I.The KpnⅠand ClaⅠdigested fragment was engineered at the corresponding sites,resulting in Rluc-ZIKV-Rep.A non-replicative NS5 mutation GDD-GAA was introduced into the Rluc-ZIKV-Rep by site-directed mutagenesis,producing Rluc-ZIKV-Rep NS5△GDD[17].All the constructs were verified by DNA sequencing.

1.3 Luciferase assay

Vero cells were seeded into a 24-well culture plate.Rluc-ZIKV-Rep or Rluc-ZIKV-Rep NS5△-GDD was then transfected into the Vero cells using the Lipofectamine 2000 reagent according to the manufacturer’s instructions(Invitrogen,USA).Cells were washed with PBS at the specified time(0 h,2 h,12 h,24 h,36 h,48 h,60 h and 72 h)and then lysed with Renilla Luciferase Assay Lysis Buffer(Promega,USA).And Rluc luciferase activity was measured according to the manufacturer’s instructions.The specific operation included 1)adding 100 μLof Renilla Luciferase Assay Reagent to the EP tube；2)adding 20 μL of cell lysate and mixing quickly by flicking the tube with a finger or vortexing to thoroughly mix(1～2 seconds)；3)placing tube in luminometer and initiating measurement(luminescence is normally integrated over 10 seconds with a 2-second delay)；4)recording the Renilla luciferase activity measurement；5)discarding the reaction tube,and proceeding to the next Renilla Luciferase Assay reaction(steps 1～4 were repeated).

Table 1 Primer sequences used in this study

1.4 Reverse transcription quantitative PCR(RT-qPCR)

Vero cells were seeded into 35 mm capsules and then transfected with Rluc-ZIKV-Rep or Rluc-ZIKV-Rep NS5△GDD.Cells were harvested for viral RNA determination at 0 h,2 h,12 h,24 h,36 h,48 h,60 h and 72 h post-transfection.Total RNA was extracted from the replicon-transfected cells using Trizol reagent(Life Technologies,USA).The RNA was then transcribed into cDNA using the PrimeScriptTMRT Reagent Kit with gDNA Eraser(TaKaRa,Japan)according to the manufacturer’s instructions.Lastly,RT-qPCR analysis of mRNA expression was performed using the SYBR Fast qPCR Mix in a CFX Connect Real-Time System(Bio-Rad,USA).Primers used for the reactions were synthesized by Wuhan Tianyi Huiyuan Biotechnology Co.,Ltd(Wuhan,China),and the sequences were as follows：NS5 forward：5′-AARTACACATACCARAACAAAGTG-3′,and NS5 reverse：5′-TCCRCTCCCYCTYTGGTCTTG-3′.

1.5 Rluc-ZIKV-Rep inhibition assay

The drug inhibition experiment was performed according to Wang et al[21].Vero cells were seeded into 24-well culture plates.Rluc-ZIKV-Rep was transfected into Vero cells using the Lipofectamine 2000 Reagent according to the manufacturer’s instructions(Invitrogen,USA).Six hours post-transfection,the cells were incubated with various concentrations of manidipine or cilnidipine(dissolved using DMSO).Experiments were carried out in triplicate.The cells were lysed at 48 h post-transfection.The Renilla luciferase signal was measured using the Renilla Luciferase Assay System(Promega,USA)according to the manufacturer’s instructions.

1.6 Statistical analysis

All data were expressed as the mean±standard deviation(SD)from at least two independent experiments(in each experiment n=3)and were statistically analyzed using GraphPad Prism 6.00 software(San Diego,CA,USA).Mean separations were determined using two-way ANOVA and unpaired t-test.P≤0.05 was considered statistically significant.*represents P≤0.05,**represents P≤0.01,***represents P≤0.001,and****represents P≤0.000 1.

2 Results

2.1 Construction of the Rluc-ZIKV-Rep

As illustrated in Fig.1,the ZIKV infectious clone was used as a template to construct a ZIKV replicon(Rluc-ZIKV-Rep).The majority of sequences that encode ZIKV structural proteins were replaced in the Rluc-ZIKV-Rep with Rluc2A,however,sequences encoding the 22 N-terminal amino acids of the C protein and the 30 C-terminal amino acids of the E protein were preserved.As a negative control,a non-replicative NS5 mutation GDD-GAA was introduced into the Rluc-ZIKV-Rep by sitedirected mutagenesis,thereby producing Rluc-ZIKVRep NS5△GDD.

2.2 Rluc-ZIKV-Rep can transfect and replicate in Vero cells

The ZIKV was isolated from serum of rhesus monkeys initially,so this experiment chose African green monkey kidney cell line(Vero),which was also from monkeys,to test the function of the Zika replicon as other experiments did[22～23].Vero cells were transfected with either Rluc-ZIKV-Rep or Rluc-ZIKV-Rep NS5△GDD.After transfection,cells were collected and lysed at 0 h,2 h,12 h,24 h,36 h,48 h,60 h,and 72 h post-transfection to assay for Renilla luciferase activity.Results showed that Renilla luciferase activity increased exponentially.Renilla luciferase activity of the Rluc-ZIKV-Rep was similar to that generated by the Rluc-ZIKV-Rep NS5△GDD from 0 h to 12 h post-transfection.Subsequently,however,the luciferase activity in the Rluc-ZIKV-Rep continually increased from 12 h to 60 h post-transfection,and then decreased from 60 h to 72 h post-transfection.In contrast,Renilla luciferase activity of Rluc-ZIKV-Rep NS5△GDD was stable and exhibited a declining trend over the 12～72 h post-transfection period(Fig.2A).

Fig.1 Diagrammatic representation of the ZIKV replicon(Rluc-ZIKV-Rep)construction

Vero cells transfected with either Rluc-ZIKVRep or Rluc-ZIKV-Rep NS5△GDD were also assayed for replication of Rluc-ZIKV-Rep from the mRNA level.The amplification of the NS5 region of the replicon was used in the replication assay.The results showed that the copy numbers of the two replicons changed identically to their luciferase activities(Fig.2B).This indicated that Rluc-ZIKV-Rep can replicate in cells and that the replication of the replicon was not affected by the inclusion of the reporter gene in the replicon.

2.3 Validation of Rluc-ZIKV-Rep based assays for screening antivirals

Two viral inhibitor drugs,manidipine and cilnidipine,were used to determine if the replicon system could be used for screening antiviral drugs.Manidipine and cilnidipine are Ca2+inhibitors that are useful for treating viral infections or determining the utility of replicon-based screening systems,including JEV infection,JEV replicon,DENV-2 replicon,WNV replicon,and ZIKV infection[21].Vero cells were transfected with the Rluc-ZIKV-Rep and then incubated with different concentrations of manidipine and cilnidipine.Cells were collected 48 h posttransfection and lysed to measure Renilla luciferase activity in the lysed cells.Results showed that inhibition of luciferase activity increased with the increasing concentrations of manidipine and cilnidipine.Both drugs powerfully inhibited the replication of Rluc-ZIKV-Rep in a dose-dependent manner(Figs.3A and 3B).These results suggest that manidipine and cilnidipine could be potential candidates for treatment of ZIKV.

3 Discussion

Fig.2 Renilla luciferase activity and the replication of NS5 detected in cells transfected with either Rluc-ZIKV-Rep or Rluc-ZIKV-Rep NS5△GDD

Fig.3 Antiviral activity assay using Rluc-ZIKV-Rep

DNA-based flaviviral replicon systems have been previously reported,including a DNA-based West Nile virus replicon[17],a dengue virus replicon system[6,18],and a YFV replicon[19].Replicons proved to be a powerful tool in flaviviral studies.In the present study,a replicon of ZIKV was constructed and evaluated.In this replicon,a Renilla luciferase gene was included in order to monitor virus replication.The Renilla luciferase system is a convenient reporter system that is designed to yield reliable and linear results over a concentration range of at least seven orders of magnitude.In our study,the replication of Rluc-ZIKV-Rep was monitored by measuring Renilla luciferase activity.Results indicated that the constructed replicon(Rluc-ZIKV-Rep)can efficiently replicate and readily express foreign genes in a stable manner.Thus,the ZIKV replicon system can be used to reliably study the life cycle of the virus.

People infected with ZIKV can lack any apparent symptoms.Individuals whose immunity is relatively weak,however,often develop severe disease or symptoms.Fever,rashes,arthralgia,conjunctivitis,fatigue,headache,and myalgia are the main symptoms of ZIKV infection[24～25].The screening and identification of antiviral drugs that are effective against ZIKV are urgently needed to address this global disease.The replicon system designed in the current study lacks structural genes,and thus is not infectious.It could provide a safe and efficient way to detect ZIKV replication in the course of screening antiviral drugs.A high-throughput screening system has been designed to identify small molecular inhibitors of the ZIKV replicon,which could potentially be used for antiviral therapy.

Ca2+has a rapid and strong effect on the induction of many cell processes in response to some sensor signals.Calcium is involved in rapid responses such as cytoskeleton reconstruction,as well as slow responses including proliferation and apoptosis.The concentration of intracellular calcium is generally four times greater than that of extracellular calcium.Increases in intracellular Ca2+occur during endocytosis-mediated entry of several enveloped viruses[26].Ca2+appears to affect the entry of the Ebola virus into host cells[27],viral replication[26],and hemorrhagic fever virus budding[28].Both of the compounds manidipine and cilnidipine evaluated in the present study are Ca2+inhibitors.Our results indicated that both compounds had strong inhibitory activities against the replication of Rluc-ZIKVRep.In summary,a simple,safe and efficient ZIKV replicon system was constructed in the present study,and it can be reliably used to screen effective anti-ZIKV compounds and also to study the life cycle of the virus.

Acknowledgements

We thank Dr.Wang Han-zhong and Dr.Xiao Geng-fu(Wuhan Institute of Virology,Chinese A-cademy of Sciences)for generously providing the infectious cDNA clone of ZIKV and the antiviral drugs manidipine and cilnidipine,respectively.