Zika Virus NS2A Protein Induces The Degradation Of KPNA2 Via Chaperone-mediated Autophagy

ZIKV is a mosquito-borne RNA virus of the genus Flavivirus,the family Flaviviridae.ZIKV was first isolated from a monkey in the Zika forest of Uganda in 1947.The majority of the ZIKV-infected are asymptomatic or have only mild symptoms.ZIKV infection of pregnant women,however,can pass the virus to their fetuses,leading to congenital severe microcephaly and neurological deficiency.Infection can also cause premature delivery or miscarriage in pregnant women.ZIKV infected adults can cause Guillain-Barre syndrome(GBS),a rare but serious autoimmune disorder.The virus did not draw much attention until the first documented human outbreak occurred in the Pacific Islands and the recent epidemic in South America.The virus has been transmitted to over 60 countries.The recent outbreak in the central and south Americas has drawn wide public attention to ZIKV infection and led to the latest research on ZIKV.However,ZIKV-host cells interactions are not well understood.There is no vaccine or treatment medicine for Zika virus infection or related diseases.Our earlier study demonstrated that ZIKV infection induces the elevation of KPNA6,an isoform of karyopherin ? and that KPNA6 knockout dampens ZIKV replication.We performed mass spectrometry on samples of ZIKV infection and noninfection to screen out a number of significantly reduced proteins.The results showed a significant reduction of KPNA2 in ZIKV infected cells.Karyopherin ?2(KPNA2,also known as importin-?1)is known as the primary transporter for some transcription factors to activate cellular proliferation and differentiation.Aberrant KPNA2 expression levels have been found in a variety of cancers.This research is divided into the following four parts.First,to determine the influence ZIKV has on KPNA2 in host cells,we detected the protein level and m RNA level of cells with ZIKV infection.We found that the protein level of ZIKV after infection was significantly reduced,while its m RNA level has not,and the protein level of KPNA2 was decreased in a time and dose dependent manner.It was also found that ZIKV could induce the reduction of KPNA2 in Hela and SK-N-SH cells.Second,in order to determine the degradation pathway of KPNA2,we applied protease inhibitor MG132 and hydrolase inhibitor NH4 CL to cells.We found that both inhibitors could restore the level of KPNA2 caused by ZIKV infection.Detection of the half-life of KPNA2 after ZIKV infection showed that ZIKV could shorten the half-life of KPNA2 by promoting its degradation.We also detected the level of ubiquitination.It was found that ZIKV infection did not cause significant change in the level of ubiquitination.We speculated that KPNA2 was degraded through lysosomal pathway.After analyzing the sequence of KPNA2,we found it had a KFERQ-like motif,which is characteristic for Chaperon mediated Autophagy,CMA.Then we studied the interaction between KPNA2 and Lysosome membrane-associated protein type 2A,LAMP2 A.It was confirmed that there is interaction between KPNA2 and LAMP2 A and that LAMP2 A knockdown can restore the level of KPNA2 caused by ZIKV infection.Third,to clarify the mechanisms of KPNA2 degradation induced by ZIKV,we screened the proteins of ZIKV and its non-structural protein NS2 A was identified as the key protein leading to the degradation of KPNA2.We first confirmed that there is an interaction between KPNA2 and NS2 A.In LAMP2 A knockdown cells,the expression of NS2 A did not induce the reduction of KPNA2.NS2 A truncations were constructed to screen the key site that cause the reduction of KPNA2,and it was found that both NS2A-D1 and NS2A-D2 could cause the reduction.To construct the mutants,point mutation was carried out on the amino acid at position 90-100 aa of NS2 A.It was found that NS2A-M4,which amino acid at position 100 is mutated,could restore the level of KPNA2.Studies on the interaction between NS2A-M4 and KPNA2 showed that NS2A-M4 cannot interact with KPNA2 efficiently.In order to determine the effect of NS2 A on ZIKV replication,reverse genetics was used to rescue the mutant virus.It was found that the replication of the mutant virus with mutated amino acid at 100 aa was significantly reduced,and the infection of the mutant virus can not induce a significant reduction of KPNA2.Fourth,to figure out the influence of KPNA2 on the replication of ZIKV in host cells.We constructed mutants of KPNA2 by inactivated amino acid of KFERQ-like sequence of KPNA2,LSREKQ,and found that KPNA2-M4 with amino acid at position 109 mutated could save the reduction of KPNA2 after ZIKV infection.We speculated that the amino acid at position 109 plays an important role in CMA pathway.We studied the interaction between KPNA2-M4 and LAMP2 A,we found that KPNA2-M4 almost lost the ability to interact with LAMP2 A.In addition,the increase of replication ability of ZIKV in KPNA2 knockout cells indicted the potential antiviral effect of KPNA2.Our research focus on the interaction mechanisms between ZIKV and KPNA2 in host cells by employing techniques in molecular biology,cytobiology,virology,and reverse genetics.Here,we demonstrate that KPNA2 is degraded via the chaperonemediated autophagy(CMA)and that Zika virus(ZIKV)enhances the KPNA2 degradation.The CMA motif of KPNA2 was identified by site-directed mutagenesis.The residue Q109 of the motif was shown to be indispensable for the degradation.The knockdown of LAMP2 A,a vital component of the CMA pathway,led to a higher level of KPNA2.Moreover,ZIKV NS2 A protein reduced KPNA2 via the CMA pathway.The residue T100 of NS2 A was shown to be essential for inducing KPNA2 degradation.Notably,mutant ZIKV with T100 A in NS2 A replicates much weaker than the wildtype virus.Also,knockdown of KPNA2 led to a higher level of ZIKV replication,which indicates that KPNA2 mediates some antiviral effects.This research lays a foundation for the further study on the replication and pathogenetic mechanisms of Zika virus,and provides a theoretical basis for the development of vaccine and targeted medicine against Zika virus.