The Molecular Epidemiology Analysis Of Coxsackievirus A16 And The Mechanism Study Of CA16-induced Autophagy

Backgroud:Coxsackievirus A16 (CA16) is one of the main pathogens of hand, foot and mouth disease (HFMD). In the past ten years, large outbreaks and epidemics of CA16 infection associated HFMD have occurred repeatedly in mainland China. Although in most cases, the clinical symptoms are mild. But in recent years, increasing evidences show that CA16 infection may also lead to serious complications such as aseptic meningitis, lung syndrome, natural abortion and lethal myocarditis. Currently there is no effective drugs or vaccines for CA16. Although many researches have reported the molecular epidemiological characteristics of CA16 in different regions in mainland China, few reports have been about the molecular epidemiology of CA16 in Hubei province.Autophagy is a substance metabolism process that widely exists in eukaryotic cells. Under normal circumstances, cells keep low levels of autophagy to remove the damaged and excess organelles, maintaining homeostasis in the cells. When cells confronted with environmental stresses or invading pathogens, autophagy is induced. Autophagy plays different roles in the processes of different viruses infection and the regulatory mechanisms of different viruses on cell autophagy are not the same. On one hand, autophagy can capture viruses, present antigen, activate the innate immunity and eliminate the viruses; on the other hand viruses have evolved some strategies to suppress or escape autophagy degradation and maintain their own survival. Currently the influence of CA16 infection on autophagy has not been reported. To further clarify the relationship between CA16 infection and cell autophagy not only can help to elucidate the pathogenic mechanism of CA16, but also provide the theory basis and new targets for the research and development of new drugs and vaccines.Objective:(1) To analyze the molecular epidemiological characteristics of CA16 strains circulating in Hubei province of China in 2010-2011;(2) To characterize the relationship between CA16 infection and autophagy;(3) To clarify the influence of autophagy induction on virus replication;(4) To detect the nonstructural proteins of CA16 which play key roles in autophagy induction;(5) To detect the important proteins and signaling pathways which are associated with CA16-induced autophagy.Methods:(1) The throat swab specimens of HFMD were collected from Wuhan region and Enshi region in Hubei province in 2010-2011. The viruses were amplified by cell culture. The viral genome was extracted and then was reverse transcribed into the cDNA. The positive specimens with CA16 were identified by RT-PCR. The VP1 clones were constructed by PCR and TA cloning and then were sequenced. The genotype of CA16 strains in Hubei province in 2010-2011 was analyzed by the phylogenetic analysis. On the basis of the CA16 genome sequences deposited in Gen Bank, four pairs of primers were designed to amplify the fragments spanning the entire genome of ensh01-CHN-12, and each fragment amplified by RT-PCR shared overlapping sequences with its neighbouring fragments. Through sequencing and editing, ensh01-CHN-12 genome sequence was obtained. Simplot software was used to analysis CA16 recombination.(2) To investigate whether CA16 infection can or can not induce autophagy, Western blot was taken to detect the expression of LC3 which is an important autophagy marker. GFP-LC3 was transfected into HeLa cells and the distribution of LC3 dots were detected by confocal microscopy. Transmission electron microscope was used to detect the autophagosomes in CA16-infected and mock-infected HeLa cells.(3) In order to detect whether the CA16 infection can or can not induce complete autophagy flow, the P62 expression levels was tested by Western blot. Moreover, the expression of LC3II was detected by Western blot after autophagy inhibitor chloroquine (CQ) treatment. Based on the theory that the GFP is sensitive to the acidic environment of lysosomes, the mRFP-GFP-LC3 plasmid was used to transfect cells. The confocal microscopy was used to reveal the co-localization of red and green fluorescent proteins and detect the formation of autophagolysosomes.(4) In order to study the effect of CA16-induced autophagy on viral replication, the autophagy inhibitor CQ was used to suppress autophagolysosomes mature, leading to the accumulation of autophagosomes. Then the TCID50 detection was adopted to detect the extracellular virus titer. In addition, the Atg5-shRNA and Beclin-1-shRNA were used to down-regulate the expression of Atg5 and Beclin-1. The extracellular virus titer was measured by TCID50 after decreasing the expression of Atg5 and Beclin-1 which play key roles in the formation of autophagosomes.(5) In order to further explore the key viral proteins involved in autophagy regulation, the eukaryotic expression plasmids of CA16 non structural proteins (2A,2B,2C,3AB, 3C,3D) were constructed. Next the viral non-structural proteins were transfected into the RD and HeLa cells. Western blot was taken to detect the expression of LC3 which is an important autophagy marker. GFP-LC3 and viral protein (2C,3C) were cotransfected into HeLa cells and the distribution of LC3 was detected by confocal microscopy. Transmission electron microscope was used to detect the formation of autophagosomes.(6) In order to further explore the influence of viral proteins (2C,3C) transfection on autophagy flow, the autophagy inhibitor CQ was used to pretreat viral proteins plasmids transfected cells. Then Western blot was adopted to detect the expression of LC3. The mRFP-GFP-LC3 and viral proteins (2C,3C) plasmids were cotransfected into HeLa cells and the formation of autophagosomes and autophagolysosomes were detected by confocal microscope.(7) In order to explore whether IRGM is involved in CA16-induced autophagy, Western blot was used to detect the protein expression of IRGM and QPCR was adopted to detect the mRNA expression of IRGM after CA16 challenge. The activation of viral protein (2C,3C) plasmids transfection on IRGM promoter was detected by luciferase assay. The interaction between IRGM and autophagy related proteins (Atg5, Atg10) was tested by Co-Immunoprecipitation (Co-IP). Meanwhile, Co-IP also was used to detect the interaction between IRGM and viral proteins (2C, 3C). The eukaryotic expression plasmid of IRGM was constructed and then transfected into HeLa cells to detect the effect of IRGM overexpression on autophagy. Meanwhile, the IRGM siRNA was synthesized and transfected into HeLa cells to detect the influence of IRGM on autophagy.(8) To explore whether the AKT-mTOR and MEK-ERK signaling pathways are involved in autophagy, Western blot was used to detect the expression of p-AKT, p-mTOR, p-S6K, p-MEK and p-ERK. Furthermore, myr-Akt (constitutively active myristoylated Akt) plasmid was transfected into HeLa cells to detect the effect of activating AKT on CA16 induced autophagy. PD98059, a specific repressor of MKE/ERK, also was used to investigate the influence of MEK/ERK inhibition on autophagy.Results:(1) This study collected 189 cases of HFMD throat swab specimens. Seven CA16 strains were successfully isolated and all of them were classified into B1 subgenotype. The late spring and early summer was the peak period of HFMD incidence. Children less than five years old were the primary patient populations and morbidity rate of male was higher than female. The Simplot result showed that the CA16 strain circulating in Enshi region maybe the recombination strains and some genetic regions of CA16 were highly similar to EV71.(2) We collected the RD and HeLa protein samples and detected the LC3II expression levels by Western blot. We found that the LC3II expression levels increased significantly after CA16 infection. Meanwhile, the confocal microscopy results showed that intracellular LC3 dots obviously increased. Under the electron microscope, we observed the typical autophagy structures.(3) The results of Western blot showed that the expression of P62 was not decrease along with the infection process of CA16. Moreover, we detected the expression of LC3II upon CQ treatment and found that the expression of LC3Ⅱ was not obviously increased. The confocal microscopy results showed that there were lots of yellow dots (autophagosomes) and fewer red dots (autophagolysosomes) in CA16-infected HeLa cells.(4) We used the CQ to restrain the autophagolysosomes mature and increase the accumulation of autophagosomes. The TCID50 results showed that the extracellular virus titer increased significantly with the increase of autophagosomes. Conversely, the extracellular virus titers were significantly reduced by using Atg5-shRNA and Beclinl - shRNA to suppress the formation of autophagosomes.(5) We constructed the CA16 nonstructural protein expression plasmids and transfected these plasmids to RD and HeLa cells. The Western blot results showed that in HeLa cells the expression levels of LC3Ⅱ/Actin was significantly increased after 2C,3C and 3D viral proteins transfection. In RD cells the expression levels of LC3II/Actin increased significantly after 2C,3AB and 3C viral proteins transfection. Moreover, we observed the increase of LC3 dots in viral proteins (2C,3C) plasmids transfected HeLa cells by using confocal microscopy. In addition, typical autophagy double membrane structures also were detected by electron microscopy.(6) The Western blot results showed that the expression of LC3II was not obviously increase upon CQ treatment in viral protein (2C,3C) plasmids transfected HeLa cells. The confocal microscopy results showed that large numbers of yellow autophagosomes were observed and the red autophagolysosomes were much fewer in mRFP-GFP-LC3 and viral protein (2C,3C) cotransfected HeLa cells.(7) The Western blot and QPCR results showed that the protein and mRNA expression levels of IRGM increased significantly upon CA16 infection. At the same time the IRGM promoter could be activated by 2C protein. The results of Co-IP revealed the interaction between IRMG and autophagy related proteins (Atg5, Atg10). But we did not detect the interaction between viral proteins (2C,3C) and IRGM. The Western blot results demonstrated that the up-regulation expression of IRMG could promote autophagy and down-regulating the expression of IRGM could suppress autophagy.(8) The Western blot results showed that the expression levels of p-AKT, p-mTOR and p-S6K decreased and the expression levels of p-MEK, p-ERK increased in CA16-infected HeLa cells. After transfecting myr-Akt plasmid into HeLa cells, the CA16-induced autophagy was suppressed. Meanwhile, the CA16-induced autophagy was inhibited upon PD98059 treatment.Conclusions:(1) The CA16 strains circulating in 2010-2011 in Hubei province of China belong to B1 genotype.(2) CA16 infection can induce autophagy in RD and HeLa cells, but the autophagy process is incomplete.(3) The increase of autophagosomes can promote CA16 infection and increase the extracellular virus titers.(4) The viral nonstructural proteins 2C and 3C can induce incomplete autophagy.(5) IRGM protein plays an important role in the autophagy process induced by CA16 infection or viral proteins transfection. In addition, the CA16-induced autophagy is dependent on the inhibition of AKT/mTOR signaling pathway and the activation of MEK/ERK signaling pathway.