| Porcine reproductive and respiratory syndrome virus(PRRSV)is the pathogen of the highly contagious disease PRRS.PRRSV can cause reproductive failure in pregnant pigs,respiratory symptoms in piglets and growing pigs,and is a disease of high mortality rate which can also cause persistent infection and immune suppression.PRRSV has mutated into the highly pathogenic strains and brought great economic losses to the pig industry all over the world.PRRSV is a member of Arteriviridae,with envelope in the surface and single-stranded and positive-sense RNA genome inside.The RNA virus just need to transport its genome to the perinuclear region for translation and replication,while the DNA virus’ s genome must be transported to enter the nucleus for replication.Since there are protein network of cytoskeleton and organelles with different functions the cytoplasm of the host cells,the free diffusion of molecular that is greater than 500 k Da is prevented.Without the help of the host’s cytoskeleton and the inner membrane transport system,it is difficult for the virus to reach a specific destination through a gelatinous cytoplasm.At present,the mutation of the virus and its immune suppression effect,persistent infection and the approach of invasion,have been widely researched.While the molecular mechanism on the way of PRRSV transport its genome to the region around the nucleus of host cells and complete the entire multiplication has not been studied well.Rab is the largest family of small GTP enzymes which play a major role in the regulation of the cell membrane transport system,it is difficult for the virus to reach a specific destination through a gelatinous cytoplasm.At present,the mutation of the virus and its immune suppression effect,persistent infection and the approach of invasion have been widely researched,while the molecular mechanism of PRRSV genome transport to the area around the nucleus of host cells and that of the viral multiplication cycle have not yet been studied intensively.The family of Rab proteins is the largest family of small GTP enzymes which play a major role in the regulation of the cell membrane transport system of eukaryotes.There are nearly 70 kinds of Rab proteins in mammals,different types of Rab proteins are regulated by different factors along the upstream,and effect on different factors downstream.They play important roles in vesicle formation,transport,adhesion,anchoring and integration,formming a complex regulatory network in the cells.The important roles of Rab in different virus replication have already been reported increasingly.For example,the functional Rab5 in early endosomes is involved in the invasion of Bernard virus.Rab7 is recruited to early endosomes and then mediate the transport of simliki virus from early endosomes to late endosomes.Rab11 which is an import GTP binding protein in thecycling endosomes is essential for the assembly of respiratory syncytial virus,Sendai virus,Andes virus,and so on.Based on these studies,it’s high possible that Rab play important roles in the intracellular transport,shelling and assemble and release of PRRSV.In this study,we used the monkey embryonic kidney epithelial cells Marc-145 which are susceptible to PRRSV in vitro to investigate the role of Rab protein in the multiplication of PRRSV after its invasion into the host cells.Firstly,we established the sh RNA library for Chlorocebus sabaeus Rab genes by searching on NCBI.The sh RNA library includes 62 Rab genes and 187 clones.Secondly,the sh RNAs were cloned into the p LKO.1 vector and then cotransfected into HEK293T/17 cells with the packaging plasmids--p MD2.G and ps PAX2 for lentivirus production.The collected lentivirus was used to infect Marc-145.Then the cell lines with Rab genes stably knockdown were selected by puromycine and the relative expression of Rab m RNA in different cells lines was detected by real-time quantitative PCR.By these ways,we screened out different stable cell lines called sh Rab-Marc145 cells,which can effectively knockdown the expression of different Rab genes.Then we used green fluorescent protein labeled PRRSV strains infected all these different sh Rab-Marc145 cells,and tested the fluorescence intensity of different infected cells by a multifunctional microplate scanner,and observed through the fluorescence microscopy at the same time.Then the specific Rabs which can make obvious effect on the multiplication of PRRSV can be initially determined.The results showed that we successfully estabilished the sh RNA Library of Rab gene,and screened out all of the stable transfected cell lines.RT-q PCR analysis showed that the expression levels of the Rab m RNAs in these 62 stable cell lines were significantly decreased with specific Rab gene expression.The test that PRRSV-GFP infect sh Rab-Marc145 showed that,14 sh Rab-Marc145 cell lines could significantly inhibit the multiplication of PRRSV-GFP,while 15 sh Rab-Marc145 cell lines showed a weak inhibitory effect,6 sh Rab-Marc145 cell lines can obviously promote the multiplication of PRRSV-GFP,and 7 sh Rab-Marc145 cell lines showed weak effect in promoting multiplication.Among 62 cell lines,20 kinds of different sh Rab-Marc145 cells lines showed no significant effects on the multiplication of PRRSV-GFP.These results suggest that Rabs exactly play important role in PRRSV multiplication.This study provides a basis for the further exploration of the specific function of Rab protein in the life cycle of PRRSV and the molecular mechanism of how PRRSV in the host cells can reach the perinuclear region. |
