hepatitis b virus ( hbv ) can cause chronic and acute infection and remains a global health problem with a considerable morbidity and mortality .
it was estimated that there are currently more than 350 million carriers all over the world .
chronic hepatitis b virus ( hbv ) infection is a major cause of hepatocellular carcinoma ( hcc ) .
hbv chronic carriers , including those carrying vaccine - escape mutants , have a greater than 100-fold increased relative risk of developing the tumor .
despite characterization of integrated hbv genome in chromosomes of hcc , a comprehensive understanding of the underlying mechanism needs further elucidation .  
tumor progression is a complex multistage process , making the cells from normal to malignant by a series of alterations including cell adhesion and movements .
one of the important characteristics of the tumor cells is that they have the ability to escape from the normal controls during proliferation , differentiation , apoptosis , and migration . in metastasis ,
tumor cells detached from their place of formation , move , and form new tumors in distant healthy tissues .  
cell migration is a key activity of many important normal and abnormal biological processes , including tumor cell metastasis .
adhesion of tumor cells to host cell layers and subsequent transcellular migration are important processes in cancer invasion and metastasis , which involves the extracellular matrix ( ecm ) degradation [ 810 ] .
high motility and cell migration might be the premonition of malignancy of chronic viral infection .
the rho small gtpases , which contains the rho , rac , cdc42 subfamilies , regulate cell migration through the reorganization of actin cytoskeleton , which is considered as a driving force of cell motility [ 11 , 12 ] .
the rho gtpases act as molecular switches cycling between active gtp - bound forms and inactive gdp - bound forms .
such cycling is regulated by guanosines nucleotide exchange factors ( gefs ) and gtpase - activating proteins ( gaps ) .
in addition , rho gtpases activation has been linked to virus replication such as hiv-1 , hsv ( herpes simplex virus ) and hbv [ 1517 ] .
rho a , rho c , and rac1 have been reported   significantly elevated in a variety of tumors , especially in the more metastatic forms [ 18 , 19 ] .   during the tumor progression , the loss of the cell characteristics results in morphological changes , such as cell - cell adhesion , gene expression , and motility rate .
for example , the adhesion velocity has been shown to be affected by hbv replication .
our laboratory has recently reported that hbv - replicating hepg2 cells display similar morphology with those expressing the constitutively activated rac1 , mediated by hbx .
hbx can directly interact with a rac1 nucleotide exchange factor pix , by an sh3 binding motif and affect the cell morphology .
our data are in line with earlier reports on the potential invasion ability of hbx through indirect activation of matrix metalloproteinase ( mmps ) [ 21 , 22 ] .
we now report that the rac1 gtpase was involved synergistically with hbx in the migration of hbv - replicating cells .
the replicative hbv genome comprising a 3.5 kb linear fragment was constructed as described   in mammalian expression vector pcdna3.1 ( invitrogen , usa ) , using a two step cloning strategy .  
wild - type replicative hbv genome was used as template to generate proline to alanine mutation by site - directed mutagenesis as described in .
four prolines in hbx on the replicative genome were mutated into alanine : esrgr29plpg33plgalppas42p43pivptdh  to esrgr29alpg33algalppas42a43aivptdh .
the plasmids of rac1 wide type , constitutively activated rac1 ,   and dominant negative rac1 were constructed into a mammalian vector pxj40 with a ha tag at n - terminal as described in [ 24 , 25 ] .
hepg2 cells were maintained and passaged in gibco dulbecco 's minimal essential medium , supplemented with 10% fetal bovine serum , 1% antimycotic , and controlled 5% co2 .
rna of the hepg2 cells transfected with various plasmids was extracted using rneasy mini kit ( qiagen , usa ) as described in .
the concentration and quality of rna were obtained by the absorbance at od260 and the absorbance ratio of od260 and od280 .  
rt - pcr was performed to confirm gene expression from the replicative hbv genome as described in .
the primers used for rt - pcr were as follows :  
 -actin forward : 5-cttagttgcgttacaccctttc-3 , 
 -actin reverse : 5-accttcaccgttccagtttt-3 , hbsag forward : 5-tcaccatattcttgggaacaa-3 , hbsag reverse : 5-gttttgttagggtttaaatg-3 , hbcag forward : 5-atctcctagacaccgcctca-3 , hbcag reverse : 5-ttccaaattattacccaccc-3. hepg2 cells transfected with individual constructs were collected 48 hours after transfection .
western blot analysis was carried out using specific anti - ha or anti - actin antibodies as described .
cell motility was assessed by transwell migration assay kit ( bd falcon , usa ) .
the inserts were put into the 24-well plate and both sides of the membrane of the inserts were coated with collagen ( bioscience , usa ) for more than 2 hours .
36 to 48 hours after transfection , the hepg2 cells were trypsinized and cell number was adjusted to 20,00030,000 cells per 200 l volume by cell numbering .
the cell suspension was deposited onto the collagen - coated membrane in each insert in the 24-well plate .
the plate was put into 37c , 5% co2 incubator and cultured for 8 hours to overnight .
this was needed to allow cell migration to take place , from the upper side to the lower side of the collagen - coated membrane .
the medium was then removed and the upside cells of the inserts were gently mopped with cotton bars . the cells at the lower side of the membrane were fixed with 100% methanol for 15 minutes .
the cells were stained with 0.04% giemsa solution ( sigma aldrich , usa ) for 1015 minutes .
four fields were randomly selected in each insert for counting and microscopic images were taken .
hepg2 cells were transfected using nucleofector to achieve high efficiency of transfection , as indicated by the high percentage of cells expressing egfp ( figure 1 ) .  
the expression of hbsag from the replicative genome in transfected hepg2 cells was assessed by rt - pcr , whereas the expression of wild - type rac1 , rac1 , rac1 was determined by western blot analysis using anti - ha antibody.how ever -actin was used as an internal control .
results were shown in figure 2 and table 1 showed the normalized densities of the bands shown in figure 2(a ) .
the density levels of rac1 wt were used as standards , other bands were normalized based on it .
it indicated that all three constructs of rac1 gtpase were successfully expressed in hepg2 cells at comparable level .
in addition , both hbsag and hbv core antigen ( hbcag ) could be detected in hepg2 cells transfected by replicative hbv genome ( lane 2 , figure 2(b ) ) and p - a mutant hbv ( lane 3 , figure 2(b ) ) , with the empty vector pcdna3.1 used as control ( lane 1 , figure 2(b ) ) .  
the motility of hepg2 cells expressing rac1 alone , and those expressing both rac1 and replicative hbv genome was then analysed by transwell assay , as indicated by the number of cells migrated to the lower side of the collagen - coated membrane ( see section 2 ) .
seven sets of constructs were used in this analysis : wild - type rac1 alone ; constitutively activated rac1 alone ; dominant negative rac1 alone ; wild - type rac1 and wild - type hbv genome ; wild - type rac1 and p - a mutant hbv genome ; wild - type hbv genome alone ; and p - a mutant hbv genome alone .
results shown in figure 3 indicated that hepg2 cells displayed differential motility depending on the transfected constructs , with the highest number of cells ( indicated by arrow heads ) in cells transfected either with rac1 alone ( figure 3(b ) ) or with wild - type rac1/wild - type hbv genome ( figure 3(d ) ) .
a graphical representation of the motility assay was summarized in figure 4 after cell counting and analysis .
hepg2 cells transfected with the constitutively activated rac1 showed a 2.5-fold increase in cell motility ( lane 2 , figure 4 ) as compared with those expressing wild - type rac1 alone ( lane 1 , figure 4 ) or those expressing the dominant negative rac1 ( lane 3 , figure 4 ) .
interestingly , cells co - transfected with wild - type rac1 and replicative hbv genome showed similar motility as those transfected with constitutively activated rac1 .
this suggested that the cell motility was enhanced by hbv replication , likely via activation of wild - type rac1 to its gtp - bound state as recently reported . to validate our findings , the p - a mutant hbv genome ( with four proline residues mutated to alanine residues in the proline rich domain ) was co - transfected with the wild - type rac1 .
results shown in figure 4 ( lane 5 ) indicated that the p - a mutant hbv genome abrogated the cell motility significantly , to a level comparable with expressing cells .
meanwhile , the expression of replicative hbv genome itself enhanced the cell migration also ( lane 6 ) , which may imply the endogenous rac1 activation by the hbv replication .
not surprisingly , the p - a mutant hbv genome abrogated the cell motility ( lane 7 ) .
how ever the cell migration level caused by endogenous rac1 was less than that caused by the overexpressed rac1 v12 , which suggested that endogenous wild - type rac1 was not sufficient for the activation caused by hbv replication . and
such activation was confirmed by the augmentation of cell movements induced by overexpression of rac1 wt .
in other words , the endogenous rac1 was enough to be activated by hbv replication and passed away cellular signal inducing high cell motility , but the over - expressed rac1 can even increase such activation in a certain degree .  
taken together , our results suggested that the cell migration can be caused by hbv replication through the activation of rac1 gtpase .
rho gtpases comprise a family of proteins which are highly conserved from lower eukaryotes to plants and animals .
rho gtpases were implicated in various cellular processes including cytoskeletal reorganization , cell cycle and division , motility , angiogenesis , and phagocytosis . for cytoskeletal reorganization ,
processes such as cell adhesion , cell migration , cell polarity , spindle formation and locomotion were covered .  
it has been reported that the hbx activates erks and pi-3k / akt pathways25 , and further activates matrix metalloproteinase-919 with a potential function of invasion and metastasis .
we have recently demonstrated the same activation of erk1/2 and akt by hbx using sh3 binding domain located in hbx .
our recent investigation has also indicated that rac1 gtpase can be activated by hbv replication , which in turn sustains hbv replication .
interestingly , the activation of rac1 and hbv replication has been found to be significantly decreased in cells transfected with p - a mutant hbv genome .
our findings can be further validated by careful design of hbx knockdown in the context of the whole hbv genome , without affecting the proper viral replication . furthermore , the involvement of rac1 can be supported by titration experiments in which the same amount of hbv genome is introduced into the cells with various amount of rac1 ( activated v or inactivated n ) .  
findings in this report have provided an extension on the consequences of the rac1 activation by hbv replication , in terms of cell motility . to our knowledge , this is the first report to show the potential relationship between proline rich domain in hbx ( in the context of hbv replication ) and cell motility , and should shed new lights on the understanding of the mechanism of hcc development .
 hepatitus b virus ( hbv ) is a major cause of the development of hepatpcellular carcinoma ( hcc ) . 
 one of the significant characteristics of tumor progression is cell migration which is reflective of cytoskeletal dynamics . 
 the rho gtpases contribute to a multiple cellular processes , including the cellular cytoskeletal reorganization and motility . 
 it has been found that some rho gtpases have oncogenic activity and can promote cancer cell invasion . 
 here we discuss one of the rho gtpases , rac1 can be activated by hbv replication and such activation results in the high motility of hbv - replicating cells . 
 the enhanced cell motility can be interestingly alleviated by the mutation at the sites of proline rich domain located in hbx . 
 our findings may provide new insights on the mechanism of hcc development associated with chronic hbv infection . 