Involvement Of Rab8 And Myosin Vc In DV2 Infection In HepG2 Cells

Dengue virus (DV) is an enveloped, single-stranded RNA virus belonging to the family Flaviviridae. The viruses are comprised of four distinct serotypes, DV1 through DV4. DV infection causes a wide range of symptoms from an unapparent or mild disease (dengue fever, DF) to severe, life-threatening complications (dengue hemorrhagic fever/dengue shock syndrome, DHF/DSS). This virus has spread throughout the tropical and subtropical regions worldwide over the past several decades by two mosquito species: Aedes aegypti and A. albopictus. According to the WHO's reports, almost 10 million of DV cases occurs annually, most of which take place in Southeast Asia. And recently its infection has reemerged as a more and more severe threat against human health. However, the pathogenesis about DF and DHF/DSS remains unclear.Some ultrastructural data has been reported that DV virions are observed in the lumen of the rough endoplasmic reticulum (rER), rER-derived vesicles and the Golgi region not only in infected mosquito and Vero cells but also in neurons. After nucleocapsids are assembled and acquire their envelopes and associated structures, a part of virus particles are transferred to the Golgi system for maturation and are delivered from the cell by exocytosis. As it parasitizes in cells, DV is supposed to utilize the living cell's exocytosis pathway to release from hosts. Thus, which regulator of cellular transports would be involved in the maturation of the newly biosynthesized virions? The answer would provide more details on life cycle of DV and maybe provide further insight into controlling DF and DHF/DSS and therapy.Rab proteins are a Ras-related family of low-molecular weight monomeric GTP-binding proteins (~20 to 29 kDa), which are key regulators of vesicular transports within eukaryotic cells. The Rab8 GTPase localizes predominantly on the trans-Golgi network (TGN) region and transport vesicles and regulates vesicular traffics from Golgi to plasma membrane. It is involved in transports and secretion of many proteins, for example the actin-based movement of melanosomes. Recently, some reports refine our understanding on Rab8. Apical peptidases and transporters abnormally localize to lysosomes in the small intestine of Rab8-deficient mice. Their mislocalization and degradation in lysosomes lead to a marked reduction in the absorption rate of nutrients in the small intestine. In general, Rab8 is involved in biosynthesis and traffics of plasma membrane and some membrane proteins and its abnormal function have great effect on not only exocytosis but also endocytosis. Therefore, we suppose that DV2 virions which mature at the region from ER to Golgi may employ the exocytosis pathways associated with Rab8 to be released from host cells, on the other hand, DV receptors or components involved in endocytic pathways for DV may also need Rab8 to be properly secreted or located on plasma membrane.For actin-mediated movement of Rab8-positive vesicles are largely dependent on Rab8 activity, Rab8 would be ideal to regulate specific recruitment of motor proteins to defined vesicles. MyosinⅤc (Myo5c) is a novel member of classⅤmyosins, which class is the most efficient and processive in actin-mediated transport, and selectively co-localizes with a membrane compartment containing Rab8. When Myo5c tail, as a dominant negative mutant of Myo5c, is expressed in cells, transferrin accumulates in some cellular compartment, suggesting that Myo5c is involved in transferrin trafficking. Thus Myo5c may drive actin-mediated membrane trafficking pathway in many physiologically crucial tissues of the human body. Because we have confirmed the contribution of actin during DV2 infection, we suppose whether Myo5c, which is a motor of actin and interacts with Rab8, would be also involved in this infection process.Although the liver is not a major target organ, the involvement of liver cells in pathogenesis of DV infection has been indicated by the abnormal liver function, pathological findings, and detection of viral antigen in Hepatocytes and Kupffer's cells at biopsies. It is reported that DV can replicate in a human hepatocarcinoma cell line, HepG2, and infectious particles are released into the culture medium. In this paper, HepG2 cells were used to study the effects of Rab8 and Myo5c on DV2 infection. Results and conclusions:1. Preparation of anti-Rab8 polyclonal antibodyRab8 gene was TA-cloned from HepG2 cells by RT-PCR and the prokaryotic expression plasmid, pQE31-Rab8-122, was constructed with the gene segment encoding 122 amino acids of Rab8 in C terminal. After expression in E. coil and purification by Ni affinity chromatograph, truncated Rab8 protein, which was a 15 kDa fusion protein containing 6×His in N terminal, was used to immunize rabbits to obtain the antiserum. The anti-Rab8 polyclonal antibody showed highly titer and specificity, and could recognize endogenous Rab8 antigens in HepG2 cells. And it might support the experiments on the involvement of Rab8 in viral infection.2. Preparation of anti-myo5c polyclonal antibodypQE31-Myo5c-297 was constructed with the DNA fragment encoding 297 amino acids of myo5c inα-helical coiled-coil tail domain. After expression and purification, truncated myo5c protein, which was a 42 kDa fusion protein containing 6×His in N terminal, was used to immunize Balb/c mouse to obtain the antiserum. The anti-myo5c polyclonal antibody showed highly titer and specificity, and could recognize endogenous myo5c antigens in HepG2 cells and human gastric mucosa. And it might support the experiments on the involvement of myo5c in viral infection.3. Involvement of Rab8 in life cycle of DV2A. Highly co-localization of Rab8 with DV2 in HepG2 cells.After infection, although there were no visible changes in distribution pattern of endogenous Rab8 antigen, which predominantly localized in the cytoplasm from the perinuclear region to plasma membrane in HepG2 cells, some obvious puncta-like structure of Rab8 was revealed in the cytoplasma. Rab8 showed highly co-localization with DV2 antigen and the rate of their co-localization was up to about 90% in the puncta-like components analyzed by confocal laser microscope. This result may suggest close association of Rab8 and DV2.B. Establishment of HepG2 cells stably expressing Rab8 mutants.HepG2 cells were transfected with plasmids of pcDNA3.1, pcDNA3.1-Rab8Q67L and pcDNA3.1-Rab8T22N by lipofectamine followed by G418 selection. HepG2p3.1, HepG2Rab8AM and HepG2Rab8DN cells were obtained and then assessed by flow cytometric analysis. The result showed that HepG2Rab8AM cells stably expressed Rab8Q67L, a constitutively active mutant of Rab8, and HepG2Rab8DN cells stably expressed Rab8T22N, a dominant negative mutant of Rab8.C. The process of DV2 infection is inhibited by expressing Rab8 mutants.More than 60 % HepG2p3.1 cells were infected by DV2 at 24 h p.i., while only about 20-30% DV2 antigen-positive cells were observed in HepG2Rab8AM or HepG2Rab8DN cells. The result by immuno-staining assay indicates that the process of DV2 infection should be impacted by interrupting the function of Rab8 in cells.D. Supernatant progeny virions are reduced by expressing Rab8 mutants.The supernatant virus released from HepG2Rab8AM, HepG2Rab8DN and HepG2p3.1 cells at 8, 24 and 48 h p.i. were detected by plaque assay. Expression of Rab8Q67L reduced virus released from HepG2Rab8AM cells by 97.0 % and 82.2 % at 24 and 48 h p.i. respectively, and expression of Rab8T22N reduced virus released from HepG2Rab8DN cells by 77.2 % and 53.3 % at 24 and 48 h p.i. respectively. Thus, our results indicate that Rab8 is most likely to be necessary for DV2 release or production and expression of Rab8Q67L has stronger inhibitory effects than expression of Rab8T22N.E. Productions of infectious virions are inhibited by expressing Rab8 mutants.The intracellular virus titers of HepG2Rab8AM, HepG2Rab8DN and HepG2p3.1 cells at 8, 24 and 48 h p.i. were assayed by plaque assay. Expression of Rab8Q67L reduced production of infectious virus in HepG2Rab8AM cells by 96.4 % and 78.8% at 24 and 48 h p.i. respectively, while expression of Rab8T22N reduced production of infectious virus in HepG2Rab8DN cells by 86.4 % and 80.2 % at 24 and 48 h p.i. respectively. Our data indicate that expression of Rab8 mutants significantly inhibits the production of progeny virions and Rab8 may be an important host factor for the formation of infectious DV2 in HepG2 cells.F. Viral RNA replication was down-regulated by a different level.To further understand the interruption of DV2 production by expressing Rab8 mutants, the replication levels of viral RNA in HepG2Rab8AM, HepG2Rab8DN and HepG2p3.1 cells were detected by real-time RT-PCR. We found expression of Rab8Q67L inhibited DV2 replication in HepG2Rab8AM cells by 55.3 % and 28.5% at 24 and 48 h p.i., respectively. The similar results were obtained in HepG2Rab8DN cells, and expression of Rab8T22N inhibited replication by 33 % and 36 % at 24 and 48 h p.i., respectively. Those data indicate that viral replication is down-regulated by expressing Rab8 mutants. While as compared with intracellular titers at the same time points, levels of viral RNA decrease not as significantly as the production of infectious virions, suggesting that viral assembly, not replication, is most likely to be interrupted by expression of Rab8 mutants.G. Expressions of Rab8 mutants inhibit the viral entry into HepG2 cell.The amounts of DV2 entry into HepG2Rab8AM, HepG2Rab8DN and HepG2p3.1 cells were detected by viral entry assay. The results showed that expression of Rab8T22N reduced DV2 entry into HepG2Rab8DN cells by 79.7 % and expression of Rab8Q67L even reduced viral entry into HepG2Rab8AM by 83.1 %, compared with those in HepG2p3.1 cells. The data show the inhibition of DV2 entry into HepG2 cells by expressing Rab8 mutants, suggesting the involvement of Rab8 in DV2 entry into cells.4. Modulation of DV2 release by Myo5cA. Establishment of HepG2 cells stably expressing Myo5c tail.HepG2 cells were transfected with plasmids of pCI-neo and pCI-Myo5c-tail by lipofectamine. HepG2pCI and HepG2Myo5c-tail cells were obtained by G418 selection and then assessed by Western Bolt. The rabbit anti-Myo5c pAb specifically reacted with both the endogenous Myo5c proteins (203 kDa) in HepG2pCI and with Myo5c-tail protein (93 kDa) expressed in HepG2Myo5c-tail cells. The result showed that HepG2Myo5c-tail cells stably expressed Myo5c tail.B. Association of Rab8 with Myo5c in HepG2 cells.By double staining analysis, both Rab8 and Myo5c localized in the cytoplasm from the perinuclear region to plasma membrane in HepG2 cells, and about 90% co-localization of Myo5c with Rab8 was confirmed in HepG2 cells by confocal laser microscope, suggesting the highly co-localization of Rab8 with Myo5c.By flow cytometric analysis, Rab8 was up-regulated by expressing Myo5c tail in HepG2Myo5c-tail cells, which indicated that compensated increase of Rab8 was induced by functional inhibition of Myo5c.C. Myo5c indirectly modulates the release of DV2.The result of double staining showed that although both Myo5c and DV2 localized in the cytoplasm from the perinuclear region to plasma membrane in infected HepG2 cells at 24 h p.i., the rate of their co-localization was poor. Thus, there is rare direct interaction of Myo5c with DV2.Virus titers of supernatants and cell fraction from HepG2pCI and HepG2Myo5c-tail cells were assayed by plaque assay. As compared with the control, expression of Myo5c tail reduced progeny virus release by 32.5 %, 39.4 % and 47.3 % at 8, 24 and 48 h p.i., respectively. However, viral titers in HepG2Myo5c-tail cells fraction only showed a tendency of slightly decreasing. The data indicate that Myo5c indirectly modulate the release of DV2 from HepG2 cells but not the formation of infectious virions.