Secretion of HBV from the late endosome is regulated by the small GTPase Rab7
Jun Inoue1, Eugene W. Krueger1, Mark A. McNiven1,2;
1Center for Basic Research in Digestive Diseases, Mayo Clinic, Rochester, MN; 2Department of Biochemistry and Molecular Biology, Mayo Clinic, Rochester, MN
Background: The infectious life cycle of the hepatitis B virus (HBV) from endocytic internalization to infectious secretion is poorly defined. It has been shown that viral assembly appears to occur in the late endosomes/multivesicular bodies (MVBs) of the hepatocyte where mature virons are packaged and subsequently secreted into the blood space. Understanding the regulation of this trafficking step utilized by this virus is essential toward disrupting its growth and infection. The small GTPase Rab7 is known to act as a regulatory switch in mediating the transport of cargo from the MVB to the lysosome for degradation. The central GOAL of this study was to define the role of the MVB in HBV propagation, and, test if Rab7 regulates the life cycle of this virus by controlling its traffic between the MVB, the lysosome, and the cell surface during secretion/infection. Results: Confocal immunofluorescence microscopy (IF) of HBV genome-transfected HepG2.2.15 cells showed that two different HBV proteins (LHBs and HBc) localized with Rab7 and LAMP1 at the MVB and lysosome, respectively. Importantly, depletion of Rab7 by siRNA decreased the colocalization with LAMP1 and significantly increased both the retained levels of cytoplasmic LHBs protein and the HBV DNA secreted into the culture supernatant. This effect was rescued by overexpression of wild type Rab7. In support of these findings, electron microscopy (EM) of these cells showed that Rab7 depletion led to a striking increase in the number and size of virus-containing MVBs compared to the non-transfected parental HepG2 cells. Remarkably, many of the MVBs in the HBV-transfected cells exhibited a large number of tubules extending outward into the cytoplasm. Accordingly, Rab7 activity was markedly increased (7-fold) in the HBV-expressing HepG2.2.15 cells compared to parental HepG2 cells. To define which viral components are responsible for the increased Rab7 activity and the subsequent changes in MVB morphology, all five individual HBV proteins were expressed in HuH7, Hep3B, and Hela cells. Importantly, a 2-4 fold increase in Rab7 activation was observed in all 3 cell types expressing the exogenous HBeAg protein alone while the other proteins had no effect. Conclusion: These findings suggest that membrane traffic from the MVB plays an essential role in the infectious secretion cycle of HBV. Most surprising is that the virus itself appears to regulate Rab7 activity and MVB dynamics to aid in its own propagation.
The following people have nothing to disclose: Jun Inoue, Eugene W. Krueger, Mark A. McNiven