Persistent hepatitis C virus infection in microscale primary human hepatocyte cultures.

Hepatitis C virus (HCV) remains a major public health problem, affecting approximately 130 million people worldwide. HCV infection can lead to cirrhosis, hepatocellular carcinoma, and end-stage liver disease, as well as extrahepatic complications such as cryoglobulinemia and lymphoma. Preventative and therapeutic options are severely limited; there is no HCV vaccine available, and nonspecific, IFN-based treatments are frequently ineffective. Development of targeted antivirals has been hampered by the lack of robust HCV cell culture systems that reliably predict human responses. Here, we show the entire HCV life cycle recapitulated in micropatterned cocultures (MPCCs) of primary human hepatocytes and supportive stroma in a multiwell format. MPCCs form polarized cell layers expressing all known HCV entry factors and sustain viral replication for several weeks. When coupled with highly sensitive fluorescence- and luminescence-based reporter systems, MPCCs have potential as a high-throughput platform for simultaneous assessment of in vitro efficacy and toxicity profiles of anti-HCV therapeutics.

Human occludin is a hepatitis C virus entry factor required for infection of mouse cells.

Hepatitis C virus (HCV) is a leading cause of liver disease worldwide. The development of much needed specific antiviral therapies and an effective vaccine has been hampered by the lack of a convenient small animal model. The determinants restricting HCV tropism to human and chimpanzee hosts are unknown. Replication of the viral RNA has been demonstrated in mouse cells, but these cells are not infectable with either lentiviral particles bearing HCV glycoproteins (HCVpp) or HCV produced in cell culture (HCVcc) (A.P., M.E. and C.M.R., unpublished observations), suggesting that there is a block at the level of entry. Here we show, using an iterative complementary DNA library screening approach, that human occludin (OCLN) is an essential HCV cell entry factor that is able to render murine cells infectable with HCVpp. Similarly, OCLN is required for the HCV-susceptibility of human cells, because its overexpression in uninfectable cells specifically enhanced HCVpp uptake, whereas its silencing in permissive cells impaired both HCVpp and HCVcc infection. In addition to OCLN, HCVpp infection of murine cells required expression of the previously identified HCV entry factors CD81 (ref. 4), scavenger receptor class B type I (SR-BI, also known as SCARB1) and claudin-1 (CLDN1). Although the mouse versions of SR-BI and CLDN1 function at least as well as the human proteins in promoting HCV entry, both OCLN and CD81 must be of human origin to allow efficient infection. The species-specific determinants of OCLN were mapped to its second extracellular loop. The identification of OCLN as a new HCV entry factor further highlights the importance of the tight junction complex in the viral entry process, and provides an important advance towards efforts to develop small animal models for HCV.

Formation and function of the polar body contractile ring in Spisula.

Initial studies suggested that spatial organization of the putative polar body contractile ring was determined by the peripheral aster in Spisula [Biol. Bull. 205 (2003) 192]. Here we report detailed supporting observations, including testing of aster and ring function with inhibitors. The metaphase peripheral aster was confirmed to spread cortically in an umbrella-like pattern, with microtubule-poor center. The aster disassembled during anaphase, leaving the spindle docked at the F-actin-poor center of a newly generated cortical F-actin ring that closely approximated the aster in location, measured diameter range, and pattern. Cytochalasin D and latrunculin-B permitted all events except ring and polar body formation. Nocodazole disassembly or taxol stabilization of the peripheral aster produced poorly defined rings or bulging anaphase asters within the ring center, respectively, inhibiting polar body formation. Polar body extrusion occurred at the ring center, the diameter of which diminished. Ring contractility-previously assumed-was verified using blebbistatin, a myosin-II ATPase inhibitor that permitted ring assembly but blocked polar body extrusion. The data support the hypothesis that peripheral aster spreading, perhaps dynein-driven, is causally related to polar body contractile ring formation, with anaphase entry and aster disassembly also required for polar body biogenesis. Previously reported astral spreading during embryonic micromere formation suggests that related mechanisms are involved in asymmetric somatic cytokinesis.