Entry of pseudotyped hepatitis C virus into primary human hepatocytes depends on the scavenger class B type I receptor

Summary. Entry of the hepatitis C virus (HCV) into the cell seems to be a complex multi‐step process involving several cellular factors such as the scavenger class B type I receptor (SRBI). Until now, all investigations conducted to assess the involvement of SRBI have been based on in vitro infection models which use human hepatoma‐derived cell lines. However, the HCV entry pathway may be altered in these types of cells because of the impairment of some hepatic characteristics. In this study, we showed that SRBI also plays an essential role in HCV entry into primary human hepatocytes with two distinct approaches: gene extinction and antibodies neutralization assays.     

A human monoclonal antibody that recognizes viral polypeptides and in vitro translation products of the genome of the hepatitis D virus

Peripheral blood lymphocytes from a patient chronically infected with hepatitis D virus (HDV) were immortalized by Epstein-Barr virus transformation. Two stable monoclonal cell lines, derived from the same parent culture, were established and produced antibodies of the IgG isotype that were specific for the hepatitis delta antigen (HDAg). Both monoclonal antibodies (MAbs) recognized the major HDAg polypeptides of 24 kilodaltons and 27 kilodaltons that were previously detected by polyclonal antibodies to HDAg in both liver and serum from HDV-infected humans, chimpanzees, and woodchucks. This result indicates that the major polypeptides of HDAg share common epitopes. The MAbs also reacted with minor polypeptides of lower molecular weight, which were present in infected liver. In vitro translation products of HDV-specific RNA from infected liver were also detected by the MAbs; these polypeptides were 24 kilodaltons and 27 kilodaltons, respectively, and comigrated with liver- or serum-derived HDAg. In contrast, HDV RNA isolated from virions in serum was not translated into HDAg polypeptides in the in vitro system.     

Activation of the rat scavenger receptor class B type I gene by PPARalpha

Peroxisomal proliferator activated receptor alpha (PPARalpha) is activated by fibrate drugs which are known to protect against atherosclerosis. The present study examines the effects of PPARalpha on SR-BI expression. For this study, a rat SR-BI promoter-luciferase reporter gene construct was co-transfected into different cell lines with expression vectors that encode for PPARalpha+/-retinoic X receptor alpha (RXRalpha). PPARalpha/RXR increased the activity of the SR-BI promoter, an effect that was enhanced by clofibrate. Sequence analysis of the rat SR-BI promoter revealed the presence of a putative peroxisomal proliferator response element (PPRE) at bp -1,622. Electrophoretic mobility shift assays demonstrated that PPARalpha and RXRalpha are able to bind to the SR-BI PPRE motif. In addition, mutational analysis studies confirmed that this PPRE motif is responsible for the PPARalpha/RXRalpha-dependent activation of the rat SR-BI promoter in the cell lines examined.     

Ethnic differences in viral dominance patterns in patients with hepatitis B virus and hepatitis C virus dual infection

Studies of hepatitis B virus (HBV)/hepatitis C virus (HCV) dual infection are limited. Most are small, conducted outside the United States, and compare dual infection with HCV monoinfection. The goal of this study was to characterize HBV/HCV dual infection in a large multiethnic, matched, case-control study of dual-infected and HBV-monoinfected patients at two United States centers. Using an International Classification of Disease Version 9 electronic query and chart review, we identified 115 HBV/HCV dual-infected patients with serial HBV DNA, HCV RNA, and alanine aminotransferase (ALT) levels. As a control, 115 HBV-monoinfected patients were chosen randomly and matched with cases by age ±10 years, sex, Asian versus non-Asian ethnicity, and study site. Both groups had similar sex, ethnic, and age distributions (68% male, 83% Asian, age 52 ± 14 years). The median follow-up times were 33 and 38 months for the dual-infected and monoinfected groups, respectively. More monoinfected patients received HBV antiviral therapy than dual-infected patients (43% versus 24%; P = 0.002). No significant difference was detected between the proportion of monoinfected versus dual-infected patients with ALT above 40 U/L at presentation or during follow-up. Dual infection patients exhibited very little HBV/HCV codominance at baseline and throughout follow-up: patients had either HBV viremia with low or absent HCV RNA or detectable HCV RNA with low or absent HBV DNA. Asian ethnicity was predictive of HBV dominance after adjusting for sex, age, and baseline ALT elevation (odds ratio 7.35; P = 0.01). CONCLUSION: HBV/HCV dual-infected and HBV-monoinfected patients had similar clinical characteristics. Asian ethnicity is a major independent predictor of HBV-dominant disease, and HCV dominance with undetectable HBV DNA is more common in non-Asian individuals. Larger studies are needed to further characterize the natural history of HBV/HCV dual infection in Asian and non-Asian individuals.     

Intrafamilial spread of hepatitis B virus infection in Greece

OBJECTIVE: No study has investigated the intrafamilial spread of hepatitis B virus (HBV) in Greece. We conducted a 9-year prospective study to determine the rate of HBV spread in family members when a member is identified as an HBV carrier, the possible routes and risk factors for transmission of HBV and the family members with the highest risk of infection according to kinship degrees. METHODS: A total of 387 family members of 166 hepatitis B surface antigen (HBsAg) carriers were investigated for the detection of HBV infection markers using standard enzyme immunoassays; 6.696 blood donors from the same area were used as controls. RESULTS: Serological markers of past or current HBV infection were detected significantly more frequently among family members of HBsAg carriers (23.2 and 15.8%, respectively) compared with blood donors (14.1 and 0.85%, respectively). The prevalence of the above markers was higher among siblings, husbands and parents of the carriers. Offspring of the female index cases had higher rates of current or past infection. HBV infection markers were significantly increased in family members who reported common use of syringes (P<0.001), birth in rural areas (P<0.001) and a low level of education (P<0.001). CONCLUSIONS: We demonstrated a high risk of HBV transmission among family members of HBsAg carriers, which was associated with special risk factors for contracting HBV. Our findings indicate the need for strict adherence to the universal guidelines of vaccination against HBV and also the need for an immediate investigation of other potentially infected relatives among family members of HBsAg carriers.     

Dual chronic hepatitis B virus and hepatitis C virus infection

Dual hepatitis B virus (HBV) and hepatitis C virus (HCV) infection are common in HBV or HCV endemic areas. However, several clinical and pathogenetic issues remain unresolved. First, clinical and in vitro studies suggest the interactions between two viruses. The dynamics of the interaction in untreated setting versus treated setting and its influence on the long-term outcomes await further studies. A key issue regarding viral interactions is whether modulation of infection occurs in the same dually infected individual hepatocyte of the liver. Clarifying this issue may help to understand the reciprocal interference between HCV and HBV and provide clues for future immunopathogenetic studies. Second, the prevalence and clinical significance of coexisting occult HBV infection in patients with chronic HCV infection need further investigations. Third, combination therapy of peginterferon alfa-2a and ribavirin appears to be just as effective and safe for the treatment of hepatitis B surface antigen (HBsAg)-positive patients chronically infected with active chronic hepatitis C as it is in patients with HCV monoinfection. Nevertheless, one-third of dually infected patients with nondetectable serum HBV DNA-level pretreatment developed HBV reactivation posttreatment. How to prevent and treat this reactivation should be clarified. Furthermore, about 10% of the dually infected patients lost HBsAg. Underlying mechanisms await further investigations. Finally, the optimal treatment strategies for dually infected patients with hepatitis B e antigen-positive chronic hepatitis B should be identified in future clinical trials.     

A mouse monoclonal antibody against Epstein-Barr virus envelope glycoprotein 350 prevents infection both in vitro and in vivo

A mouse monoclonal antibody (MAb) against Epstein-Barr virus (EBV) envelope glycoprotein 350, 72A1, inhibited EBV infection of B lymphocytes in vitro. When severe combined immunodeficient mice were injected with EBV-seronegative donors' peripheral-blood mononuclear cells and challenged with EBV, 72A1 MAb prevented development of EBV-positive tumors: none of the test mice (0/12) developed EBV-positive tumors. In contrast, 67% (8/12) of control mice developed EBV-positive tumors (P=.001). Purified 72A1 MAb was infused into 1 healthy adult and 4 EBV-seronegative children after liver transplant. No adverse reactions were seen in the adult or in 3 of the transplant recipients. The remaining patient developed a hypersensitivity reaction, thus underlining the need to humanize the MAb.     

Robust hepatitis C virus infection in vitro

The absence of a robust cell culture model of hepatitis C virus (HCV) infection has severely limited analysis of the HCV life cycle and the development of effective antivirals and vaccines. Here we report the establishment of a simple yet robust HCV cell culture infection system based on the HCV JFH-1 molecular clone and Huh-7-derived cell lines that allows the production of virus that can be efficiently propagated in tissue culture. This system provides a powerful tool for the analysis of host-virus interactions that should facilitate the discovery of antiviral drugs and vaccines for this important human pathogen.     

Isolated antibody to hepatitis B core antigen in patients with chronic hepatitis C virus infection

AIM: To evaluate the prevalence of isolated anti-HBc in patients with chronic hepatitis C virus (HCV) infection, and its relation to disease severity. METHODS: We screened all patients with chronic HCV infection referred to King Faisal Specialist Hospital and Research Center for hepatitis B surface antigen (HBsAg), antibody to hepatitis B surface antigen (anti-HBs), and anti-HBc. One hundred and sixty nine patients who tested negative for both HBsAg and anti-HBs were included in this study. RESULTS: Pathologically, 59 had biopsy-proven cirrhosis and 110 had chronic active hepatitis (CAH). Of these 169 patients, 85 (50.3%) tested positive for anti-HBc. Patients with CAH had significantly higher prevalence of isolated anti-HBc than patients with cirrhosis, 71 (64.5%) and 14 (23.7%) respectively (P < 0.001). Twenty-five patients were tested for HBV DNA by qualitative PCR. The test was positive in 3 of them (12%; occult HBV infection). CONCLUSION: Isolated anti-HBc alone is common in Saudi patients with chronic HCV infection, and is significantly more common in those with CAH than those with cirrhosis. Therefore, a screening strategy that only tests for HBsAg and anti-HBs in these patients will miss a large number of individuals with isolated anti-HBc, who may be potentially infectious.     

Phosphorylation-dependent human immunodeficiency virus type 1 infection and nuclear targeting of viral DNA.

In the replication of human immunodeficiency virus type 1 (HIV-1), gag MA (matrix), a major structural protein of the virus, carries out opposing targeting functions. During virus assembly, gag MA is cotranslationally myristoylated, a modification required for membrane targeting of gag polyproteins. During virus infection, however, gag MA, by virtue of a nuclear targeting signal at its N terminus, facilitates the nuclear localization of viral DNA and establishment of the provirus. We now show that phosphorylation of gag MA on tyrosine and serine prior to and during virus infection facilitates its dissociation from the membrane, thus allowing it to translocate to the nucleus. Inhibition of gag MA phosphorylation either on tyrosine or on serine prevents gag MA-mediated nuclear targeting of viral nucleic acids and impairs virus infectivity. The requirement for gag MA phosphorylation in virus infection is underscored by our finding that a serine/threonine kinase is associated with virions of HIV-1. These results reveal a novel level of regulation of primate lentivirus infectivity.     

Liver/kidney microsome antibody type 1 and hepatitis C virus infection.

Recent studies have shown that hepatitis C virus antibodies are present in a large proportion of patients with autoimmune hepatitis type 2. We have studied 83 patients with liver/kidney microsome antibody-positive type 1 hepatitis. Hepatitis C virus antibodies were sought in every case by second-generation tests (hepatitis C virus enzyme-linked immunosorbent assay and recombinant immunoblot assay). Hepatitis C virus RNA sequences were sought in 22 patients (12 with recombinant immunoblot assay-positive results and 10 with recombinant immunoblot assay-negative results) by means of polymerase chain reaction and by use of primers located in the 5' noncoding region. Sixty-four patients (77%) had positive results for hepatitis C virus antibodies in the enzyme-linked immunosorbent assay test, and 41 (49.3%) were confirmed by recombinant immunoblot assay. Hepatitis C virus RNA sequences were found in all the recombinant immunoblot assay-positive patients but in none of the 10 who were recombinant immunoblot assay-negative. The recombinant immunoblot assay-negative patients were younger than those who were positive (13 +/- 11 vs. 50 +/- 11 years) and had higher gamma-globulin levels and liver/kidney microsome antibody-positive type 1 titers (61% had a titer of 1:1,000 or more, vs. only 17% of the recombinant immunoblot assay-positive patients).(ABSTRACT TRUNCATED AT 250 WORDS)     

Identification of a PDZ-domain-containing protein that interacts with the scavenger receptor class B type I

The scavenger receptor class B type I (SR-BI) mediates the selective uptake of cholesteryl esters from high-density lipoprotein (HDL) and cholesterol secretion into bile in the liver. In this study, we identified an SR-BI-associated protein from rat liver membrane extracts by using an affinity chromatography technique. This protein of 523 amino acids contains four PDZ domains and associates with the C terminus of SR-BI by using its N-terminal first PDZ domain. Therefore, we denoted this protein as CLAMP (C-terminal linking and modulating protein). CLAMP was located mostly in the sinusoidal membranes, whereas SR-BI was detected in both sinusoidal and canalicular membranes. After the solubilization of the liver membranes with Triton X-100, SR-BI was immunoprecipitated with anti-CLAMP monoclonal antibody, suggesting the association of these proteins in vivo. By coexpressing SR-BI with CLAMP in Chinese hamster ovary cells, we observed (i) the increase in the expression level of SR-BI, (ii) the reduction in the deacylation rate of the cholesteryl esters taken up from HDL, and (iii) the change in the intracellular distribution of fluorescent lipid 1,1'-dioctadecyl-3,3, 3',3'-tetramethylindocarbocyanine percholate taken up from HDL. Taken together, these data suggest that CLAMP, a four-PDZ-domain-containing protein, is associated with SR-BI in the liver sinusoidal plasma membranes and may modulate the intracellular transport and metabolism of cholesteryl esters taken up from HDL.     

The scavenger receptor class B type I adaptor protein PDZK1 maintains endothelial monolayer integrity

Circulating levels of high-density lipoprotein (HDL) cholesterol are inversely related to the risk of cardiovascular disease, and HDL and the HDL receptor scavenger receptor class B type I (SR-BI) initiate signaling in endothelium through src that promotes endothelial NO synthase activity and cell migration. Such signaling requires the C-terminal PDZ-interacting domain of SR-BI. Here we show that the PDZ domain-containing protein PDZK1 is expressed in endothelium and required for HDL activation of endothelial NO synthase and cell migration; in contrast, endothelial cell responses to other stimuli, including vascular endothelial growth factor, are PDZK1-independent. Coimmunoprecipitation experiments reveal that Src interacts with SR-BI, and this process is PDZK1-independent. PDZK1 also does not regulate SR-BI abundance or plasma membrane localization in endothelium or HDL binding or cholesterol efflux. Alternatively, PDZK1 is required for HDL/SR-BI to induce Src phosphorylation. Paralleling the in vitro findings, carotid artery reendothelialization following perivascular electric injury is absent in PDZK1-/- mice, and this phenotype persists in PDZK1-/- mice with genetic reconstitution of PDZK1 expression in liver, where PDZK1 modifies SR-BI abundance. Thus, PDZK1 is uniquely required for HDL/SR-BI signaling in endothelium, and through these mechanisms, it is critically involved in the maintenance of endothelial monolayer integrity.