A molecularly cloned hepatitis B virus produced in vitro is infectious in a chimpanzee.

The infectivity of hepatitis B virus (HBV) produced in vitro by HepG2 cells transfected with HBV DNA (HepG2T14) has been assayed in a chimpanzee. Following inoculation, the chimpanzee underwent a typical course of type B hepatitis infection, characterized by elevation of serum aminotransferases and by histological identification of hepatic damage. Hepatitis B surface antigen and core-related antigen appeared in the serum at weeks 5 and 7, respectively, after infection. HBV DNA was detected in serum samples, and replicative forms of the HBV genome were identified in liver biopsies. Subtype identification of hepatitis B surface antigen and restriction enzyme analysis of HBV DNA in both the inoculum and the serum of the infected chimpanzee confirmed that the hepatitis B infection observed in this animal was caused by viral particles produced by HepG2T14 cells. These findings indicate that, although HepG2 cells do not seem to be susceptible to infection by HBV in vitro, they can produce biologically active infectious virions after transfection with cloned HBV DNA.     

Cloned hepatitis delta virus cDNA is infectious in the chimpanzee.

A head-to-tail trimer of a full-length cDNA clone of the hepatitis delta virus (HDV) genome was examined for infectivity by direct inoculation into the liver of a chimpanzee that was already infected with hepatitis B virus. Five weeks after inoculation, a marked elevation of serum alanine aminotransferase activity was observed, followed by the appearance of high levels of HDV RNA and antigen in both liver and serum and a high level of viral particles in the serum. A transient suppression of hepatitis B virus replication was evident during the acute phase of HDV infection. Seroconversion for antibodies to delta antigen occurred 3 weeks after the onset of the disease. These results demonstrate that a typical HDV infection can be initiated by inoculation of a susceptible animal with recombinant HDV cDNA.     

Production of infectious hepatitis delta virus in vitro and neutralization with antibodies directed against hepatitis B virus pre-S antigens.

Hepatitis delta virus (HDV) particles were produced in Huh7 human hepatoma cells by transfection with cloned hepatitis B virus (HBV) DNA and HDV cDNA. The particles were characterized by their buoyant density, the presence of encapsidated viral RNA, and their ability to infect primary cultures of chimpanzee hepatocytes. Successful infection was evidenced by the appearance of increasing amounts of intracellular HDV RNA after exposure to particles. Infection was prevented when particles were incubated with antibodies directed against synthetic peptides specific for epitopes of the pre-S1 or pre-S2 domains of the HBV envelope proteins before exposure to hepatocytes. These data demonstrate that HDV particles produced in vitro are infectious and indicate (i) that infectious particles are coated with HBV envelope proteins that contain the pre-S1 and pre-S2 regions, (ii) that epitopes of the pre-S1 and pre-S2 domains of HBV envelope proteins are exposed at the surface of HDV particles, and (iii) that antibodies directed against those epitopes have neutralizing activity against HDV.     

SNARF-1 as an intracellular pH indicator in laser microspectrofluorometry: a critical assessment

The use of SNARF-1-AM (seminaphtorhodafluor-1-acetoxymethylester) to measure the internal pH of a single living cell by laser microspectrofluorometry has been analyzed with a lymphocyte murine B cell line A20. After incubation of the cells at 37 degrees C in the presence of 10 microM SNARF-1-AM, the internal concentration of SNARF-1 was approximately 200 microM. The enhancement of fluorescent intensity of the probe is concomitant with its leakage out of the cells. During the measurement period, this induces a continuous increase of the contribution of the external probe to the total fluorescence intensity. This prevented classical spectrofluorometry measurements, but did not preclude microspectrofluorometry measurements of internal pH. The ratio R was calculated from fluorescence intensities at 635 and 590 nm and used as an indicator of the intracellular pH. Calibration curves of the intracellular pH were obtained in the presence of nigericin and valinomycin. It appeared that both the fluorescence intensity and the ratio R were lower inside the cell than those values obtained in aqueous solutions. Possible interactions with the main biological macromolecules (i.e., DNA, proteins, membranes) were investigated as well as a possible compartmentation of the probe in cellular organelles. The modifications of probe characteristics inside the cells were attributed to the binding of the probe to cellular proteins. The intracellular pH of A20 cells, measured by SNARF-1 on 84 cells, was found to be 7.18 +/- 0.10 (with an external pH of 7.40 +/- 0.05), which corresponded with values obtained by conventional fluorometric methods.     

Mutations in the carboxyl-terminal domain of the small hepatitis B virus envelope protein impair the assembly of hepatitis delta virus particles

The carboxyl-terminal domain of the small (S) envelope protein of hepatitis B virus was subjected to mutagenesis to identify sequences important for the envelopment of the nucleocapsid during morphogenesis of hepatitis delta virus (HDV) virions. The mutations consisted of carboxyl-terminal truncations of 4 to 64 amino acid residues and small combined deletions and insertions spanning the entire hydrophobic domain between residues 163 and 224. Truncation of as few as 14 residues partially inhibited glycosylation and secretion of S and prevented assembly or stability of HDV virions. Short internal combined deletions and insertions were tolerated for secretion of subviral particles with the exceptions of those affecting residues 164 to 173 and 219 to 223. However, mutants competent for subviral particle secretion had a reduced capacity for HDV assembly compared to that of the wild type. One exception was a mutant carrying a deletion of residues 214 to 218, which exhibited a twofold increase in HDV assembly (or stability), whereas deletions of residues 179 to 183, 194 to 198, and 199 to 203 were the most inhibitory. Substitutions of single amino acids between residues 194 and 198 demonstrated that HDV assembly deficiency could be assigned to the replacement of the tryptophan residue at position 196. We concluded that assembly of stable HDV particles requires a specific function of the carboxyl terminus of S which is mediated at least in part by Trp-196.     

Functional coupling of acoustic and chemical signals in the courtship behaviour of the male Drosophila melanogaster

During courtship, the male Drosophila melanogaster sends signals to the female through two major sensory channels: chemical and acoustic. These signals are involved in the stimulation of the female to accept copulation. In order to determine the respective importance in the courtship of these signals, their production was controlled using genetical and surgical techniques. Males deprived of the ability to emit both signals are unable to mate, demonstrating that other (e.g. visual or tactile) signals are not sufficient to stimulate the female. If either acoustic or chemical signals are lacking, the courtship success is strongly reduced, the lack of the former having significantly more drastic effects. However, the accelerated matings of males observed with males bearing wild-type hydrocarbons compared with defective ones, whichever the modality of acoustic performance (wing vibration or playback), strongly support the role of cuticular compounds to stimulate females. We can conclude that among the possible factors involved in communication during courtship, acoustic and chemical signals may act in a synergistic way and not separately in D. melanogaster.     

Hepatitis B virus subviral envelope particle morphogenesis and intracellular trafficking

Hepatitis B virus (HBV) is unusual in that its surface proteins (small [S], medium, and large [L]) are not only incorporated into the virion envelope but they also bud into empty subviral particles in great excess over virions. The morphogenesis of these subviral envelope particles remains unclear, but the S protein is essential and sufficient for budding. We show here that, in contrast to the presumed model, the HBV subviral particle formed by the S protein self-assembles into branched filaments in the lumen of the endoplasmic reticulum (ER). These long filaments are then folded and bridged for packing into crystal-like structures, which are then transported by ER-derived vesicles to the ER-Golgi intermediate compartment (ERGIC). Within the ERGIC, they are unpacked and relaxed, and their size and shape probably limits further progression through the secretory pathway. Such progression requires their conversion into spherical particles, which occurred spontaneously during the purification of these filaments by affinity chromatography. Small branched filaments are also formed by the L protein in the ER lumen, but these filaments are not packed into transport vesicles. They are transported less efficiently to the ERGIC, potentially accounting for the retention of the L protein within cells. These findings shed light on an important step in the HBV infectious cycle, as the intracellular accumulation of HBV subviral filaments may be directly linked to viral pathogenesis.     

hnRNP A1 and the SR proteins ASF/SF2 and SC35 have antagonistic functions in splicing of beta-tropomyosin exon 6B

Mutually exclusive splicing of exons 6A and 6B from the chicken beta-tropomyosin gene involves numerous regulatory sequences. Previously, we identified a G-rich intronic sequence (S3) downstream of exon 6B. This element consists of six G-rich motifs, mutations of which abolish splicing of exon 6B. In this paper, we investigated the cellular factors that bind to this G-rich element. By using RNA affinity chromatography, we identified heterogeneous nuclear ribonucleoprotein (hnRNP) A1, the SR proteins ASF/SF2 and SC35, and hnRNP F/H as specific components that are assembled onto the G-rich element. By using hnRNP A1-depleted HeLa nuclear extract and add-back experiments, we show that hnRNP A1 has a negative effect on splicing of exon 6B. In agreement with in vitro data, artificial recruitment of hnRNP A1, as a fusion with the MS2 coat protein, also represses splicing of exon 6B ex vivo. In contrast, ASF/SF2 and SC35 activate splicing of exon 6B. As observed with other systems, hnRNP A1 counteracts the stimulating effect of the SR proteins. Moreover, cross-linking experiments show that both ASF/SF2 and SC35 are able to displace binding of hnRNP A1 to the G-rich element, suggesting that the binding sites for these proteins are overlapping. These data indicate that the G-rich sequence is a composite element that acts as an enhancer or as a silencer, depending on which proteins bind to them.     

The polypyrimidine tract binding protein (PTB) represses splicing of exon 6B from the beta-tropomyosin pre-mRNA by directly interfering with the binding of the U2AF65 subunit

Splicing of exon 6B from the beta-tropomyosin pre-mRNA is repressed in nonmuscle cells and myoblasts by a complex array of intronic elements surrounding the exon. In this study, we analyzed the proteins that mediate splicing repression of exon 6B through binding to the upstream element. We identified the polypyrimidine tract binding protein (PTB) as a component of complexes isolated from myoblasts that assemble onto the branch point region and the pyrimidine tract. In vitro splicing assays and PTB knockdown experiments by RNA interference demonstrated that PTB acts as a repressor of splicing of exon 6B. Using psoralen experiments, we showed that PTB acts at an early stage of spliceosome assembly by preventing the binding of U2 snRNA on the branch point. Using UV cross-linking and immunoprecipitation experiments with site-specific labeled RNA in PTB-depleted nuclear extracts, we found that the decrease in PTB was correlated with an increase in U2AF65. In addition, competition experiments showed that PTB is able to displace the binding of U2AF65 on the polypyrimidine tract. Our results strongly support a model whereby PTB competes with U2AF65 for binding to the polypyrimidine tract.