RESUMO
Hepatitis B virus (HBV) and woolly monkey hepatitis B virus (WMHBV) are primate hepadnaviruses that display restricted tissue and host tropisms. Hepatitis D virus (HDV) particles pseudotyped with HBV and WMHBV envelopes (HBV-HDV and WM-HDV) preferentially infect human and spider monkey hepatocytes, respectively, thereby confirming host range bias in vitro. The analysis of chimeric HBV and WMHBV large (L) envelope proteins suggests that the pre-S1 domain may comprise two regions that affect infectivity: one within the amino-terminal 40 amino acids of pre-S1 and one downstream of this region. In the present study, we further characterized the role of the amino terminus of pre-S1 in infectivity by examining the ability of synthetic peptides to competitively block HDV infection of primary human and spider monkey hepatocytes. A synthetic peptide representing the first 45 residues of the pre-S1 domain of the HBV L protein blocked infectivity of HBV-HDV and WM-HDV, with a requirement for myristylation of the amino terminal residue. Competition studies with truncated peptides suggested that pre-S1 residues 5 to 20 represent the minimal domain for inhibition of HDV infection and, thus, presumably represent the residues involved in virus-host receptor interaction. Recombinant pre-S1 proteins expressed in insect cells blocked infection with HBV-HDV and WM-HDV at a concentration of 1 nanomolar. The ability of short pre-S1 peptides to efficiently inhibit HDV infection suggests that they represent suitable ligands for identification of the HBV receptor and that a pre-S1 mimetic may represent a rational therapy for the treatment of HBV infection.
Assuntos
Vírus da Hepatite B/fisiologia , Receptores Virais/análise , Proteínas do Envelope Viral/genética , Sequência de Aminoácidos , Animais , Cebidae , Células Cultivadas , Hepadnaviridae/genética , Hepadnaviridae/fisiologia , Vírus da Hepatite B/genética , Vírus Delta da Hepatite/genética , Humanos , Dados de Sequência Molecular , Pegadas de Proteínas , Estrutura Terciária de Proteína/genética , Receptores Virais/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Especificidade da Espécie , Proteínas do Envelope Viral/metabolismo , Replicação ViralRESUMO
Hepatitis B virus (HBV) and woolly monkey hepatitis B virus (WMHBV) have natural host ranges that are limited to closely related species. The barrier for infection of primates seems to be at the adsorption and/or entry steps of the viral replication cycle, since a human hepatoma cell line is permissive for HBV and WMHBV replication following transfection of cloned DNA. We hypothesized that the HBV and WMHBV envelope proteins contain the principal viral determinants of host range. As previously shown by using the hepatitis D virus (HDV) system, recombinant HBV-HDV particles were infectious in chimpanzee as well as human hepatocytes. We extended the HDV system to include HDV particles pseudotyped with the WMHBV envelope. In agreement with the natural host ranges of HBV and WMHBV, in vitro infections demonstrated that HBV-HDV and WM-HDV particles preferentially infected human and spider monkey cells, respectively. Previous studies have implicated the pre-S1 region of the large (L) envelope protein in receptor binding and host range; therefore, recombinant HDV particles were pseudotyped with the hepadnaviral envelopes containing chimeric L proteins with the first 40 amino acids from the pre-S1 domain exchanged between HBV and WMHBV. Surprisingly, addition of the human amino terminus to the WMHBV L protein increased infectivity on spider monkey hepatocytes but did not increase infectivity for human hepatocytes. Based upon these data, we discuss the possibility that the L protein may be comprised of two domains that affect infectivity and that sequences downstream of residue 40 may influence host range and receptor binding or entry.
Assuntos
Cebidae/virologia , Vírus da Hepatite B/fisiologia , Vírus Delta da Hepatite/fisiologia , Hepatócitos/virologia , Sequência de Aminoácidos , Animais , Humanos , Dados de Sequência Molecular , Fenótipo , Polietilenoglicóis/farmacologia , RNA Viral/análise , Especificidade da Espécie , Proteínas do Envelope Viral/fisiologia , Vírion/fisiologiaRESUMO
Members of the Hepadnaviridae family have been isolated from birds, rodents, and primates. A new hepadnavirus isolated from the woolly monkey, a New World primate, is phylogenetically distinct from other primate isolates. An animal model has been established for woolly monkey hepatitis B virus (WMHBV) by using spider monkeys, since woolly monkeys are endangered. In this study, a greater-than-genome length construct was prepared without amplification by using covalently closed circular DNA extracted from the liver of an infected woolly monkey. Transfection of the human liver cell line Huh7 with WMHBV DNA resulted in the production of viral transcripts, DNA replicative intermediates, and secreted virions at levels similar to those obtained with an infectious human HBV clone, demonstrating that the host range restriction of WMHBV is not at the level of genome replication. WMHBV particles from the medium of transfected cultures initiated an infection in a spider monkey similar to that obtained with virions derived from woolly monkey serum. In an attempt to adapt the virus for higher levels of replication in spider monkeys, immunosuppressed and newborn animals were inoculated. Neither procedure produced persistent infections, and the level of viral replication remained several logs lower than that observed in persistently infected woolly monkeys. These data demonstrate the production of an infectious clone for WMHBV and extend the characterization of the spider monkey animal model.