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Functional chimeras of human immunodeficiency virus type 1 Gp120 and influenza A virus (H3) hemagglutinin.

ABSTRACT: In an attempt to produce a protein that will allow determination of the native human immunodeficiency virus type 1 (HIV-1) gp120 (Env) structure in its trimeric state, we fused the globular head of gp120 to the stalk region of influenza virus A (X31) hemagglutinin (HA). The chimeric protein (EnvHA) has been expressed by using a recombinant vaccinia virus system, and its functional characteristics were determined. EnvHA is expressed as a 120- to 150-kDa protein that can oligomerize to form dimers and trimers. It retains the low-pH (5.2 to 5.4) requirement of X31-HA to trigger membrane fusion but, unlike X31-HA, it is not absolutely dependent on exogenously added trypsin for protein processing to release the HA2 fusion peptide. In terms of receptor binding the chimeric protein retains specificity for human CD4 but, in relation to the membrane fusion event, it appears to lose the Env coreceptor specificity of the parental HIV-1 strains: NL43 for CXCR4 and JRFL for CCR5. These properties suggest that stable, functional EnvHAs are being produced and that they may be exploited in terms of structural studies. Further, the potential of introducing the envHA genes into influenza viruses, by use of reverse genetics, and their use as a therapeutic vaccine for HIV are discussed.

SUBMITTER: Copeland KM

PROVIDER: S-EPMC1091698 | biostudies-literature | 2005 May

REPOSITORIES: biostudies-literature

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Functional chimeras of human immunodeficiency virus type 1 Gp120 and influenza A virus (H3) hemagglutinin.

Copeland K M KM Elliot A J AJ Daniels R S RS

Journal of virology 20050501 10

In an attempt to produce a protein that will allow determination of the native human immunodeficiency virus type 1 (HIV-1) gp120 (Env) structure in its trimeric state, we fused the globular head of gp120 to the stalk region of influenza virus A (X31) hemagglutinin (HA). The chimeric protein (EnvHA) has been expressed by using a recombinant vaccinia virus system, and its functional characteristics were determined. EnvHA is expressed as a 120- to 150-kDa protein that can oligomerize to form dimers ...[more]

PMID: 15858029

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Project description:The nucleotide and amino acid sequences of 40 influenza virus hemagglutinin genes of the H3 serotype from mammalian and avian species and 9 genes of the H4 serotype were compared, and their evolutionary relationships were evaluated. From these relationships, the differences in the mutational characteristics of the viral hemagglutinin in different hosts were examined and the RNA sequence changes that occurred during the generation of the progenitor of the 1968 human pandemic strain were examined. Three major lineages were defined: one containing only equine virus isolates; one containing only avian virus isolates; and one containing avian, swine, and human virus isolates. The human pandemic strain of 1968 was derived from an avian virus most similar to those isolated from ducks in Asia, and the transfer of this virus to humans probably occurred in 1965. Since then, the human viruses have diverged from this progenitor, with the accumulation of approximately 7.9 nucleotide and 3.4 amino acid substitutions per year. Reconstruction of the sequence of the hypothetical ancestral strain at the avian-human transition indicated that only 6 amino acids in the mature hemagglutinin molecule were changed during the transition between an avian virus strain and a human pandemic strain. All of these changes are located in regions of the molecule known to affect receptor binding and antigenicity. Unlike the human H3 influenza virus strains, the equine virus isolates have no close relatives in other species and appear to have diverged from the avian viruses much earlier than did the human virus strains. Mutations were estimated to have accumulated in the equine virus lineage at approximately 3.1 nucleotides and 0.8 amino acids per year. Four swine virus isolates in the analysis each appeared to have been introduced into pigs independently, with two derived from human viruses and two from avian viruses. A comparison of the coding and noncoding mutations in the mammalian and avian lineages showed a significantly lower ratio of coding to total nucleotide changes in the avian viruses. Additionally, the avian virus lineages of both the H3 and H4 serotypes, but not the mammalian virus lineages, showed significantly greater conservation of amino acid sequence in the internal branches of the phylogenetic tree than in the terminal branches. The small number of amino acid differences between the avian viruses and the progenitor of the 1968 pandemic strain and the great phenotypic stability of the avian viruses suggest that strains similar to the progenitor strain will continue to circulate in birds and will be available for reintroduction into humans.

Project description:Membrane-associated RING-CH-type 8 (MARCH8) strongly blocks human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein (Env) incorporation into virions by downregulating its cell surface expression, but the mechanism is still unclear. We now report that MARCH8 also blocks the Ebola virus (EBOV) glycoprotein (GP) incorporation via surface downregulation. To understand how these viral fusion proteins are downregulated, we investigated the effects of MARCH8 on EBOV GP maturation and externalization via the conventional secretion pathway. MARCH8 interacted with EBOV GP and furin when detected by immunoprecipitation and retained the GP/furin complex in the Golgi when their location was tracked by a bimolecular fluorescence complementation (BiFC) assay. MARCH8 did not reduce the GP expression or affect the GP modification by high-mannose N-glycans in the endoplasmic reticulum (ER), but it inhibited the formation of complex N-glycans on the GP in the Golgi. Additionally, the GP O-glycosylation and furin-mediated proteolytic cleavage were also inhibited. Moreover, we identified a novel furin cleavage site on EBOV GP and found that only those fully glycosylated GPs were processed by furin and incorporated into virions. Furthermore, the GP shedding and secretion were all blocked by MARCH8. MARCH8 also blocked the furin-mediated cleavage of HIV-1 Env (gp160) and the highly pathogenic avian influenza virus H5N1 hemagglutinin (HA). We conclude that MARCH8 has a very broad antiviral activity by prohibiting different viral fusion proteins from glycosylation and proteolytic cleavage in the Golgi, which inhibits their transport from the Golgi to the plasma membrane and incorporation into virions.IMPORTANCE Enveloped viruses express three classes of fusion proteins that are required for their entry into host cells via mediating virus and cell membrane fusion. Class I fusion proteins are produced from influenza viruses, retroviruses, Ebola viruses, and coronaviruses. They are first synthesized as a type I transmembrane polypeptide precursor that is subsequently glycosylated and oligomerized. Most of these precursors are cleaved en route to the plasma membrane by a cellular protease furin in the late secretory pathway, generating the trimeric N-terminal receptor-binding and C-terminal fusion subunits. Here, we show that a cellular protein, MARCH8, specifically inhibits the furin-mediated cleavage of EBOV GP, HIV-1 Env, and H5N1 HA. Further analyses uncovered that MARCH8 blocked the EBOV GP glycosylation in the Golgi and inhibited its transport from the Golgi to the plasma membrane. Thus, MARCH8 has a very broad antiviral activity by specifically inactivating different viral fusion proteins.

Dataset Information

Functional chimeras of human immunodeficiency virus type 1 Gp120 and influenza A virus (H3) hemagglutinin.

Publications

Functional chimeras of human immunodeficiency virus type 1 Gp120 and influenza A virus (H3) hemagglutinin.

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